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  • 1.
    Abelein, Axel
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Bolognesi, Benedetta
    Dobson, Christopher M.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lendel, Christofer
    Hydrophobicity and conformational change as mechanistic determinants for nonspecific modulators of amyloid β self-assembly2012In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 51, no 1, p. 126-137Article in journal (Refereed)
    Abstract [en]

    The link between many neurodegenerative disorders, including Alzheimer's and Parkinson's diseases, and the aberrant folding and aggregation of proteins has prompted a comprehensive search for small organic molecules that have the potential to inhibit such processes. Although many compounds have been reported to affect the formation of amyloid fibrils and/or other types of protein aggregates, the mechanisms by which they act are not well understood. A large number of compounds appear to act in a nonspecific way affecting several different amyloidogenic proteins. We describe here a detailed study of the mechanism of action of one representative compound, lacmoid, in the context of the inhibition of the aggregation of the amyloid β-peptide (Aβ) associated with Alzheimer's disease. We show that lacmoid binds Aβ(1-40) in a surfactant-like manner and counteracts the formation of all types of Aβ(1-40) and Aβ(1-42) aggregates. On the basis of these and previous findings, we are able to rationalize the molecular mechanisms of action of nonspecific modulators of protein self-assembly in terms of hydrophobic attraction and the conformational preferences of the polypeptide.

  • 2. Adase, Christopher A.
    et al.
    Draheim, Roger R.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Manson, Michael D.
    The Residue Composition of the Aromatic Anchor of the Second Transmembrane Helix Determines the Signaling Properties of the Aspartate/Maltose Chemoreceptor Tar of Escherichia coli2012In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 51, no 9, p. 1925-1932Article in journal (Refereed)
    Abstract [en]

    Repositioning of the tandem aromatic residues (Trp-209 and Tyr-210) at the cytoplasmic end of the second transmembrane helix (TM2) modulates the signal output of the aspartate/maltose chemoreceptor of Escherichia coli (Tar(Ec)). Here, we directly assessed the effect of the residue composition of the aromatic anchor by studying the function of a library of Tar(Ec) variants that possess all possible combinations of Ala, Phe, Tyr, and Trp at positions 209 and 210. We identified three important properties of the aromatic anchor. First, a Trp residue at position 209 was required to maintain clockwise (CW) signal output in the absence of adaptive methylation, but adaptive methylation restored the ability of all of the mutant receptors to generate CW rotation. Second, when the aromatic anchor was replaced with tandem Ala residues, signaling was less compromised than when an Ala residue occupied position 209 and an aromatic residue occupied position 210. Finally, when Trp was: present at position 209, the identity of the residue at position 210 had little effect on baseline signal output or aspartate chemotaxis, although maltose taxis was significantly affected by some substitutions at position 210. All of the mutant receptors we constructed supported some level of aspartate and maltose taxis in semisolid agar swim plates, but those without Trp at position 209 were overmethylated in their baseline signaling state. These results show the importance of the cytoplasmic aromatic anchor of TM2 in maintaining the baseline Tar(Ec) signal output and responsiveness to attractant signaling.

  • 3. Adase, Christopher A.
    et al.
    Draheim, Roger R.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Goethe University .
    Rueda, Garrett
    Desai, Raj
    Manson, Michael D.
    Residues at the Cytoplasmic End of Transmembrane Helix 2 Determine the Signal Output of the Tar(Ec) Chemoreceptor2013In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 52, no 16, p. 2729-2738Article in journal (Refereed)
    Abstract [en]

    Baseline signal output and communication between the periplasmic and cytoplasmic domains of the Escherichia colt aspartate chemoreceptor Tar(Ec) are both strongly influenced by residues at the C-terminus of transmembrane helix 2 (TM2). In particular, the cytoplasmic aromatic anchor, composed of residues Trp-209 and Tyr-210 in wild type Tar(Ec) is important for determining the CheA kinase-stimulating activity of the receptor and its ability to respond to chemoeffector-induced stimuli. Here, we have studied the effect on Tar(Ec) function of the six residue sequence at positions 207-212 Moving various combinations of aromatic residues among these positions generates substantial changes M receptor activity. Trp has the largest effect on function, both in maintaining normal activity and in altering activity when it is moved. Tyr has a weaker effect, and Phe has the weakest; however, all three aromatic residues can alter signal output when they are placed in novel positions. We also find that Gly-211 plays an important role in receptor function, perhaps because of the flexibility it introduces into the TM2-HAMP domain connector. The conservation of this Gly residue in the high-abundance chemoreceptors of E. coli and Salmonella enterica suggests that it may be important for the nuanced, bidirectional transmembrane signaling that occurs in these proteins.

  • 4.
    Adrait, Annie
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Öhrström, Maria
    Barra, Anne-Laure
    The High Field Laboratory, CNRS/MPI, Grenoble, France.
    Thelander, Lars
    Department of Medical Biochemistry and Biophysicis, Umeå University.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    EPR studies on a stable sulfinyl radical observed in the iron-oxygen reconstituted Y177F/I263C protein double mutant of ribonucleotide reductase from mouse2002In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 41, no 20, p. 6510-6516Article in journal (Refereed)
    Abstract [en]

    Ribonucleotide reductase (RNR) catalyzes the biosynthesis of deoxyribonucleotides. The active enzyme contains a diiron center and a tyrosyl free radical required for enzyme activity. The radical is located at Y177 in the R2 protein of mouse RNR. The radical is formed concomitantly with the μ-oxo-bridged diferric center in a reconstitution reaction between ferrous iron and molecular oxygen in the protein. EPR at 9.6 and 285 GHz was used to investigate the reconstitution reaction in the double-mutant Y177F/I263C of mouse protein R2. The aim was to produce a protein-linked radical derived from the Cys residue in the mutant protein to investigate its formation and characteristics. The mutation Y177F hinders normal radical formation at Y177, and the I263C mutation places a Cys residue at the same distance from the iron center as Y177 in the native protein. In the reconstitution reaction, we observed small amounts of a transient radical with a probable assignment to a peroxy radical, followed by a stable sulfinyl radical, most likely located on C263. The unusual radical stability may be explained by the hydrophobic surroundings of C263, which resemble the hydrophobic pocket surrounding Y177 in native protein R2. The observation of a sulfinyl radical in RNR strengthens the relationship between RNR and another free radical enzyme, pyruvate formate-lyase, where a similar relatively stable sulfinyl radical has been observed in a mutant. Sulfinyl radicals may possibly be considered as stabilized forms of very short-lived thiyl radicals, proposed to be important intermediates in the radical chemistry of RNR.

  • 5.
    Aguilar, Ximena
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Weise, Christoph
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sparrman, Tobias
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wolf-Watz, Magnus
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Macromolecular crowding extended to a heptameric system: the co-chaperonin protein 102011In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 50, no 14, p. 3034-3044Article in journal (Refereed)
    Abstract [en]

    Experiments on monomeric proteins have shown that macromolecular crowding can stabilize toward heat perturbation and also modulate native-state structure. To assess the effects of macromolecular crowding on unfolding of an oligomeric protein, we here tested the effects of the synthetic crowding agent Ficoll 70 on human cpn10 (GroES in E. coli), a heptameric protein consisting of seven identical β-barrel subunits assembling into a ring. Using far-UV circular dichroism (CD), tyrosine fluorescence, nuclear magnetic resonance (NMR), and cross-linking experiments, we investigated thermal and chemical stability, as well as the heptamer-monomer dissociation constant, without and with crowding agent. We find that crowding shifts the heptamer-monomer equilibrium constant in the direction of the heptamer. The cpn10 heptamer is both thermally and thermodynamically stabilized in 300 mg/mL Ficoll 70 as compared to regular buffer conditions. Kinetic unfolding experiments show that the increased stability in crowded conditions, in part, is explained by slower unfolding rates. A thermodynamic cycle reveals that in presence of 300 mg/mL Ficoll the thermodynamic stability of each cpn10 monomer increases by over 30%, whereas the interfaces are stabilized by less than 10%. We also introduce a new approach to analyze the spectroscopic data that makes use of multiple wavelengths: this provides robust error estimates of thermodynamic parameters.

  • 6.
    Alkemar, Gunnar
    et al.
    Södertörn University, School of Life Sciences. Stockholm University.
    Nygård, Odd
    Södertörn University, School of Life Sciences.
    Probing the secondary structure of expansion segment ES6 in 18S ribosomal RNA2006In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 45, no 26, p. 8067-8078Article in journal (Refereed)
    Abstract [en]

    Expansion segment ES6 in 18S ribosomal RNA is, unlike many other expansion segments, present in all eukaryotes. The available data suggest that ES6 is located on the surface of the small ribosomal subunit. Here we have analyzed the secondary structure of the complete ES6 sequence in intact ribosomes from three eukaryotes, wheat, yeast, and mouse, representing different eukaryotic kingdoms. The availability of the ES6 sequence for modification and cleavage by structure sensitive chemicals and enzymatic reagents was analyzed by primer extension and gel electrophoresis on an ABI 377 automated DNA sequencer. The experimental results were used to restrict the number of possible secondary structure models of ES6 generated by the folding software MFOLD. The modification data obtained from the three experimental organisms were very similar despite the sequence variation. Consequently, similar secondary structure models were obtained for the ES6 sequence in wheat, yeast, and mouse ribosomes. A comparison of sequence data from more than 6000 eukaryotes showed that similar structural elements could also be formed in other organisms. The comparative analysis also showed that the extent of compensatory base changes in the suggested helices was low. The in situ structure analysis was complemented by a secondary structure analysis of wheat ES6 transcribed and folded in vitro. The obtained modification data indicate that the secondary structure of the in vitro transcribed sequence differs from that observed in the intact ribosome. These results suggest that chaperones, ribosomal proteins, and/or tertiary rRNA interactions could be involved in the in vivo folding of ES6.

  • 7.
    Almlöf, Martin
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Andér, Martin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Energetics of codon-anticodon recognition on the small ribosomal subunit2007In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 46, no 1, p. 200-209Article in journal (Refereed)
    Abstract [en]

    Recent crystal structures of the small ribosomal subunit have made it possible to examine the detailed energetics of codon recognition on the ribosome by computational methods. The binding of cognate and near-cognate anticodon stem loops to the ribosome decoding center, with mRNA containing the Phe UUU and UUC codons, are analyzed here using explicit solvent molecular dynamics simulations together with the linear interaction energy (LIE) method. The calculated binding free energies are in excellent agreement with experimental binding constants and reproduce the relative effects of mismatches in the first and second codon position versus a mismatch at the wobble position. The simulations further predict that the Leu2 anticodon stem loop is about 10 times more stable than the Ser stem loop in complex with the Phe UUU codon. It is also found that the ribosome significantly enhances the intrinsic stability differences of codon-anticodon complexes in aqueous solution. Structural analysis of the simulations confirms the previously suggested importance of the universally conserved nucleotides A1492, A1493, and G530 in the decoding process.

  • 8.
    Almqvist, Jonas
    et al.
    Stockholm University, Faculty of Science, Department of Physical, Inorganic and Structural Chemistry.
    Huang, Yafei
    Hovmöller, Sven
    Wang, Da-Neng
    Homology Modeling of the Human Microsomal Glucose 6-Phosphate Transporter Explains the Mutations That Cause the Glycogen Storage Disease Type Ib2004In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 43, p. 9289-9297Article in journal (Refereed)
  • 9.
    Almstedt, Karin
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Mårtensson, Lars-Göran
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Carlsson, Uno
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Hammarström, Per
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Thermodynamic interrogation of a folding disease. Mutant mapping of position 107 in human carbonic anhydrase II linked to marble brain disease.2008In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 47, no 5, p. 1288-1298Article in journal (Refereed)
    Abstract [en]

    Marble brain disease (MBD) also known as Guibaud−Vainsel syndrome is caused by autosomal recessive mutations in the human carbonic anhydrase II (HCA II) gene. HCA II is a 259 amino acid single domain enzyme and is dominated by a 10-stranded β-sheet. One mutation associated with MBD entails the H107Y substitution where H107 is a highly conserved residue in the carbonic anhydrase protein family. We have previously demonstrated that the H107Y mutation is a remarkably destabilizing folding mutation [Almstedt et al. (2004) J. Mol. Biol. 342, 619−633]. Here, the exceptional destabilization by the H107Y mutation has been further investigated. A mutational survey of position H107 and a neighboring conserved position E117 has been performed entailing the mutants H107A, H107F, H107N, E117A and the double mutants H107A/E117A and H107N/E117A. All mutants were severely destabilized versus GuHCl and heat denaturation. Thermal denaturation and GuHCl phase diagram and ANS analyses showed that the mutants shifted HCA II toward populating ensembles of intermediates of molten globule type under physiological conditions. The native state stability of the mutants was in the following order:  wt > H107N > E117A > H107A > H107F > H107Y > H107N/E117A > H107A/E117A. In conclusion:  (i) H107N is least destabilizing likely due to compensatory H-bonding ability of the introduced Asn residue. (ii) Double mutant cycles surprisingly reveal additive destabilization of H107N and E117A showing that H107 and E117 are independently stabilizing the folded protein. (iii) H107Y and H107F are exceptionally destabilizing due to bulkiness of the side chains whereas H107A is more accommodating, indicating long-range destabilizing effects of the natural pathogenic H107Y mutation.

  • 10.
    Almstedt, Karin
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Rafstedt, Therese
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Supuran, Claudiu T
    University of Florence.
    Carlsson, Uno
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Hammarström, Per
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Small-Molecule Suppression of Misfolding of Mutated Human Carbonic Anhydrase II Linked to Marble Brain Disease2009In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, no 23, p. 5358-5364Article in journal (Refereed)
    Abstract [en]

    Carbonic anhydrase II deficiency syndrome or Marble brain disease (MBD) is caused by autosomal recessive mutations in the human carbonic anhydrase II (HCA II) gene. Here we report a small-molecule stabilization study of the exceptionally destabilized HCA II mutant H107Y employing inhibitors based on p-aminobenzoyisulfonamide compounds and 1,3,4-thiadiazolylsulfonamides as well as amino acid activators. Protein stability assays showed a significant stabilization by the aromatic sulfonamide inhibitors when present at 10 mu M concentration, providing shifts of the midpoint of thermal denaturation between 10 degrees C and 16 degrees C and increasing the free energies of denaturation 0.5-3.0 kcal/mol as deduced from GuHCl denaturation. This study could be used as a starting point for the design of small-molecule folding modulators and possibly autoactivatable molecules for suppression of misfolding of destabilized HCA II mutants.

  • 11. Andersson, D.
    et al.
    Hammarström, Per
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Carlsson, Uno
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Cofactor-induced refolding: Refolding of molten globule carbonic anhydrase induced by Zn(II) and Co(II)2001In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 40, no 9, p. 2653-2661Article in journal (Refereed)
    Abstract [en]

    The stability versus unfolding to the molten globule intermediate of bovine carbonic anhydrase II (BCA II) in guanidine hydrochloride (GuHCl) was found to depend on the metal ion cofactor [Zn(II) or Co(II)], and the apoenzyme was observed to be least stable. Therefore, it was possible to find a denaturant concentration (1.2 M GuHCl) at which refolding from the molten globule to the native state could be initiated merely by adding the metal ion to the apo molten globule. Thus, refolding could be performed without changing the concentration of the denaturant. The molten globule intermediate of BCA II could still bind the metal cofactor. Cofactor-effected refolding from the molten globule to the native state can be summarized as follows: (1) initially, the metal ion binds to the molten globule, (2) compaction of the metal-binding site region is then induced by the metal ion binding, (3) a functioning active center is formed, and (4) finally, the native tertiary structure is generated in the outer parts of the protein.

  • 12.
    Andér, Martin
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Does glutamine methylation affect the intrinsic conformation of the universally conserved GGQ motif in ribosomal release factors?2009In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, no 15, p. 3483-3489Article in journal (Refereed)
    Abstract [en]

    The GGQ motif is the only universally conserved feature of ribosomal class 1 release factors. Mutational experiments and structural studies have suggested that the glutamine residue of the GGQ motif Q 185 in human eRF1 numbering) is critical for catalysis of the termination   reaction on the ribosome. Furthermore, it has been established that Q185 is NE methylated in prokaryotes as well as eukaryotes, and that methylation significantly enhances the catalytic activity. It is, however, not known whether this methylation affects the intrinsic   structure of the free release factor, which could be important for its interaction with the ribosome. In this work, we report molecular dynamics simulations, starting from 25 different NMR structures of human eRF1, in addressing this problem. The results show that there is   no such structural effect on the free release factor caused by the NE methylation of Q185, suggesting that its role is intimately associated with the ribosome environment.

  • 13.
    Antzutkin, Oleg
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Leapman, Richard D.
    Division of Physical Science, Office of Research Services, National Institutes of Health, Bethesda.
    Balbach, John J.
    Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda.
    Tycko, Robert
    Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda.
    Supramolecular structural constraints on Alzheimer's β-amyloid fibrils from electron microscopy and solid-state nuclear magnetic resonance2002In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 41, no 51, p. 15436-15450Article in journal (Refereed)
    Abstract [en]

    We describe electron microscopy (EM), scanning transmission electron microscopy (STEM), and solid-state nuclear magnetic resonance (NMR) measurements on amyloid fibrils formed by the 42-residue β-amyloid peptide associated with Alzheimer's disease (Aβ1-42) and by residues 10-35 of the full-length peptide (Aβ10-35). These measurements place constraints on the supramolecular structure of the amyloid fibrils, especially the type of β-sheets present in the characteristic amyloid cross-β structural motif and the assembly of these β-sheets into a fibril. EM images of negatively stained Aβ10-35 fibrils and measurements of fibril mass per length (MPL) by STEM show a strong dependence of fibril morphology and MPL on pH. Aβ10-35 fibrils formed at pH 3.7 are single "protofilaments" with MPL equal to twice the value expected for a single cross-β layer. Aβ10-35 fibrils formed at pH 7.4 are apparently pairs of protofilaments or higher order bundles. EM and STEM data for Aβ1-42 fibrils indicate that protofilaments with MPL equal to twice the value expected for a single cross-β layer are also formed by Aβ1-42 and that these protofilaments exist singly and in pairs at pH 7.4. Solid-state NMR measurements of intermolecular distances in Aβ10-35 fibrils, using multiple-quantum 13C NMR, 13C-13C dipolar recoupling, and 15N-13C dipolar recoupling techniques, support the in-register parallel β-sheet organization previously established by Lynn, Meredith, Botto, and co-workers [Benzinger et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 13407-13412; Benzinger et al. (2000) Biochemistry 39, 3491-3499] and show that this β-sheet organization is present at pH 3.7 as well as pH 7.4 despite the differences in fibril morphology and MPL. Solid-state NMR measurements of intermolecular distances in Aβ1-42 fibrils, which represent the first NMR data on Aβ1-42 fibrils, also indicate an in-register parallel β-sheet organization. These results, along with previously reported data on Aβ1-40 fibrils, suggest that the supramolecular structures of Aβ10-35, Aβ1-40, and Aβ1-42 fibrils are quite similar. A schematic structural model of these fibrils, consistent with known experimental EM, STEM, and solid-state NMR data, is presented

  • 14.
    Apostolov, Rossen
    et al.
    KTH, School of Computer Science and Communication (CSC), Centres, Centre for High Performance Computing, PDC.
    Yonezawa, Yasushige
    Standley, Daron M
    Kikugawa, Gota
    Takano, Yu
    Nakamura, Haruki
    Membrane attachment facilitates ligand access to the active site in monoamine oxidase A2009In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, no 25, p. 5864-5873Article in journal (Refereed)
    Abstract [en]

    Monoamine oxidase membrane enzymes are responsible for the catalytic breakdown of extra- and intracellular neurotransmitters and are targets for the development of central nervous system drugs. We analyzed the dynamics of rat MAOA by performing multiple independent molecular dynamics simulations of membrane-bound and membrane-free forms to clarify the relationship between the mechanics of the enzyme and its function, with particular emphasis on the significance of membrane attachment. Principal component analysis of the simulation trajectories as well as correlations in the fluctuations of the residues pointed to the existence of three domains that define the global dynamics of the protein. Interdomain anticorrelated movements in the membrane-bound system facilitated the relaxation of interactions between residues surrounding the substrate cavity and induced conformational changes which expanded the active site cavity and opened putative pathways for substrate uptake and product release. Such events were less pronounced in the membrane-free system due to differences in the nature of the dominant modes of motion. The presence of the lipid environment is suggested to assist in decoupling the interdomain motions, consistent with the observed reduction in enzyme activity under membrane-free conditions. Our results are also in accordance with mutational analysis which shows that modifications of interdomain hinge residues decrease the activity of rat MAOA in solution.

  • 15.
    Ariöz, Candan
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ye, Weihua
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    al Bakali, Amin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ge, Changrong
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Liebau, Jobst
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Götzke, Hansjörg
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wieslander, Åke
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mäler, Lena
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Anionic Lipid Binding to the Foreign Protein MGS Provides a Tight Coupling between Phospholipid Synthesis and Protein Overexpression in Escherichia coli2013In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 52, no 33, p. 5533-5544Article in journal (Refereed)
    Abstract [en]

    Certain membrane proteins involved in lipid synthesis can induce formation of new intracellular membranes in Escherichia coli, i.e., intracellular vesicles. Among those, the foreign monotopic glycosyltransferase MGS from Acholeplasma laidlawii triggers such massive lipid synthesis when overexpressed. To examine the mechanism behind the increased lipid synthesis, we investigated the lipid binding properties of MGS in vivo together with the correlation between lipid synthesis and MGS overexpression levels. A good correlation between produced lipid quantities and overexpressed MGS protein was observed when standard LB medium was supplemented with four different lipid precursors that have significant roles in the lipid biosynthesis pathway. Interestingly, this correlation was highest concerning anionic lipid production and at the same time dependent on the selective binding of anionic lipid molecules by MGS. A selective interaction with anionic lipids was also observed in vitro by P-31 NMR binding studies using bicelles prepared with E. coli lipids. The results clearly demonstrate that the discriminative withdrawal of anionic lipids, especially phosphatidylglycerol, from the membrane through MGS binding triggers an in vivo signal for cells to create a feed-forward stimulation of lipid synthesis in E. coil. By this mechanism, cells can produce more membrane surface in order to accommodate excessively produced MGS molecules, which results in an interdependent cycle of lipid and MGS protein synthesis.

  • 16. Bai, Shi
    et al.
    Jain, Mahendra K.
    Berg, Otto G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Contiguous binding of decylsulfate on the interface-binding surface of pancreatic phospholipase A22008In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 47, no 9, p. 2899-2907Article in journal (Refereed)
    Abstract [en]

    Pig pancreatic IB phospholipase A(2) (PLA2) forms three distinguishable premicellar E-i(#) (i = 1, 2, and 3) complexes at successively higher decylsulfate concentrations. The Hill coefficient for E-1(#) is n(1) = 1.6, and n(2) and n(3) for E-2(#) and E-3(#) are about 8 each. Saturation-transfer difference nuclear magnetic resonance (NMR) and other complementary results with PLA2 show that decylsulfate molecules in E-2(#) and E-3(#) are contiguously and cooperatively clustered on the interface-binding surface or i-face that makes contact with the substrate interface. In these complexes, the saturation-transfer difference NMR signatures of H-1 in decylsulfate are different. The decylsulfate epitope for the successive E, complexes increasingly resembles the micellar complex formed by the binding of PLA2 to preformed micelles. Contiguous cooperative amphiphile binding is predominantly driven by the hydrophobic effect with a modest electrostatic shielding of the sulfate head group in contact with PLA2. The formation of the complexes is also associated with structural change in the enzyme. Calcium affinity of E-2(#) appears to be modestly lower than that of the free enzyme and Ell. Binding of decylsulfate to the i-face does not require the catalytic calcium required for the substrate binding to the active site and for the chemical step. These results show that E-i(#) complexes are useful to structurally characterize the cooperative sequential and contiguous binding of amphiphiles on the i-face. We suggest that the allosteric changes associated with the formation of discrete E-i(#) complexes are surrogates for the catalytic and allosteric states of the interface activated PLA2.

  • 17.
    Balaz, Martina
    et al.
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Sundberg, Mark
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Persson, Malin
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Kvassman, Jan-Olov
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Månsson, Alf
    University of Kalmar, School of Pure and Applied Natural Sciences.
    "Effects of surface adsorption on catalytic activity of heavy meromyosin studied using fluorescent ATP analogue"2007In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 24, p. 4917-4934Article in journal (Refereed)
  • 18.
    Balbach, John J.
    et al.
    National Institutes of Health.
    Ishii, Yoshitaka
    National Institutes of Health.
    Antzutkin, Oleg
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Leapman, Richard D.
    National Institutes of Health.
    Rizzo, Nancy W.
    National Institutes of Health.
    Dyda, Fred
    National Institutes of Health.
    Reed, Jennifer
    National Institutes of Health.
    Tycko, Robert
    University of California.
    Amyloid Fibril Formation by Aβ16-22, a Seven-Residue Fragment of the Alzheimer's β-Amyloid Peptide, and Structural Characterization by Solid State NMR2000In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 39, no 45, p. 13748-13759Article in journal (Refereed)
    Abstract [en]

    The seven-residue peptide N-acetyl-Lys-Leu-Val-Phe-Phe-Ala-Glu-NH2, called Aβ16-22 and representing residues 16-22 of the full-length β-amyloid peptide associated with Alzheimer's disease, is shown by electron microscopy to form highly ordered fibrils upon incubation of aqueous solutions. X-ray powder diffraction and optical birefringence measurements confirm that these are amyloid fibrils. The peptide conformation and supramolecular organization in Aβ16-22 fibrils are investigated by solid state 13C NMR measurements. Two-dimensional magic-angle spinning (2D MAS) exchange and constant-time double-quantum-filtered dipolar recoupling (CTDQFD) measurements indicate a β-strand conformation of the peptide backbone at the central phenylalanine. One-dimensional and two-dimensional spectra of selectively and uniformly labeled samples exhibit 13C NMR line widths of <2 ppm, demonstrating that the peptide, including amino acid side chains, has a well-ordered conformation in the fibrils. Two-dimensional 13C-13C chemical shift correlation spectroscopy permits a nearly complete assignment of backbone and side chain 13C NMR signals and indicates that the β-strand conformation extends across the entire hydrophobic segment from Leu17 through Ala21. 13C multiple-quantum (MQ) NMR and 13C/15N rotational echo double-resonance (REDOR) measurements indicate an antiparallel organization of β-sheets in the Aβ16-22 fibrils. These results suggest that the degree of structural order at the molecular level in amyloid fibrils can approach that in peptide or protein crystals, suggest how the supramolecular organization of β-sheets in amyloid fibrils can be dependent on the peptide sequence, and illustrate the utility of solid state NMR measurements as probes of the molecular structure of amyloid fibrils. Aβ16-22 is among the shortest fibril-forming fragments of full-length β-amyloid reported to date, and hence serves as a useful model system for physical studies of amyloid fibril formation.

  • 19. Balogh, Larissa M.
    et al.
    Le Trong, Isolde
    Kripps, A
    Tars, Kaspars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Stenkamp, E
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Atkins, William M.
    Structural Analysis of a Glutathione Transferase A1-1 Mutant Tailored for High Catalytic Efficiency with Toxic Alkenals2009In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, no 32, p. 7698-7704Article in journal (Refereed)
    Abstract [en]

    The specificity of human glutathione transferase (GST) A1-1 is drastically altered to favor alkenal substrates in the GIMFhelix mutant designed to mimic first-sphere interactions utilized by GSTA4-4. This redesign serves as a model for improving our understanding of the structural determinants that contribute to the distinct specificities of alpha class GSTs. Herein we report the first crystal structures of GIMFhelix, both in complex with GSH and in apo form at 1.98 and 2.38 angstrom resolution. In contrast to the preorganized hydrophobic binding pocket that accommodates alkenals in GSTA4-4, GSTA1-1 includes a dynamic alpha 9 helix that undergoes a ligand-dependent localization to complete the active site. Comparisons of the GIMFhelix structures with previously reported structures show a striking similarity with the GSTA4-4 active site obtained within an essentially GSTA1-1 scaffold and reveal the 0 helix assumes a similar localized structure regardless of active site occupancy in a manner resembling that of GSTA4-4. However, Are cannot fully account for all the structural elements important in GSTA4-4 within the mutant's active site. The contribution of Phe10 to the Tyr212-Phe10-Phe220 network prevents complete C-terminal Closure and demonstrates that the presence of Phe10 within the context of a GSTA4-4-like active site may ultimately hinder Phe220, a key C-terminal residue, from effectively contributing to the active site. In total, these results illustrate the remaining structural differences presumably reflected in the previously reported catalytic efficiencies of GIMFhelix and GSTA4-4 and emphasize the F10P mutation as being necessary to completely accomplish the transformation to a highly specific GST from the more promiscuous GSTA1-1 enzyme.

  • 20. Balogh, Larissa M.
    et al.
    Le Trong, Isolde
    Kripps, Kimberly A.
    Shireman, Laura M.
    Stenkamp, Ronald E.
    Zhang, Wei
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Atkins, William M.
    Substrate Specificity Combined with Stereopromiscuity in Glutathione Transferase A4-4-Dependent Metabolism of 4-Hydroxynonenal2010In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 49, no 7, p. 1541-1548Article in journal (Refereed)
    Abstract [en]

    Conjugation to glutathione (GSH) by glutathione transferase A4-4 (GSTA4-4) is a major route of elimination for the lipid peroxidation product 4-hydroxynonenal (HNE), a toxic compound that contributes to numerous diseases. Both enantiomers of HNE are presumed to be toxic, and GSTA4-4 has negligible stereoselectivity toward them, despite its high catalytic chemospecificity for alkenals. In contrast to the highly flexible, and substrate promiscuous, GSTA1-1 isoform that has poor catalytic efficiency with HNE, GSTA4-4 has been postulated to be a rigid template that is preorganized for HNE metabolism. However, the combination of high substrate chemoselectivity and low substrate stereoselectivity is intriguing. The mechanism by which GSTA4-4 achieves this combination is important, because it must metabolize both enantiomers of HNE to efficiently detoxify the biologically formed mixture. The crystal structures of GSTA4-4 and ail engineered variant of GSTA1-1 with high catalytic efficiency toward HNE, cocrystallized with a GSH-HNE conjugate analogue, demonstrate that GSTA4-4 undergoes no enantiospecific induced fit; instead, the active site residue Arg15 is ideally located to interact with the 4-hydroxyl group of either HNE enantiomer. The results reveal an evolutionary strategy for achieving biologically useful stereopromiscuity toward a toxic racemate, concomitant with high catalytic efficiency and substrate specificity toward ail endogenously formed toxin.

  • 21.
    Bavdek, Andrej
    et al.
    Molecular Immunology, Helmholtz Centre for Infection Research, Braunschweig, Germany.
    Gekara, Nelson O
    Priselac, Dragan
    Gutiérrez Aguirre, Ion
    Darji, Ayub
    Chakraborty, Trinad
    Macek, Peter
    Lakey, Jeremy H
    Weiss, Siegfried
    Anderluh, Gregor
    Sterol and pH interdependence in the binding, oligomerization, and pore formation of Listeriolysin O.2007In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 46, no 14, p. 4425-4437Article in journal (Refereed)
    Abstract [en]

    Listeriolysin O (LLO) is the most important virulence factor of the intracellular pathogen Listeria monocytogenes. Its main task is to enable escape of bacteria from the phagosomal vacuole into the cytoplasm. LLO belongs to the cholesterol-dependent cytolysin (CDC) family but differs from other members, as it exhibits optimal activity at low pH. Its pore forming ability at higher pH values has been largely disregarded in Listeria pathogenesis. Here we show that high cholesterol concentrations in the membrane restore the low activity of LLO at high pH values. LLO binds to lipid membranes, at physiological or even slightly basic pH values, in a cholesterol-dependent fashion. Binding, insertion into lipid monolayers, and permeabilization of calcein-loaded liposomes are maximal above approximately 35 mol % cholesterol, a concentration range typically found in lipid rafts. The narrow transition region of cholesterol concentration separating low and high activity indicates that cholesterol not only allows the binding of LLO to membranes but also affects other steps in pore formation. We were able to detect some of these by surface plasmon resonance-based assays. In particular, we show that LLO recognition of cholesterol is determined by the most exposed 3beta-hydroxy group of cholesterol. In addition, LLO binds and permeabilizes J774 cells and human erythrocytes in a cholesterol-dependent fashion at physiological or slightly basic pH values. The results clearly show that LLO activity at physiological pH cannot be neglected and that its action at sites distal to cell entry may have important physiological consequences for Listeria pathogenesis.

  • 22.
    Belogurov, G A
    et al.
    A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia.
    Fabrichniy, I P
    A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia.
    Pohjanjoki, P
    Department of Biochemistry, University of Turku, Turku, Finland.
    Kasho, V N
    Center for Ulcer Research and Education, Department of Medicine, University of California, Los Angeles, California, USA.
    Lehtihuhta, E
    Department of Biochemistry, University of Turku, Turku, Finland.
    Turkina, Maria V
    A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia.
    Cooperman, B S
    Department of Chemistry, University of Pennsylvania, Pennsylvania, USA.
    Goldman, A
    Institute of Biotechnology, University of Helsinki, Helsinki, Finland.
    Baykov, A A
    A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia.
    Lahti, R
    Department of Biochemistry, University of Turku, Turku, Finland.
    Catalytically important ionizations along the reaction pathway of yeast pyrophosphatase2000In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 39, no 45, p. 13931-13938Article in journal (Refereed)
    Abstract [en]

    Five catalytic functions of yeast inorganic pyrophosphatase were measured over wide pH ranges: steady-state PP(i) hydrolysis (pH 4. 8-10) and synthesis (6.3-9.3), phosphate-water oxygen exchange (pH 4. 8-9.3), equilibrium formation of enzyme-bound PP(i) (pH 4.8-9.3), and Mg(2+) binding (pH 5.5-9.3). These data confirmed that enzyme-PP(i) intermediate undergoes isomerization in the reaction cycle and allowed estimation of the microscopic rate constant for chemical bond breakage and the macroscopic rate constant for PP(i) release. The isomerization was found to decrease the pK(a) of the essential group in the enzyme-PP(i) intermediate, presumably nucleophilic water, from >7 to 5.85. Protonation of the isomerized enzyme-PP(i) intermediate decelerates PP(i) hydrolysis but accelerates PP(i) release by affecting the back isomerization. The binding of two Mg(2+) ions to free enzyme requires about five basic groups with a mean pK(a) of 6.3. An acidic group with a pK(a) approximately 9 is modulatory in PP(i) hydrolysis and metal ion binding, suggesting that this group maintains overall enzyme structure rather than being directly involved in catalysis.

  • 23.
    Belogurov, Georgiy A
    et al.
    Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland; A. N. Belozersky Institute of Physico-Chemical Biology and School of Chemistry, Moscow State University, Moscow, Russia.
    Malinen, Anssi M
    Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland.
    Turkina, Maria V
    A. N. Belozersky Institute of Physico-Chemical Biology and School of Chemistry, Moscow State University, Moscow, Russia.
    Jalonen, Ulla
    Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland.
    Rytkönen, Kalle
    Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland.
    Baykov, Alexander A
    A. N. Belozersky Institute of Physico-Chemical Biology and School of Chemistry, Moscow State University, Moscow, Russia.
    Lahti, Reijo
    Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland.
    Membrane-bound pyrophosphatase of Thermotoga maritima requires sodium for activity2005In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 44, no 6, p. 2088-2096Article in journal (Refereed)
    Abstract [en]

    Membrane-bound pyrophosphatase of the hyperthermophilic bacterium Thermotoga maritima(Tm-PPase), a homologue of H(+)-translocating pyrophosphatase, was expressed in Escherichia coli and isolated as inner membrane vesicles. In contrast to all previously studied H(+)-PPases, both native and recombinant Tm-PPases exhibited an absolute requirement for Na(+) but displayed the highest activity in the presence of millimolar levels of both Na(+) and K(+). Detergent-solubilized recombinant Tm-PPase was thermostable and retained the monovalent cation requirements of the membrane-embedded enzyme. Steady-state kinetic analysis of pyrophosphate hydrolysis by the wild-type enzyme suggested that two Na(+) binding sites and one K(+) binding site are involved in enzyme activation. The affinity of the site that binds Na(+) first is increased with increasing K(+) concentration. In contrast, only one Na(+) binding site (K(+)-dependent) and one K(+) binding site were involved in activation of the Asp(703) --> Asn variant. Thus, Asp(703) may form part of the K(+)-independent Na(+) binding site. Unlike all other membrane and soluble PPases, Tm-PPase did not catalyze oxygen exchange between phosphate and water. However, solubilized Tm-PPase exhibited low but measurable PP(i)-synthesizing activity, which also required Na(+) but was inhibited by K(+). These results demonstrate that T. maritima PPase belongs to a previously unknown subfamily of Na(+)-dependent H(+)-PPase homologues and may be an analogue of Na(+),K(+)-ATPase.

  • 24. Benach, J
    et al.
    Filling, C
    Oppermann, U C T
    Roversi, P
    Bricogne, G
    Berndt, Kurt D
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institutet.
    Jörnvall, H
    Ladenstein, R
    Structure of bacterial 3 beta/17 beta-hydroxysteroid dehydrogenase at 1.2 angstrom resolution: A model for multiple steroid recognition2002In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 41, no 50, p. 14659-14668Article in journal (Refereed)
    Abstract [en]

    The enzyme 3beta/17beta-hydroxysteroid dehydrogenase (3beta/17beta-HSD) is a steroid-inducible component of the Gram-negative bacterium Conramonas testosteroni. It catalyzes the reversible reduction/ dehydrogenation of the oxo/beta-hydroxy groups at positions 3 and 17 of steroid compounds, including hormones and isobile acids. Crystallographic analysis at 1.2 Angstrom resolution reveals the enzyme to have nearly identical subunits that form a tetramer with 222 symmetry. This is one of the largest oligomeric structures refined at this resolution. The subunit consists of a monomer with a single-domain structure built around a seven-stranded beta-sheet flanked by six alpha-helices. The active site contains a Ser-Tyr-Lys triad, typical for short-chain dehydrogenases/reductases (SDR). Despite their highly diverse substrate specificities, SDR members show a close to identical folding pattern architectures and a common catalytic mechanism. In contrast to other SDR apostructures determined, the substrate binding loop is well-defined. Analysis of structure-activity relationships of catalytic cleft residues, docking analysis of substrates and inhibitors, and accessible surface analysis explains how 3beta/17beta-HSD accommodates steroid substrates of different conformations.

  • 25.
    Berg, Otto G.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Yu, Bao-Zhu
    Jain, Mahendra K.
    Thermodynamic Reciprocity of the Inhibitor Binding to the Active Site and the Interface Binding Region of IB Phospholipase A22009In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, no 14, p. 3209-3218Article in journal (Refereed)
    Abstract [en]

    Interfacial activation of pig pancreatic IB phospholipase A(2) (PLA2) is modeled in terms of the three discrete premicellar complexes (E-i(#), i = 1, 2, or 3) consecutively formed by the cooperative binding of a monodisperse amphiphile to the i-face (the interface binding region of the enzyme) without or with an occupied active site. Monodisperse PCU, the sn-2-amide analogue of the zwitterionic substrate, is a competitive inhibitor. PCU cooperatively binds to the i-face to form premicellar complexes ((E) over tilde (i), i = 1 or 2) and also binds to the active site of the premicellar complexes in the presence of calcium. In the (E) over tilde I-i complex formed in the presence of PCU and calcium, one inhibitor molecule is bound to the active site and a number of others are bound to the i-face. The properties of the (E) over tilde (i) complexes with PCU are qualitatively similar to those of E-i(#) formed with decylsulfate. Decylsulfate binds to the i-face but does not bind to the active site in the presence of calcium, nor does it interfere with the binding of PCU to the active site in the premicellar complexes. Due to the strong coupling between binding at the i-face and at the active site, it is difficult to estimate the primary binding constants for each site in these complexes. A model is developed that incorporates the above boundary conditions in relation to a detailed balance between the complexes. A key result is that a modest effect on cooperative amphiphile binding corresponds to a large change in the affinity of the inhibitor for the active site. We suggest that besides the binding to the active site, PCU also binds to another site and that full activation requires additional amphiphiles on the i-face. Thus, the activation of the inhibitor binding to the active site of the E-2(#) complex or, equivalently, the shift in the E-1(#) to E-2(#) equilibrium by the inhibitor is analogous to the allosteric activation of the substrate binding to the enzyme bound to the interface.

  • 26.
    Berg, Otto
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Yu, Bao-Zhu
    Apitz-Castro, Rafael J.
    Jain, Mahendra K.
    Phosphatidylinositol-specific phospholipase C forms different complexes with monodisperse and micellar phosphatidylcholine2004In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 43, no 7, p. 2080-2090Article in journal (Refereed)
    Abstract [en]

    Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus forms a premicellar complex E# with monodisperse diheptanoylphosphatidylcholine (DC7PC) that is distinguishable from the E* complex formed with micelles. Results are interpreted with the assumption that in both cases amphiphiles bind to the interfacial binding surface (i-face) of PI-PLC but not to the active site. Isothermal calorimetry and fluorescence titration results for the binding of monodisperse DC7PC give an apparent dissociation constant of K2 = 0.2 mM with Hill coefficient of 2. The gel-permeation, spectroscopic, and probe partitioning behaviors of E# are distinct from those of the E* complex. The aggregation and partitioning behaviors suggest that the acyl chains in E# but not in E* remain exposed to the aqueous phase. The free (E) and complexed (E# and E*) forms of PI-PLC, each with distinct spectroscopic signatures, readily equilibrate with changing DC7PC concentration. The underlying equilibria are modeled and their significance for the states of the PI-PLC under monomer kinetic conditions is discussed to suggest that the Michaelis−Menten complex formed with monodisperse DC7PC is likely to be E#S or its aggregate rather than the classical monodisperse ES complex.

  • 27.
    Berg, Otto
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Yu, Bao-Zhu
    Chang, Cherry
    Koehler, Karl A.
    Jain, Mahendra K.
    Cooperative binding of monodisperse anionic amphiphiles to the i-Face: Phospholipase A2-paradigm for interfacial binding2004In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 43, no 25, p. 7999-8013Article in journal (Refereed)
    Abstract [en]

    Equilibrium parameters for the binding of monodisperse alkyl sulfate along the i-face (the interface binding surface) of pig pancreatic IB phospholipase A2 (PLA2) to form the premicellar complexes (Ei#) are characterized to discern the short-range specific interactions. Typically, Ei# complexes are reversible on dilution. The triphasic binding isotherm, monitored as the fluorescence emission from the single tryptophan of PLA2, is interpreted as a cooperative equilibrium for the sequential formation of three premicellar complexes (Ei#, i = 1, 2, 3). In the presence of calcium, the dissociation constant K1 for the E1# complex of PLA2 with decyl sulfate (CMC = 4500 μM) is 70 μM with a Hill coefficient n1 = 2.1 ± 0.2; K2 for E2# is 750 μM with n2 = 8 ± 1, and K3 for E3# is 4000 μM with an n3 value of about 12. Controls show that (a) self-aggregation of decyl sulfate alone is not significant below the CMC; (b) occupancy of the active site is not necessary for the formation of Ei#; (c) Ki and ni do not change significantly due to the absence of calcium, possibly because alkyl sulfate does not bind to the active site of PLA2; (d) the Ei# complexes show a significant propensity for aggregation; and (e) PLA2 is not denatured in Ei#. The results are interpreted to elaborate the model for atomic level interactions along the i-face: The chain length dependence of the fit parameters suggests that short-range specific anion binding of the headgroup is accompanied by desolvation of the i-face of Ei#. We suggest that allosteric activation of PLA2 results from such specific interactions of the amphiplies and the desolvation of the i-face. The significance of these primary interfacial binding events and the coexistence of the E* and Ei# aggregates is discussed.

  • 28.
    Berndt, Kurt D
    et al.
    Eidgenössische Technische Hochschule-Hönggerberg, Zürich, Switzerland.
    Beunink, J
    Schröder, W
    Wüthrich, K
    Designed replacement of an internal hydration water molecule in BPTI: structural and functional implications of a glycine-to-serine mutation.1993In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 32, p. 4564-4570Article in journal (Refereed)
    Abstract [en]

    The three-dimensional structure of the basic pancreatic trypsin inhibitor (BPTI) contains four internal water molecules, which form a total of nine intermolecular hydrogen bonds with the BPTI polypeptide chain. To investigate the effect of such internal hydration on protein structure and stability, we displaced one of the internal water molecules in a recombinant BPTI analogue, BPTI(G36S), in which Gly 36 is replaced by serine. The replacement of a water molecule by the seryl side chain was established by the absence of the protein-water nuclear Overhauser effects (NOE) that had been attributed to the water molecule near Gly 36 in wild-type BPTI and by the presence of new, intramolecular NOEs to the hydroxyl proton of Ser 36. BPTI(G36S) has slightly reduced thermal stability compared to BPTI, corresponding to a destabilization by delta (delta G) approximately 0.7 kcal/M in 6 M guanidinium hydrochloride solution. Additionally, the stabilities of the complexes formed between BPTI(G36S) and trypsin, plasmin, or kallikrein are significantly reduced when compared to the corresponding complexes with wild-type BPTI.

  • 29.
    Bjelic, Sinisa
    et al.
    Uppsala University.
    Aqvist, Johan
    Computational prediction of structure, substrate binding mode, mechanism, and rate for a malaria protease with a novel type of active site.2004In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 43, no 46, p. 14521-14528Article in journal (Refereed)
    Abstract [en]

    The histo-aspartic protease (HAP) from the malaria parasite P. falciparum is one of several new promising targets for drug intervention. The enzyme possesses a novel type of active site, but its 3D structure and mechanism of action are still unknown. Here we use a combination of homology modeling, automated docking searches, and molecular dynamics/reaction free energy profile simulations to predict the enzyme structure, conformation of bound substrate, catalytic mechanism, and rate of the peptide cleavage reaction. We find that the computational tools are sufficiently reliable both for identifying substrate binding modes and for distinguishing between different possible reaction mechanisms. It is found that the favored pathway only involves direct participation by the catalytic aspartate, with the neighboring histidine providing critical stabilization (by a factor of approximately 10000) along the reaction. The calculated catalytic rate constant of about 0.1 s(-1) for a hexapeptide substrate derived from the alpha chain of human hemoglobin is in excellent agreement with experimental kinetic data for a similar peptide fragment.

  • 30.
    Bjelic, Sinisa
    et al.
    Uppsala University.
    Brandsdal, Bjørn O
    University of Tromsø, Norway.
    Åqvist, Johan
    Uppsala University.
    Cold adaptation of enzyme reaction rates2008In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 47, no 38, p. 10049-10057Article in journal (Refereed)
    Abstract [en]

    A major issue for organisms living at extreme temperatures is to preserve both stability and activity of their enzymes. Cold-adapted enzymes generally have a reduced thermal stability, to counteract freezing, and show a lower enthalpy and a more negative entropy of activation compared to mesophilic and thermophilic homologues. Such a balance of thermodynamic activation parameters can make the reaction rate decrease more linearly, rather than exponentially, as the temperature is lowered, but the structural basis for rate optimization toward low working temperatures remains unclear. In order to computationally address this problem, it is clear that reaction simulations rather than standard molecular dynamics calculations are needed. We have thus carried out extensive computer simulations of the keto-enol(ate) isomerization steps in differently adapted citrate synthases to explore the structure-function relationships behind catalytic rate adaptation to different temperatures. The calculations reproduce the absolute rates of the psychrophilic and mesophilic enzymes at 300 K, as well as the lower enthalpy and more negative entropy of activation of the cold-adapted enzyme, where the latter simulation result is obtained from high-precision Arrhenius plots. The overall catalytic effect originates from electrostatic stabilization of the transition state and enolate and the reduction of reorganization free energy. The simulations, however, show psychrophilic, mesophilic, and hyperthermophilic citrate synthases to have increasingly stronger electrostatic stabilization of the transition state, while the energetic penalty in terms of internal protein interactions follows the reverse order with the cold-adapted enzyme having the most favorable energy term. The lower activation enthalpy and more negative activation entropy observed for cold-adapted enzymes are found to be associated with a decreased protein stiffness. The origin of this effect is, however, not localized to the active site but to other regions of the protein structure.

  • 31.
    Bjelic, Sinisa
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Brandsdal, Bjørn O
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Cold adaptation of enzyme reaction rates2008In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 47, no 38, p. 10049-10057Article in journal (Refereed)
    Abstract [en]

    A major issue for organisms living at extreme temperatures is to preserve both stability and activity of their enzymes. Cold-adapted enzymes generally have a reduced thermal stability, to counteract freezing, and show a lower enthalpy and a more negative entropy of activation compared to mesophilic and thermophilic homologues. Such a balance of thermodynamic activation parameters can make the reaction rate decrease more linearly, rather than exponentially, as the temperature is lowered, but the structural basis for rate optimization toward low working temperatures remains unclear. In order to computationally address this problem, it is clear that reaction simulations rather than standard molecular dynamics calculations are needed. We have thus carried out extensive computer simulations of the keto-enol(ate) isomerization steps in differently adapted citrate synthases to explore the structure-function relationships behind catalytic rate adaptation to different temperatures. The calculations reproduce the absolute rates of the psychrophilic and mesophilic enzymes at 300 K, as well as the lower enthalpy and more negative entropy of activation of the cold-adapted enzyme, where the latter simulation result is obtained from high-precision Arrhenius plots. The overall catalytic effect originates from electrostatic stabilization of the transition state and enolate and the reduction of reorganization free energy. The simulations, however, show psychrophilic, mesophilic, and hyperthermophilic citrate synthases to have increasingly stronger electrostatic stabilization of the transition state, while the energetic penalty in terms of internal protein interactions follows the reverse order with the cold-adapted enzyme having the most favorable energy term. The lower activation enthalpy and more negative activation entropy observed for cold-adapted enzymes are found to be associated with a decreased protein stiffness. The origin of this effect is, however, not localized to the active site but to other regions of the protein structure.

  • 32.
    Bjelic, Sinisa
    et al.
    Uppsala University.
    Åqvist, Johan
    Uppsala University.
    Catalysis and linear free energy relationships in aspartic proteases2006In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 45, no 25, p. 7709-7723Article in journal (Refereed)
    Abstract [en]

    Aspartic proteases are receiving considerable attention as potential drug targets in several serious diseases, such as AIDS, malaria, and Alzheimer's disease. These enzymes cleave polypeptide chains, often between specific amino acid residues, but despite the common reaction mechanism, they exhibit large structural differences. Here, the catalytic mechanism of aspartic proteases plasmepsin II, cathepsin D, and HIV-1 protease is examined by computer simulations utilizing the empirical valence bond approach in combination with molecular dynamics and free energy perturbation calculations. Free energy profiles are established for four different substrates, each six amino acids long and containing hydrophobic side chains in the P1 and P1' positions. Our simulations reproduce the catalytic effect of these enzymes, which accelerate the reaction rate by a factor of approximately 10(10) compared to that of the corresponding uncatalyzed reaction in water. The calculations elucidate the origin of the catalytic effect and allow a rationalization of the fact that, despite large structural differences between plasmepsin II/cathepsin D and HIV-1 protease, the magnitude of their rate enhancement is very similar. Amino acid residues surrounding the active site together with structurally conserved water molecules are found to play an important role in catalysis, mainly through dipolar (electrostatic) stabilization. A linear free energy relationship for the reactions in the different enzymes is established that also demonstrates the reduced reorganization energy in the enzymes compared to that in the uncatalyzed water reaction.

  • 33.
    Björklund, Stefan
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Skog, Sven
    Tribukait, Bernard
    Thelander, Lars
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    S-phase-specific expression of mammalian ribonucleotide reductase R1 and R2 subunit mRNAs1990In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 29, no 23, p. 5452-5458Article in journal (Refereed)
    Abstract [en]

    Ribonucleotide reductase in mammalian cells is composed of two nonidentical subunits, proteins R1 and R2, each inactive alone. The R1 protein is present in excess in proliferating cells, and its levels are constant during the cell cycle. Expression of the R2 protein, which is limiting for enzyme activity, is strictly S-phase-correlated. In this paper, we have used antisense RNA probes in a solution hybridization assay to measure the levels of R1 and R2 mRNA during the cell cycle in centrifugally elutriated cells and in cells synchronized by isoleucine or serum starvation. The levels of both transcripts were very low or undetectable in G0/G1-phase cells, showed a pronounced increase as cells progressed into S phase, and then declined when cells progressed into G2 + M phase. The R1 and R2 transcripts increased in parallel, starting slightly before the rise in S-phase cells, and reached the same levels. The relative lack of cell cycle dependent variation in R1 protein levels, obtained previously, may therefore simply be a consequence of the long half-life of the R1 protein. Hydroxyurea-resistant, R2-overproducing mouse TA3 cells showed the same regulation of the R1 and R2 transcripts as the parental cells, but with R2 mRNA at a 40-fold higher level.

  • 34.
    Björnerås, Johannes
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mäler, Lena
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Membrane Interaction of Disease-Related Dynorphin A Variants2013In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 52, no 24, p. 4157-4167Article in journal (Refereed)
    Abstract [en]

    The membrane interaction properties of two single-residue variants, R6W and L5S, of the 17-amino acid neuropeptide dynorphin A (DynA) were studied by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. Corresponding gene mutations have recently been discovered in humans and causatively linked to a neurodegenerative disorder. The peptides were investigated in buffer and in isotropic solutions of q = 0.3 bicelles with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or DMPC (0.8) and 1,2-dimyristoyl-sn-glycero-3-phospho(1'-rac-glycerol) (DMPG) (0.2). The CD results and the NMR secondary chemical shifts show that R6W-DynA has a small a-helical fraction in buffer, which increases in the presence of bicelles, while L5S-DynA is mainly unstructured under all conditions studied here. R6W-DynA has an almost complete association with zwitterionic bicelles (similar to 90%, as probed by NMR diffusion experiments), similar to the behavior of wtDynA, while L5S-DynA has a weaker association (similar to 50%). For all peptides, the level of bicelle association is increased in negatively charged bicelles. The L5A-DynA peptide adopts a very shallow position in the headgroup region of the bicelle bilayer, as studied by paramagnetic spin relaxation enhancement experiments using paramagnetic probes. Similarly, the results show that R6W-DynA is more deeply buried in the bilayer, with only the C-terminal residues exposed to solvent, again more similar to the case of wild-type DynA. We suggest that the results presented here may explain the differences in cell toxicity of these disease-related neuropeptide variants.

  • 35.
    Blasco, Pilar
    et al.
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Patel, Dhilon S.
    Engström, Olof
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Im, Wonpil
    Becker, Antonia
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Conformational Dynamics of the Lipopolysaccharide from Escherichia coli O91 Revealed by Nuclear Magnetic Resonance Spectroscopy and Molecular Simulations2017In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 56, no 29, p. 3826-3839Article in journal (Refereed)
    Abstract [en]

    The outer leaflet of the outer membrane in Gram-negative bacteria contains lipopolysaccharides (LPS) as a major component, and the outer membrane provides a physical barrier and protection against hostile environments. The enterohemorrhagic Escherichia coli of serogroup O91 has an O-antigen polysaccharide (PS) with five sugar residues in the repeating unit (RU), and the herein studied O-antigen PS contains similar to 10 RUs. H-1-C-13 HSQC-NOESY experiments on a 1-C-13-labeled PS were employed to deduce H-1-H-1 cross-relaxation rates and transglycosidic (3)J(CH) related to the psi torsional angles were obtained by H-1-H-1 NOESY experiments. Dynamical parameters were calculated from the molecular dynamics (MD) simulations of the PS in solution and compared to those from C-13 nuclear magnetic resonance (NMR) relaxation studies. Importantly, the MD simulations can reproduce the dynamical behavior of internal correlation times along the PS chain. Two-dimensional free energy surfaces of glycosidic torsion angles delineate the conformational space available to the O-antigen. Although similar with respect to populated states in solution, the O-antigen in LPS bilayers has more extended chains as a result of spatial limitations due to close packing. Calcium ions are highly abundant in the phosphate-containing core region mediating LPS LPS association that is crucial for maintaining bilayer integrity, and the negatively charged O-antigen promotes a high concentration of counterbalancing potassium ions. The ensemble of structures present for the PS in solution is captured by the NMR experiments, and the similarities between the O-antigen on its own and as a constituent of the full LPS in a bilayer environment make it possible to realistically describe the LPS conformation and dynamics from the MD simulations.

  • 36.
    Blomberg, Margareta R. A.
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Active Site Midpoint Potentials in Different Cytochrome c Oxidase Families: A Computational Comparison2019In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 58, no 15, p. 2028-2038Article in journal (Refereed)
    Abstract [en]

    Cytochrome c oxidase (CcO) is the terminal enzyme in the respiratory electron transport chain, reducing molecular oxygen to water. The binuclear active site in CcO comprises a high-spin heme associated with a Cu-B complex and a redox active tyrosine. The electron transport in the respiratory chain is driven by increasing midpoint potentials of the involved cofactors, resulting in a release of free energy, which is stored by coupling the electron transfer to proton translocation across a membrane, building up an electrochemical gradient. In this context, the midpoint potentials of the active site cofactors in the CcOs are of special interest, since they determine the driving forces for the individual oxygen reduction steps and thereby affect the efficiency of the proton pumping. It has been difficult to obtain useful information on some of these midpoint potentials from experiments. However, since each of the reduction steps in the catalytic cycle of oxygen reduction to water corresponds to the formation of an O-H bond, they can be calculated with a reasonably high accuracy using quantum chemical methods. From the calculated O-H bond strengths, the proton-coupled midpoint potentials of the active site cofactors can be estimated. Using models representing the different families of CcO's (A, B, and C), the calculations give midpoint potentials that should be relevant during catalytic turnover. The calculations also suggest possible explanations for why some experimentally measured potentials deviate significantly from the calculated ones, i.e., for Cu-B in all oxidase families, and for heme b(3) in the C family.

  • 37.
    Blomberg, Margareta R. A.
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Can Reduction of NO to N2O in Cytochrome c Dependent Nitric Oxide Reductase Proceed through a Trans-Mechanism?2017In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 56, no 1, p. 120-131Article in journal (Refereed)
    Abstract [en]

    As part of microbial denitrification, NO is reduced to N2O in the membrane bound enzyme nitric oxide reductase, NOR The N N coupling occurs in the diiron binuclear active site, BNC, and different mechanisms for this reaction step have been suggested. Computational studies have supported a so-called cis:b(3)-mechanism, in which the hyponitrite product of the reductive N N bond formation coordinates with one nitrogen to the heme iron and with both oxygens to the non-heme iron in the BNC. In contrast, experimental results have been interpreted to support a so-called trans-mechanism, in which the hyponitrite intermediate coordinates with one nitrogen atom to each of the two iron ions. Hybrid density functional theory is used here to perform an extensive search for possible intermediates of the NO reduction in the cNOR enzyme. It is found that hyponitrite structures coordinating with their negatively charged oxygens to the positively charged iron ions are the most stable ones. The hyponitrite intermediate involved in the suggested trans-mechanism, which only coordinates with the nitrogens to the iron ions, is found to be prohibitively high in energy, leading to a too slow reaction, which should rule out this mechanism. Furthermore, intermediates binding one NO molecule to each iron ion in the BNC, which have been suggested to initiate the trans-mechanism, are found to be too high in energy to be observable, indicating that the experimentally observed electron paramagnetic resonance signals, taken to support such an iron-nitrosyl dimer intermediate, should be reinterpreted.

  • 38.
    Blomberg, Margareta R. A.
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Mechanism of Oxygen Reduction in Cytochrome c Oxidase and the Role of the Active Site Tyrosine2016In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 55, no 3, p. 489-500Article in journal (Refereed)
    Abstract [en]

    Cytochrome c oxidase, the terminal enzyme in the respiratory chain, reduces molecular oxygen to water and stores the released energy through electrogenic chemistry and proton pumping across the membrane. Apart from the heme-copper binuclear center, there is a conserved tyrosine residue in the active site (BNC). The tyrosine delivers both an electron and a proton during the O-O bond cleavage step, forming a tyrosyl radical. The catalytic cycle then occurs in four reduction steps, each taking up one proton for the chemistry (water formation) and one proton to be pumped. It is here suggested that in three of the reduction steps the chemical proton enters the center of the BNC, leaving the tyrosine unprotonated with radical character. The reproprotonation of the tyrosine occurs first in the final reduction step before binding the next oxygen molecule. It is also suggested that this reduction mechanism and the presence of the tyrosine are essential for the proton pumping. Density functional theory calculations on large cluster models of the active site show that only the intermediates with the proton in the center of the BNC and with an unprotonated tyrosyl radical have a high electron affinity of similar size as the electron donor, which is essential for the ability to take up two protons per electron and thus for the proton pumping. This type of reduction mechanism is also the only one that gives a free energy profile in accordance with experimental observations for the amount of proton pumping in the working enzyme.

  • 39.
    Blomberg, Margareta R. A.
    et al.
    Stockholm University, Faculty of Science, Department of Physics. Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Siegbahn, Per E. M.
    Mechanism for N2O Generation in Bacterial Nitric Oxide Reductase: A Quantum Chemical Study2012In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 51, no 25, p. 5173-5186Article in journal (Refereed)
    Abstract [en]

    The catalytic mechanism of reduction of NO to N2O in the bacterial enzyme nitric oxide reductase has been investigated using hybrid density functional theory and a model of the binuclear center (BNC) based on the newly determined crystal structure. The calculations strongly suggest a so-called cis:b(3) mechanism, while the commonly suggested trans mechanism is found to be energetically unfavorable. The mechanism suggested here involves a stable cis-hyponitrite, and it is shown that from this intermediate one N-O bond can be cleaved without the transfer of a proton or an electron into the binuclear active site, in agreement with experimental observations. The fully oxidized intermediate in the catalytic cycle and the resting form of the enzyme are suggested to have an oxo-bridged BNC with two high-spin ferric irons antiferromagnetically coupled. Both steps of reduction of the BNC after N2O formation are found to be pH-dependent, also in agreement with experiment. Finally, it is found that the oxo bridge in the oxidized BNC can react with NO to give nitrite, which explains the experimental observations that the fully oxidized enzyme reacts with NO, and most likely also the observed substrate inhibition at higher NO concentrations.

  • 40.
    Boukharta, Lars
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Keränen, Henrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Stary-Weinzinger, Anna
    Wallin, Göran
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    de Groot, Bert L.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Computer Simulations of Structure-Activity Relationships for hERG Channel Blockers2011In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 50, no 27, p. 6146-6156Article in journal (Refereed)
    Abstract [en]

    The hERG potassium channel is of major pharmaceutical importance, and its blockade by various compounds, potentially causing serious cardiac side effects, is a major problem in drug development. Despite the large amounts of existing biochemical data on blockade of hERG by drugs and druglike compounds, relatively little is known regarding the structural basis of binding of blockers to the channel. Here, we have used a recently developed homology model of hERG to conduct molecular docking experiments with a series of channel blockers, followed by molecular dynamics simulations of the complexes and evaluation of binding free energies with the linear interaction energy method. The calculations yield a remarkably good agreement with experimental binding affinities and allow for a rationalization of three-dimensional structure-activity relationships in terms of a number of key interactions. Two main interaction regions of the channel are thus identified with implications for further mutagenesis experiments and design of new compounds.

  • 41. Branneby, Cecilia
    et al.
    Carlqvist, Peter
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Physical Chemistry (closed 20110630).
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Brinck, Tore
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Physical Chemistry (closed 20110630).
    Berglund, Per
    KTH, Superseded Departments, Biotechnology.
    Rational redesign of a lipase to an aldolase2003In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 42, no 28, p. 8633-8633Article in journal (Refereed)
  • 42. Busenlehner, Laura
    et al.
    Brändén, Gisela
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Namslauer, Ida
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Armstrong, Richard
    Structural Elements Involved in Proton Translocation by Cytochrome c Oxidase as Revealed by Backbone Amide Hydrogen-Deuterium Exchange of the E286H Mutant2008In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 47, no 1, p. 73-83Article in journal (Refereed)
    Abstract [en]

    Cytochrome c oxidase is the terminal electron acceptor in the respiratory chains of aerobic organisms and energetically couples' the reduction of oxygen to water to proton pumping across the membrane. The mechanisms of proton uptake, gating, and pumping have yet to be completely elucidated at the molecular level for these enzymes. For Rhodobacter sphaeroides CytcO (cytochrome aa<sub>3</sub>), it appears as though the E286 side chain of subunit I is a branching point from which protons are shuttled either to the catalytic site for O<sub>2</sub> reduction or to the acceptor site for pumped protons. Amide hydrogen-deuterium exchange mass spectrometry was used to investigate how mutation of this key branching residue to histidine (E286H) affects the structures and dynamics of four redox intermediate states. A functional characterization of this mutant reveals that E286H CytcO retains ∼1% steady-state activity that is uncoupled from proton pumping and that proton transfer from H286 is significantly slowed. Backbone amide H-D exchange kinetics indicates that specific regions of CytcO, perturbed by the E286H mutation, are likely to be involved in proton gating and in the exit pathway for pumped protons. The results indicate that redox-dependent conformational changes around E286 are essential for internal proton transfer. E286H CytcO, however, is incapable of these specific conformational changes and therefore is insensitive to the redox state of the enzyme. These data support a model where the side chain conformation of E286 controls proton translocation in CytcO through its interactions with the proton gate, which directs the flow of protons either to the active site or to the exit pathway. In the E286H mutant, the proton gate does not function properly and the exit channel is unresponsive. These results provide new insight into the structure and mechanism of proton transtocation by CytcO.

  • 43. Busenlehner, Laura S.
    et al.
    Alander, Johan
    Jegerscohld, Caroline
    Holm, Peter J.
    Bhakat, Priyaranjan
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Morgenstern, Ralf
    Armstrong, Richard N.
    Location of substrate binding sites within the integral membrane protein microsomal glutathione transferase-12007In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 46, no 10, p. 2812-2822Article in journal (Refereed)
    Abstract [en]

    Microsomal glutathione transferase-1 (MGST1) is a trimeric, membrane-bound enzyme with both glutathione (GSH) transferase and hydroperoxidase activities. As a member of the MAPEG superfamily, MGST1 aids in the detoxication of numerous xenobiotic substrates and in cellular protection from oxidative stress through the GSH-dependent reduction of phospholipid hydroperoxides. However, little is known about the location of the different substrate binding sites, including whether the transferase and peroxidase activities overlap structurally. Although molecular density attributed to GSH has been observed in the 3.2 A resolution electron crystallographic structure of MGST1, the electrophilic and phospholipid hydroperoxide substrate binding sites remain elusive. Amide H-D exchange kinetics and H-D ligand footprinting experiments indicate that GSH and hydrophobic substrates bind within similar, but distinct, regions of MGST1. Site-directed mutagenesis, guided by the H-D exchange results, demonstrates that specific residues within the GSH footprint effect transferase activity toward 1-chloro-2,4-dinitrobenzene. In addition, cytosolic residues surrounding the chemical stress sensor C49 but not modeled in the crystal structure appear to play an important role in the formation of the binding site for hydrophobic substrates. Although the fatty acid/phospholipid binding site structurally overlaps that for GSH, it does not appear to be localized to the same region as other hydrophobic substrates. Finally, H-D exchange mass spectrometry reveals a specific conformational transition that may mediate substrate binding and/or product release. Such structural changes in MGST1 are essential for activation of the enzyme and are important for its biological function.

  • 44. Busenlehner, L.S.
    et al.
    Codreanu, S.G.
    Holm, P.J.
    Bhakat, P.
    Hebert, Hans
    Karolinska Institutet.
    Morgenstern, R.
    Armstrong, R.N.
    Stress sensor triggers conformational response of the integral membrane protein microsomal glutathione transferase 12004In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 43, no 35, p. 11145-11152Article in journal (Refereed)
    Abstract [en]

    Microsomal glutathione (GSH) transferase 1 (MGST1) is a trimeric, integral membrane protein involved in cellular response to chemical or oxidative stress. The cytosolic domain of MGST1 harbors the GSH binding site and a cysteine residue (C49) that acts as a sensor of oxidative and chemical stress. Spatially resolved changes in the kinetics of backbone amide H/D exchange reveal that the binding of a single molecule of GSH/trimer induces a cooperative conformational transition involving movements of the transmembrane helices and a reordering of the cytosolic domain. Alkylation of the stress sensor preorganizes the helices and facilitates the cooperative transition resulting in catalytic activation.

  • 45. Cajal, Yolanda
    et al.
    Berg, Otto
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Jain, Mahendra K.
    Origins of delays in monolayer kinetics: Phospholipase A2 paradigm2004In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 43, no 28, p. 9256-9264Article in journal (Refereed)
    Abstract [en]

    The interfacial kinetic paradigm is adopted to model the kinetic behavior of pig pancreatic phospholipase A2 (PLA2) at the monolayer interface. A short delay of about a minute to the onset of the steady state is observed under all monolayer reaction progress conditions, including the PLA2-catalyzed hydrolysis of didecanoylphosphatidyl-choline (PC10) and -glycerol (PG10) monolayers as analyzed in this paper. This delay is independent of enzyme concentration and surface pressure and is attributed to the equilibration time by stationary diffusion of the enzyme added to the stirred subphase to the monolayer through the intervening unstirred aqueous layer. The longer delays of up to several hours, seen with the PC10 monolayers at >15 mN/m, are influenced by surface pressure as well as enzyme concentration. Virtually all features of the monolayer reaction progress are consistent with the assumption that the product accumulates in the substrate monolayer, although the products alone do not spread as a compressible monolayer. These results rule out models that invoke slow “activation” of PLA2 on the monolayer. The observed steady-state rate on monolayers after the delays is <1% of the rate observed with micellar or vesicles substrates of comparable substrate. Together these results suggest that the monolayer steady-state rate includes contributions from steps other than those of the interfacial turnover cycle. Additional considerations that provide understanding of the pre-steady-state behaviors and other nonideal effects at the surface are also discussed.

  • 46.
    Carlsson, Gunilla H.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Nicholls, Peter
    Svistunenko, Dimitri
    Berglund, Gunnar I.
    Hajdu, Janos
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Complexes of horseradish peroxidase with formate, acetate and carbon monoxide2005In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 44, no 2, p. 635-642Article in journal (Refereed)
    Abstract [en]

    Carbon monoxide, formate, and acetate interact with horseradish peroxidase (HRP) by binding to subsites within the active site. These ligands also bind to catalases, but their interactions are different in the two types of enzymes. Formate (notionally the “hydrated” form of carbon monoxide) is oxidized to carbon dioxide by compound I in catalase, while no such reaction is reported to occur in HRP, and the CO complex of ferrocatalase can only be obtained indirectly. Here we describe high-resolution crystal structures for HRP in its complexes with carbon monoxide and with formate, and compare these with the previously determined HRP−acetate structure [Berglund, G. I., et al. (2002) Nature 417, 463−468]. A multicrystal X-ray data collection strategy preserved the correct oxidation state of the iron during the experiments. Absorption spectra of the crystals and electron paramagnetic resonance data for the acetate and formate complexes in solution correlate electronic states with the structural results. Formate in ferric HRP and CO in ferrous HRP bind directly to the heme iron with iron−ligand distances of 2.3 and 1.8 Å, respectively. CO does not bind to the ferric iron in the crystal. Acetate bound to ferric HRP stacks parallel with the heme plane with its carboxylate group 3.6 Å from the heme iron, and without an intervening solvent molecule between the iron and acetate. The positions of the oxygen atoms in the bound ligands outline a potential access route for hydrogen peroxide to the iron. We propose that interactions in this channel ensure deprotonation of the proximal oxygen before binding to the heme iron.

  • 47. Carpenter, Katharine A.
    et al.
    Schmidt, Ralf
    von Mentzer, Bengt
    Haglund, Ulla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences.
    Roberts, Edward
    Walpole, Chris
    Turn structures in CGRP C-terminal analogues promote stable arrangements of key residue side chains2001In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 40, no 28, p. 8317-8325Article in journal (Refereed)
    Abstract [en]

    The 37-amino acid calcitonin gene-related peptide (CGRP) is a potent endogenous vasodilator thought to be implicated in the genesis of migraine attack. CGRP antagonists may thus have therapeutic value for the treatment of migraine. The CGRP C-terminally derived peptide [D(31),P(34),F(35)]CGRP(27-37)-NH(2) was recently identified as a high-affinity hCGRP(1) receptor selective antagonist. Reasonable CGRP(1) affinity has also been demonstrated for several related analogues, including [D(31),A(34),F(35)]CGRP(27-37)-NH(2). In the study presented here, conformational and structural features in CGRP(27-37)-NH(2) analogues that are important for hCGRP(1) receptor binding were explored. Structure-activity studies carried out on [D(31),P(34),F(35)]CGRP(27-37)-NH(2) resulted in [D(31),P(34),F(35)]CGRP(30-37)-NH(2), the shortest reported CGRP C-terminal peptide analogue exhibiting reasonable hCGRP(1) receptor affinity (K(i) = 29.6 nM). Further removal of T(30) from the peptide's N-terminus greatly reduced receptor affinity from the nanomolar to micromolar range. Additional residues deemed critical for hCGRP(1) receptor binding were identified from an alanine scan of [A(34),F(35)]CGRP(28-37)-NH(2) and included V(32) and F(37). Replacement of the C-terminal amide in this same peptide with a carboxyl, furthermore, resulted in a greater than 50-fold reduction in hCGRP(1) affinity, thus suggesting a direct role for the amide moiety in receptor binding. The conformational properties of two classes of CGRP(27-37)-NH(2) peptides, [D(31),X(34),F(35)]CGRP(27-37)-NH(2) (X is A or P), were examined by NMR spectroscopy and molecular modeling. A beta-turn centered on P(29) was a notable feature consistently observed among active peptides in both series. This turn led to exposure of the critical T(30) residue to the surrounding environment. Peptides in the A(34) series were additionally characterized by a stable C-terminal helical turn that resulted in the three important residues (T(30), V(32), and F(37)) adopting consistent interspatial positions with respect to one another. Peptides in the P(34) series were comparatively more flexible at the C-terminus, although a large proportion of the [D(31),P(34),F(35)]CGRP(27-37)-NH(2) calculated conformers contained a gamma-turn centered on P(34). These results collectively suggest that turn structures at both the C-terminus and N-terminus of CGRP(27-37)-NH(2) analogues may help to appropriately orient critical residues (T(30), V(32), and F(37)) for hCGRP(1) receptor binding.

  • 48.
    Chen, Eefei
    et al.
    Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064, United States.
    Christiansen, Alexander
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wang, Qian
    Department of Physics, University of Houston, Houston, Texas 77204, United States.
    Cheung, Margaret S
    Department of Physics, University of Houston, Houston, Texas 77204, United States.
    Kliger, David S
    Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064, United States.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Effects of macromolecular crowding on burst phase kinetics of cytochrome c folding2012In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 51, no 49, p. 9836-9845Article in journal (Refereed)
    Abstract [en]

    Excluded volume and viscosity effects of crowding agents that mimic crowded conditions in vivo on "classical" burst phase folding kinetics of cytochrome c are assessed in vitro. Upon electron transfer-triggered folding of reduced cytochrome c, far-UV time-resolved circular dichroism (TRCD) is used to monitor folding under different conditions. Earlier work has shown that folding of reduced cytochrome c from the guanidinium hydrochloride-induced unfolded ensemble in dilute phosphate buffer involves kinetic partitioning: one fraction of molecules folds rapidly, on a time scale identical to that of reduction, while the remaining population folds more slowly. In the presence of 220 mg/mL dextran 70, a synthetic macromolecular crowding agent that occupies space but does not interact with proteins, the population of the fast folding step for cytochrome c is greatly reduced. Increasing the viscosity with sucrose to the same microviscosity exhibited by the dextran solution showed no significant decrease in the amplitude of the fast-folding phase of cytochrome c. Experiments show that the unfolded-state heme ligation remains bis-His in the presence of dextran 70, but coarse-grained simulations suggest that the unfolded-state ensemble becomes more compact in the presence of crowders. We conclude that excluded volume effects alter unfolded cytochrome c such that access to fast-folding conformations is reduced.

  • 49.
    Chen, Guiying
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Han, Guangye
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Göransson, Erik
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Physical Chemistry.
    Mamedov, Fikret
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Styring, Stenbjörn
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Stability of the S(3) and S(2) State Intermediates in Photosystem II Directly Probed by EPR Spectroscopy2012In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 51, no 1, p. 138-148Article in journal (Refereed)
    Abstract [en]

    The stability of the S(3) and S(2) states of the oxygen evolving complex in photosystem II (PSII) was directly probed by EPR spectroscopy in PSII membrane preparations from spinach in the presence of the exogenous electron acceptor PpBQ at 1, 10, and 20 degrees C. The decay of the S(3) state was followed in samples exposed to two flashes by measuring the split S(3) EPR signal induced by near-infrared illumination at 5 K. The decay of the S(2) state was followed in samples exposed to one flash by measuring the S(2) state multiline EPR signal. During the decay of the S(3) state, the S(2) state multiline EPR signal first increased and then decreased in amplitude. This shows that the decay of the S(3) state to the S(1) state occurs via the S(2) state. The decay of the S(3) state was biexponential with a fast kinetic phase with a few seconds decay half-time. This occurred in 10-20% of the PSII centers. The slow kinetic phase ranged from a decay half-time of 700 s (at 1 degrees C) to similar to 100 s (at 20 degrees C) in the remaining 80-90% of the centers. The decay of the S(2) state was also biphasic and showed quite similar kinetics to the decay of the S(3) state. Our experiments show that the auxiliary electron donor Y(D) was oxidized during the entire experiment. Thus, the reduced form of Y(D) does not participate to the fast decay of the S(2) and S(3) states we describe here. Instead, we suggest that the decay of the S(3) and S(2) states reflects electron transfer from the acceptor side of PSII to the donor side of PSII starting in the corresponding S state. It is proposed that this exists in equilibrium with Y(Z) according to S(3)Y(2) double left right arrow S(2)Y(Z)(.) in the case of the S(3) state decay and S(2)Y(Z) double left right arrow S(1)Y(Z)(.) in the case of the S(2) state decay. Two kinetic models are discussed, both developed with the assumption that the slow decay of the S(3) and S(2) states occurs in PSII centers where Y(Z) is also a fast donor to P(680)(+) working in the nanosecond time regime and that the fast decay of the S(3) and S(2) states occurs in centers where Y(Z) reduces P(680)(+) with slower microsecond kinetics. Our measurements also demonstrate that the split S(3) EPR signal can be used as a direct probe to the S(3) state and that it can provide important information about the redox properties of the S(3) state.

  • 50. Chen, H
    et al.
    Nyström, Fredrik
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Cell biology. Östergötlands Läns Landsting, MKC-2, GE: endomed.
    Dong, LQ
    Cong, L
    Li, Y
    Liu, F
    Insulin-stimulated activation of phosphoinositide-dependent Kinase-1 (PDK1): potential role in translocation of CLUT4 in rat adipose cells.2001In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 30, p. 11851-11859Article in journal (Refereed)
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