Dissertation
Protein determinants of CLC-ec1 stoichiometry
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Summer 2021
DOI: 10.17077/etd.005992
Abstract
The efforts of this thesis are directed towards understanding the physical driving forces that mediate membrane protein self-assembly processes within the native environment of membrane proteins, the bilayer milieu. While many defined determinants contribute to the energetics of membrane protein self-assembly, such as electrostatics, non-polar side-chain packing, hydrogen bonding, and solvophobic effects, the roles that these driving forces play have not been well characterized. The inability to describe the role of these physical driving forces in processes involving membrane proteins comes from the lack of quantitative thermodynamic measurements concerning membrane protein self-assembly equilibria within the lipid bilayer environment. These thermodynamic measurements would describe the free energy of self-assembly (∆G°) and the breakdown of this term into enthalpic (∆H°) and entropic components (∆S°). The studies within this thesis bridge the gaps in our knowledge of membrane protein self-assembly by quantifying the contributions of non-polar side-chain packing in the dimerization reaction of the large poly-topic membrane protein CLC-ec1 within lipid bilayers. An emphasis on describing non-polar side-chain packing was chosen due to membrane proteins commonly exhibiting the packing of transmembrane helices and surfaces lined with non-polar residues within lipid bilayers. Furthermore, non-polar side-chain packing is thought to be a significant contributor to membrane protein self-assembly, but its quantification has been limited. An emphasis is placed within the thesis on quantifying non-polar side-chain packing interactions in terms of van der Waals (VDW) interaction energies. Chapter 1 of this thesis describes the pertinent background of the driving forces thought to be involved in membrane protein self-assembly and provides an extensive review of the membrane protein CLC-ec1. The studies within this thesis employed a novel method, called the subunit capture method, to measure CLC-ec1 dimerization within membranes. Chapter 2 describes the subunit capture method and other methods commonly used throughout the thesis.
Chapter 3 describes the first study, which characterizes the impact of removing non-polar side-chain contacts on the stoichiometry of the CLC-ec1 homodimer. Computational analysis of the dimerization interface of CLC-ec1 characterized non-polar side-chain packing by measuring the VDW interaction energies of side-chains within a structure of CLC-ec1 determined by x-ray crystallography. Subsequent experiments targeted non-polar side-chains exhibiting favorable VDW interaction energies by substituting 4-5 of the native residues at a time to the smaller residue alanine, depleting the favorable VDW interactions at the dimerization interface. The CLC-ec1 multiple alanine constructs revealed that the stoichiometry of CLC-ec1 is dependent upon the non-polar side-chains at the dimerization interface, as each construct becomes entirely monomeric in lipid bilayers. The studies also found that changes in CLC-ec1 stoichiometry occur in a solvent-dependent manner as three of the four CLC-ec1 multiple alanine constructs remain stable dimers in detergent micelles. Lastly, the study exposes a molecular hot-spot at residue 194, characterizing the substitution to alanine at that position as important for dimer stoichiometry in both detergents and lipids.
The contents of Chapter 4 present the findings from a second study that sought answers to two questions. The study begins by posing the first question: Are protein-protein interactions driving the dimerization of CLC-ec1? To answer this question, we performed long all-atom molecular dynamics (MD) simulations to quantify the interactions of CLC-ec1 in a dimeric and monomeric state while embedded in a lipid bilayer. Through the quantification of VDW interaction energies, the measurements revealed that the interactions within each state are similar in magnitude. The findings from the MD simulations present VDW interactions as important to CLC-ec1 dimerization but not as a determinant that excessively stabilizes the CLC-ec1 dimer complex.
The results from the analysis of the MD simulations allowed us to form the second question of the study: Will removing side-chain packing interactions at the dimerization interface destabilize the CLC-ec1 homodimer? The second question was thoroughly investigated by measuring the changes in dimerization free energy of CLC-ec1 constructs that contained single substitutions to alanine that generate cavities at the dimerization interface. The various changes in CLC-ec1 dimer stability due to the alanine substitutions were compared to the MD computational analysis to find no correlation between the two datasets. We conclude that while the CLC-ec1 single alanine substitutions destabilize the dimer complex, the removal of non-polar packing interactions from the dimer state of CLC-ec1 does not explain the measured changes in stability.
The final chapter in this thesis, Chapter 5, presents a discussion of how the results obtained within this thesis fit into our current understanding of membrane protein self-assembly. Included in the discussion, we reason that CLC-ec1 may not have excessively favorable non-polar packing interactions at the CLC-ec1 dimerization interface in order to retain a flexible interface for function -related activities. Finally, Chapter 5 will conclude by posing a few future directions of study to answer new questions we have encountered while performing the work presented in this thesis.
Details
- Title: Subtitle
- Protein determinants of CLC-ec1 stoichiometry
- Creators
- Kacey Nicholas Mersch
- Contributors
- Janice L Robertson (Advisor)Christopher A Ahern (Committee Member)Nikolai O Artemyev (Committee Member)Robert C Piper (Committee Member)Madeline A Shea (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Molecular Physiology and Biophysics
- Date degree season
- Summer 2021
- DOI
- 10.17077/etd.005992
- Publisher
- University of Iowa
- Number of pages
- xvii, 185 pages
- Copyright
- Copyright 2021 Kacey Nicholas Mersch
- Language
- English
- Description illustrations
- color illustrations
- Description bibliographic
- Includes bibliographical references (pages 175-185).
- Public Abstract (ETD)
Proteins are large chemical molecules that perform essential biological tasks at the cellular level that allow all living organisms to function properly. The tasks that proteins execute depend upon the structure of the protein. A specific structure enables a protein to perform particular operations for an organism that are key in processes not limited to metabolism, immunological responses, and sensing of the overall environment (e.g., sight in humans). Protein structures have been under intense investigation since the 1960s. Since then, studies have provided a detailed physical and chemical description of the rules that dictate how proteins form a particular structure. However, a special class of proteins, called membrane proteins, has not been amenable to the same studies as other proteins as they reside within the boundary of cells called the lipid bilayer. When studying membrane proteins within lipid bilayers, the technical issues that arise have disallowed a similarly detailed characterization of membrane protein structures as other protein classes. To fill the gap of knowledge in membrane protein structure, the work here describes measurements quantifying the impact of destabilizing changes made to the chemical composition of the membrane protein complex CLC-ec1, which associates with itself in a 1:1 fashion. A focused effort was placed on performing the measurements on the membrane protein CLC-ec1 within lipid bilayers. The studies here concluded that the membrane protein field should reconsider the role of the surrounding environment and the chemical makeup of membrane proteins as determinants of membrane protein structure.
- Academic Unit
- Molecular Physiology and Biophysics
- Record Identifier
- 9984124358702771
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