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Structure, dynamics and lipid interaction of membrane-associated proteins
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

A research topic within the field of molecular biophysics is the structure-function relationship of proteins. Membrane proteins are a large, diverse group of biological macromolecules that perform many different and essential functions for the cell. Despite the abundance and importance of membrane proteins, high-resolution 3D structures from this class of proteins are underrepresented among all yet determined structures. The limited amount of data for membrane proteins hints about the higher difficulty associated with studies of this group of molecules. The determination of an atomic resolution structure is often a long process in which several obstacles need to be overcome, in particular for membrane proteins.

Solution-state nuclear magnetic resonance (NMR) is a powerful measurement technique that can provide high-resolution data on the structure and dynamics of biological macromolecules, and is suitable for studies of small, dynamic membrane proteins. However, even with solution-state NMR, the membrane proteins need to be investigated in environments that are sometimes severely compromising for the protein’s native structure and function. In order to evaluate the biological significance of results obtained under such artificial conditions, supporting data from experiments in more realistic membrane models, obtained using NMR and other biophysical methods, is of great importance.

The work presented in this thesis concerns studies of four membrane proteins: WaaG, Rcf1, Rcf2 and TatA. These proteins have very different characteristics in terms of their sizes and expected membrane interactions, and were accordingly found to be differently affected by the model membranes in which they were studied. Our results illustrate both the current possibilities and limitations of solution-state NMR for studying membrane proteins, and highlight the benefits of an approach where several membrane mimicking systems and measurements techniques are used in combination to arrive at correct conclusions on the properties of proteins.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 2019. , p. 85
National Category
Biophysics
Research subject
Biophysics
Identifiers
URN: urn:nbn:se:su:diva-167352ISBN: 978-91-7797-692-9 (print)ISBN: 978-91-7797-693-6 (electronic)OAI: oai:DiVA.org:su-167352DiVA, id: diva2:1301468
Public defence
2019-05-23, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Manuscript. Paper 6: Manuscript.

Available from: 2019-04-26 Created: 2019-04-02 Last updated: 2019-04-17Bibliographically approved
List of papers
1. Membrane Interaction of the Glycosyltransferase WaaG
Open this publication in new window or tab >>Membrane Interaction of the Glycosyltransferase WaaG
2015 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 109, no 3, p. 552-563Article in journal (Refereed) Published
Abstract [en]

The glycosyltransferase WaaG is involved in the synthesis of lipopolysaccharides that constitute the outer leaflet of the outer membrane in Gram-negative bacteria such as Escherichia coli. WaaG has been identified as a potential antibiotic target, and inhibitor scaffolds have previously been investigated. WaaG is located at the cytosolic side of the inner membrane, where the enzyme catalyzes the transfer of the first outer-core glucose to the inner core of nascent lipopolysaccharides. Here, we characterized the binding of WaaG to membrane models designed to mimic the inner membrane of E. coli. Based on the crystal structure, we identified an exposed and largely a-helical 30-residue sequence, with a net positive charge and several aromatic amino acids, as a putative membrane-interacting region of WaaG (MIR-WaaG). We studied the peptide corresponding to this sequence, along with its bilayer interactions, using circular dichroism, fluorescence quenching, fluorescence anisotropy, and NMR. In the presence of dodecylphosphocholine, MIR-WaaG was observed to adopt a three-dimensional structure remarkably similar to the segment in the crystal structure. We found that the membrane interaction of WaaG is conferred at least in part by MIR-WaaG and that electrostatic interactions play a key role in binding. Moreover, we propose a mechanism of anchoring WaaG to the inner membrane of E. coli, where the central part of MIR-WaaG inserts into one leaflet of the bilayer. In this model, electrostatic interactions as well as surface-exposed Tyr residues bind WaaG to the membrane.

National Category
Biological Sciences
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-120191 (URN)10.1016/j.bpj.2015.06.036 (DOI)000359180400012 ()
Available from: 2015-09-04 Created: 2015-09-02 Last updated: 2019-04-02Bibliographically approved
2. Solution NMR structure of yeast Rcf1, a protein involved in respiratory supercomplex formation
Open this publication in new window or tab >>Solution NMR structure of yeast Rcf1, a protein involved in respiratory supercomplex formation
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2018 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 115, no 12, p. 3048-3053Article in journal (Refereed) Published
Abstract [en]

The Saccharomyces cerevisiae respiratory supercomplex factor 1 (Rcf1) protein is located in the mitochondrial inner membrane where it is involved in formation of supercomplexes composed of respiratory complexes III and IV. We report the solution structure of Rcf1, which forms a dimer in dodecylphosphocholine (DPC) micelles, where each monomer consists of a bundle of five transmembrane (TM) helices and a short flexible soluble helix (SH). Three TM helices are unusually charged and provide the dimerization interface consisting of 10 putative salt bridges, defining a charge zipper motif. The dimer structure is supported by molecular dynamics (MD) simulations in DPC, although the simulations show a more dynamic dimer interface than the NMR data. Furthermore, CD and NMR data indicate that Rcf1 undergoes a structural change when reconstituted in liposomes, which is supported by MD data, suggesting that the dimer structure is unstable in a planar membrane environment. Collectively, these data indicate a dynamic monomer-dimer equilibrium. Furthermore, the Rcf1 dimer interacts with cytochrome c, suggesting a role as an electron-transfer bridge between complexes III and IV. The Rcf1 structure will help in understanding its functional roles at a molecular level.

Keywords
charge zipper, membrane proteins, mitochondria, bicelles, lipids
National Category
Biological Sciences
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-154705 (URN)10.1073/pnas.1712061115 (DOI)000427829500069 ()29507228 (PubMedID)
Available from: 2018-04-12 Created: 2018-04-12 Last updated: 2019-04-05Bibliographically approved
3. NMR Study of Rcf2 Reveals an Unusual Dimeric Topology in Detergent Micelles
Open this publication in new window or tab >>NMR Study of Rcf2 Reveals an Unusual Dimeric Topology in Detergent Micelles
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2018 (English)In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 19, no 5, p. 444-447Article in journal (Refereed) Published
Abstract [en]

The Saccharomyces cerevisiae mitochondrial respiratory supercomplex factor2 (Rcf2) plays a role in assembly of supercomplexes composed of cytochromebc(1) (complexIII) and cytochromec oxidase (complexIV). We expressed the Rcf2 protein in Escherichia coli, refolded it, and reconstituted it into dodecylphosphocholine (DPC) micelles. The structural properties of Rcf2 were studied by solution NMR, and near complete backbone assignment of Rcf2 was achieved. The secondary structure of Rcf2 contains seven helices, of which five are putative transmembrane (TM) helices, including, unexpectedly, a region formed by a charged 20-residue helix at the Cterminus. Further studies demonstrated that Rcf2 forms a dimer, and the charged TM helix is involved in this dimer formation. Our results provide a basis for understanding the role of this assembly/regulatory factor in supercomplex formation and function.

Keywords
dimers, membrane protein, membrane topology, NMR spectroscopy, respiratory supercomplex factor
National Category
Biological Sciences
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-154787 (URN)10.1002/cbic.201700664 (DOI)000426494600009 ()29240987 (PubMedID)
Available from: 2018-04-17 Created: 2018-04-17 Last updated: 2019-04-05Bibliographically approved
4. NMR structure and dynamics studies of yeast respiratory supercomplex factor 2 in micelles
Open this publication in new window or tab >>NMR structure and dynamics studies of yeast respiratory supercomplex factor 2 in micelles
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(English)Manuscript (preprint) (Other academic)
National Category
Biological Sciences
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-167323 (URN)
Available from: 2019-03-25 Created: 2019-03-25 Last updated: 2019-04-05Bibliographically approved
5. Structure and dynamics of plant TatA in micelles and lipid bilayers studied by solution NMR
Open this publication in new window or tab >>Structure and dynamics of plant TatA in micelles and lipid bilayers studied by solution NMR
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2018 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 285, no 10, p. 1886-1906Article in journal (Refereed) Published
Abstract [en]

The twin-arginine translocase (Tat) transports folded proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of plant chloroplasts. In Gram-negative bacteria and chloroplasts, the translocon consists of three subunits, TatA, TatB, and TatC, of which TatA is responsible for the actual membrane translocation of the substrate. Herein we report on the structure, dynamics, and lipid interactions of a fully functional C-terminally truncated core TatA' from Arabidopsisthaliana using solution-state NMR. Our results show that TatA consists of a short N-terminal transmembrane helix (TMH), a short connecting linker (hinge) and a long region with propensity to form an amphiphilic helix (APH). The dynamics of TatA were characterized using N-15 relaxation NMR in combination with model-free analysis. The TMH has order parameters characteristic of a well-structured helix, the hinge is somewhat less rigid, while the APH has lower order parameters indicating structural flexibility. The TMH is short with a surprisingly low protection from solvent, and only the first part of the APH is protected to some extent. In order to uncover possible differences in TatA's structure and dynamics in detergent compared to in a lipid bilayer, fast-tumbling bicelles and large unilamellar vesicles were used. Results indicate that the helicity of TatA increases in both the TMH and APH in the presence of lipids, and that the N-terminal part of the TMH is significantly more rigid. The results indicate that plant TatA has a significant structural plasticity and a capability to adapt to local environments.

Keywords
bicelle, dynamics, membrane mimetics, micelle, model-free approach, paramagnetic relaxation enhancement, relaxation, structure, TatA, twin-arginine translocation
National Category
Biological Sciences
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-157788 (URN)10.1111/febs.14452 (DOI)000434177700011 ()29654717 (PubMedID)
Available from: 2018-07-26 Created: 2018-07-26 Last updated: 2019-04-05Bibliographically approved
6. Soluble TatA forms oligomers that interact with membranes: structure and insertion studies of a versatile protein transporter
Open this publication in new window or tab >>Soluble TatA forms oligomers that interact with membranes: structure and insertion studies of a versatile protein transporter
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(English)Manuscript (preprint) (Other academic)
National Category
Biological Sciences
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-167325 (URN)
Available from: 2019-03-25 Created: 2019-03-25 Last updated: 2019-04-05Bibliographically approved

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