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  • 1. Cebula, Marcus
    et al.
    Turan, Ilke Simsek
    Sjodin, Birgitta
    Thulasingam, Madhuranayaki
    Brock, Joseph
    Chmyrov, Volodymyr
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Abe, Hiroshi
    Mannervik, Bengt
    Haeggstrom, Jesper Z.
    Rinaldo-Matthis, Agnes
    Akkaya, Engin U.
    Morgenstern, Ralf
    Catalytic Conversion of Lipophilic Substrates by Phase constrained Enzymes in the Aqueous or in the Membrane Phase2016In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, article id 38316Article in journal (Refereed)
    Abstract [en]

    Both soluble and membrane-bound enzymes can catalyze the conversion of lipophilic substrates. The precise substrate access path, with regard to phase, has however, until now relied on conjecture from enzyme structural data only (certainly giving credible and valuable hypotheses). Alternative methods have been missing. To obtain the first experimental evidence directly determining the access paths (of lipophilic substrates) to phase constrained enzymes we here describe the application of a BODIPY-derived substrate (PS1). Using this tool, which is not accessible to cytosolic enzymes in the presence of detergent and, by contrast, not accessible to membrane embedded enzymes in the absence of detergent, we demonstrate that cytosolic and microsomal glutathione transferases (GSTs), both catalyzing the activation of PS1, do so only within their respective phases. This approach can serve as a guideline to experimentally validate substrate access paths, a fundamental property of phase restricted enzymes. Examples of other enzyme classes with members in both phases are xenobiotic-metabolizing sulphotransferases/UDP-glucuronosyl transferases or epoxide hydrolases. Since specific GSTs have been suggested to contribute to tumor drug resistance, PS1 can also be utilized as a tool to discriminate between phase constrained members of these enzymes by analyzing samples in the absence and presence of Triton X-100.

  • 2.
    Chmyrov, Volodymyr
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Fluorescence fluctuation studies of biomolecular interactions in solutions, biomembranes and live cells2016Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Fluorescence spectroscopy and imaging have a very broad spectrum of applicationswithin the life sciences, in particular for detection and characterization ofbiomolecular dynamics and interactions in different environments. This thesis comprisesprojects that strive to further expand the information content extracted fromthe detected fluorescence, leading to sensitive readout parameters for studies ofbiomolecular dynamics and interactions. Two major strategies are presented toachieve this aim. The first strategy is based on the expansion of the availablereadout parameters beyond the "traditional" fluorescence parameters: intensity,wavelength, polarization and fluorescence lifetime. The additional parameters arebased on blinking properties of fluorescent labels. In particular on transitions betweensinglet and triplet states, and transitions between the trans- and cis-isomersof fluorophores. Two publications in the thesis are based on this strategy (paperI and IV). The second strategy is based on the utilization of fluorescence intensityfluctuations in order to detect the oligomerization mechanisms of fluorescentlylabeled peptides and proteins. This strategy combines the intensity fluctuationanalysis and the readout of distance dependent energy transfer between fluorescentmolecules together with the correlation analysis of fluorescence from two labeledproteins emitting at different wavelengths. Another two publications presented inthe thesis are based on the second comprehensive strategy (papers II and III).The work presented in this thesis shows that the blinking kinetics of fluorescentlabels contain significant information that can be exploited by a combination of fluctuationsanalysis with distance dependent excitation energy transfer between thefluorescent molecules, or by analysis of fluorescence covariance between moleculesthat emit at different wavelengths. These fluorescence-based methods have a significantpotential for molecular interaction studies in the biomedical field.

  • 3.
    Chmyrov, Volodymyr
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Spielmann, Thiemo
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Hevekerl, Heike
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Trans-Cis isomerization of lipophilic dyes probing membrane microviscosity in biological membranes and in live cells2015In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 87, no 11, p. 5690-5697Article in journal (Refereed)
    Abstract [en]

    Membrane environment and fluidity can modulate the dynamics and interactions of membrane proteins and can thereby strongly influence the function of cells and organisms in general. In this work, we demonstrate that trans-cis isomerization of lipophilic dyes is a useful parameter to monitor packaging and fluidity of biomembranes. Fluorescence fluctuations, generated by trans-cis isomerization of the thiocarbocyanine dye Merocyanine 540 (MC540), were first analyzed by fluorescence correlation spectroscopy (FCS) in different alcohol solutions. Similar isomerization kinetics of MC540 in lipid vesicles could then also be monitored, and the influence of lipid polarity, membrane curvature, and cholesterol content was investigated. While no influence of membrane curvature and lipid polarity could be observed, a clear decrease in the isomerization rates could be observed with increasing cholesterol contents in the vesicle membranes. Finally, procedures to spatially map photoinduced and thermal isomerization rates on live cells by transient state (TRAST) imaging were established. On the basis of these procedures, MC540 isomerization was studied on live MCF7 cells, and TRAST images of the cells at different temperatures were found to reliably detect differences in the isomerization parameters. Our studies indicate that trans-cis isomerization is a useful parameter for probing membrane dynamics and that the TRAST imaging technique can provide spatial maps of photoinduced isomerization as well as both photoinduced and thermal back-isomerization, resolving differences in local membrane microviscosity in live cells.

  • 4.
    Chmyrov, Volodymyr
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Spielmann, Thiemo
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Hevekerl, Heike
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Trans-cis isomerization of lipophilic dyes provides a measure of membrane microviscosity in biological membranes and in live cellsManuscript (preprint) (Other academic)
  • 5.
    Chmyrov, Volodymyr
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Tornmalm, Johan
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Piguet, Joachim
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Monitoring of NBD-probes and their location in lipid membranes via their triplet state parametersManuscript (preprint) (Other academic)
    Abstract [en]

    By a combination of fluorescence correlation spectroscopy (FCS) and transient state (TRAST)imaging, the triplet state kinetics of the membrane fluorophore 7-nitrobenz-2-oxa-1,3-diazole-4-yl(NBD) was studied in small unilamellar vesicles (SUVs). It was shown that spin-labels, included inSUV membranes together with the NBD-labeled lipids, induce prominent effects on the triplet statekinetics of NBD. The relative effects on the triplet state kinetics are considerably larger than thoseobserved in traditional fluorescence quenching studies, and can provide information about thelocalization and the interactions between the lipids in the SUVs, using considerably lowerconcentrations of spin-labeled lipids in the membranes. From the effects of the spin labels on thetriplet state kinetics of NBD, we revisited the folding behavior of NBD-labeled phospholipid chains inthe membranes. Our results indicate that the NBD probe on the acyl chain of the phospholipids do notunambiguously loop back towards the membrane surface, as previously reported, but may alternatebetween a straight and a folded acyl chain, with the NBD label at the surface, or deep into themembrane bilayer. Our study suggests that the triplet state parameters of NBD can provide anadditional set of orthogonal parameters, which can increase accuracy and precision of fluorescencebasedmolecular dynamics and interaction studies with NDB as a probe molecule.

  • 6. Kronqvist, Nina
    et al.
    Otikovs, Martins
    Chmyrov, Volodymyr
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Chen, Gefei
    Andersson, Marlene
    Nordling, Kerstin
    Landreh, Michael
    Sarr, Médoune
    Jörnvall, Hans
    Wennmalm, Stefan
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Meng, Qing
    Rising, Anna
    Otzen, Daniel Erik Rik
    Knight, Stefan
    Jaudzems, Kristaps
    Johansson, Jan Ove
    Sequential pH-driven dimerization and stabilization of the N-terminal domain enables rapid spider silk formation2014In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 5, p. 3254-Article in journal (Refereed)
    Abstract [en]

    The mechanisms controlling the conversion of spider silk proteins into insoluble fibres, which happens in a fraction of a second and in a defined region of the silk glands, are still unresolved. The N-terminal domain changes conformation and forms a homodimer when pH is lowered from 7 to 6; however, the molecular details still remain to be determined. Here we investigate site-directed mutants of the N-terminal domain from Euprosthenops australis major ampullate spidroin 1 and find that the charged residues D40, R60 and K65 mediate intersubunit electrostatic interactions. Protonation of E79 and E119 is required for structural conversions of the subunits into a dimer conformation, and subsequent protonation of E84 around pH 5.7 leads to the formation of a fully stable dimer. These residues are highly conserved, indicating that the now proposed three-step mechanism prevents premature aggregation of spidroins and enables fast formation of spider silk fibres in general.

  • 7.
    Tornmalm, Johan
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Quantum and Biophotonics.
    Piguet, Joachim
    KTH, School of Engineering Sciences (SCI), Applied Physics, Quantum and Biophotonics.
    Chmyrov, Volodymyr
    KTH, School of Engineering Sciences (SCI), Applied Physics, Quantum and Biophotonics.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Quantum and Biophotonics.
    Transient state imaging of intermittent interactions between lipids and receptor proteins in artificial and live cell membranesManuscript (preprint) (Other academic)
    Abstract [en]

    Transient collisional interactions between lipids and membrane proteins play an important role in modulating cellular functions but occur at frequencies too low to be readily observable via fluorescence imaging or quenching studies. We used transient state imaging (TRAST) to quantify those interaction in living cells. This method combines sensitive detection of fluorescence from fluorophore marker molecules with the ability to monitor their long-lived dark triplet states, highly sensitive to molecular interactions in artificial and live cell membranes.

    By TRAST we first determined the dark transient state kinetics of 7-nitrobenz-2-oxa-1,3-diazole-4-yl (NBD), an extensively used biomembrane fluorophore, available as a label on a wide range of lipids and sterols. We then measured quenching of NBD triplet states by spin-labels, in the membranes of small unilamellar vesicles (SUVs), and studied how it depends on the fluorescent lipid-derivative type and on the position of the spin label in the membranes. By the same strategy, we then quantified the collisional quenching of NBD-lipid derivatives and spin-labelled stearic acids in live cell plasma membranes.

    Finally, we extended the method to study the collisional interactions between G-Protein Coupled Receptors (GPCRs), covalently labelled with the spin label TEMPO, and NBD-lipid derivatives, in the plasma membranes of living cells. Thereby, we could resolve transient interactions between the GPCRs and lipids with different hydrophilic heads or sterols, and how these interactions were changed upon activation of the GPCR by an agonist. The presented approach offers a straightforward and widely applicable means to characterize and image transient interactions in live cell membranes, of large biomedical relevance.

  • 8.
    Turdalieva, Aizat
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Chmyrov, Volodymyr
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Johansson, Eric
    Xu, Hao
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Ahniyaz, Anwar
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Fu, Ying
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Fluorescence characterization of colloidal ZnO nanoparticle synthesized by sol-gel method at room temperatureManuscript (preprint) (Other academic)
  • 9. Wegner, Maria
    et al.
    Chmyrov, Voldymyr
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Piper, Aundrea R.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Reed, Scott M.
    Brzezinski, Peter
    Studies of biological membranes on silica particles2012In: Abstract of Papers of the American Chemical Society, ISSN 0065-7727, Vol. 243Article in journal (Other academic)
  • 10.
    Wennmalm, Stefan
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Chmyrov, Volodymyr
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Tjernberg, Lars
    Highly Sensitive FRET-FCS Detects Amyloid beta-Peptide Oligomers in Solution at Physiological Concentrations2015In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 87, no 23, p. 11700-11705Article in journal (Refereed)
    Abstract [en]

    Oligomers formed by the amyloid beta-peptide (A beta) are pathogens in Alzheimers disease. Increased knowledge on the oligomerization process is crucial for understanding the disease and for finding treatments. Ideally, A beta oligomerization should be studied in solution and at physiologically relevant concentrations, but most popular techniques of today are not capable of such analyses. We demonstrate here that the combination of FOrster Resonance Energy Transfer and Fluorescence Correlation Spectroscopy (FRET-FCS) has a unique ability to detect small subpopulations of FRET-active molecules and oligomers. FRET-FCS could readily detect a FRET-active oligonucleotide present at levels as low as 0.5% compared to FRET-inactive dye molecules. In contrast, three established fluorescence fluctuation techniques (FCS, FCCS, and PCH) required fractions between 7 and 11%. When applied to the analysis of A beta, FRET-FCS detected oligomers consisting of less than 10 A beta molecules, which coexisted with the monomers at fractions as low as 2 +/- 2%. Thus, we demonstrate for the first time direct detection of small fractions of A beta oligomers in solution at physiological concentrations. This ability of FRET-FCS could be an indispensable tool for studying biological oligomerization processes, in general, and for finding therapeutically useful oligomerization inhibitors.

  • 11.
    Xu, Hao
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Chmyrov, Volodymyr
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Fu, Ying
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Mechanisms of fluorescence decays of colloidal CdSe-CdS/ZnS quantum dots unraveled by time-resolved fluorescence measurement2015In: Physical Chemistry, Chemical Physics - PCCP, ISSN 1463-9076, E-ISSN 1463-9084, Vol. 17, no 41, p. 27588-27595Article in journal (Refereed)
    Abstract [en]

    By narrowing the detection bandpass and increasing the signal-to-noise ratio in measuring the time-resolved fluorescence decay spectrum of colloidal CdSe-CdS/ZnS quantum dots (QDs), we show that directly after the photoexcitation, the fluorescence decay spectrum is characterized by a single exponential decay, which represents the energy relaxation of the photogenerated exciton from its initial high-energy state to the ground exciton state. The fluorescence decay spectrum of long decay time is in the form of beta/t(2), where beta is the radiative recombination time of the ground-state exciton and t is the decay time. Our findings provide us with a direct and quantitative link between fluorescence decay measurement data and fundamental photophysics of QD exciton, thereby leading to a novel way of applying colloidal QDs to study microscopic, physical and chemical processes in many fields including biomedicine.

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