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Protein quality control involves the regulation of functional protein concentration at an optimal level in cells. To achieve this cellular need, a variety of biomolecular phenomena including protein synthesis, protein folding, chaperone action, and protein turnover are coordinated and balanced. While many studies on protein quality control focus on water-soluble proteins, it is not well understood how the quality control of membrane proteins is maintained. However, this question has been challenging to address due to difficulties in establishing tractable model systems in the lipid bilayer environment. This dissertation aims to answer two specific problems in membrane biology: 1) How does the lipid bilayer influence the folding and cooperativity of membrane proteins? 2) How do the intrinsic folding properties of membrane proteins influence their susceptibility to degradation? Using the intramembrane protease GlpG as a model, I find that, compared to micelles, the lipid bilayer enhances the stability of the protein by facilitating residue burial in the protein interior and strengthening the cooperative interaction network. Also, I find that conformational stability is not a major determinant of degradation rates of membrane proteins, and rather, the hydrophobicity of transmembrane segments or the conformational distribution of denatured state ensembles impact more. This finding suggests that the rate-limiting step of FtsH-mediated degradation of membrane proteins is not substrate denaturation but the dislocation of the hydrophobic transmembrane segments from the membrane to water. My studies will contribute to the fundamental understanding of the lipid bilayer as a solvent mediating folding, function, and quality control of membrane proteins.
Membrane proteins are a neglected, but important class of proteins throughout the biological world. They carry out critical roles in the cell due to their unique location, such as transport across a membrane, transduction of exterior signals, and interaction between discrete aqueous regions. Despite the importance of these proteins, understanding of how they fold has lagged far behind that of soluble proteins. One of the primary challenges to studying membrane protein folding is developing methods that interrogate folding in the native environment of the lipid bilayer. Our lab has developed a method for measuring membrane protein stability under native conditions using a secondary protein that preferentially binds the unfolded state, obviating the need for harsh denaturants. Employing this method with a multimeric polytopic membrane protein, we measured an extremely slow unfolding rate, demonstrating that -helical membrane proteins can have high kinetic stability under non-denaturing conditions. Efforts were made to expand the steric trap method for single-molecule fluorescence measurements in lipid vesicles, but were ultimately stymied by the inability to preserve the trapped complexes for measurement. Our lab has also applied single-molecule techniques to membrane protein folding. We were able to map the energy landscape of a membrane protein in a lipid bilayer using forced unfolding driven by magnetic tweezers. Further advancements to this technique simplified the attachment chemistry to ready the protein for tweezing. These techniques can be applied to a wide array of membrane proteins in a broad spectrum of membrane environments.
Controlled degradation of misassembled and dispensable proteins is a crucial cellular process for maintaining the quality control of proteomes. In cells, one of the important carriers of this process is AAA+ (ATPases Associated with diverse cellular Activities) proteases, which mediate ATP-dependent proteolysis. The FtsH family proteins are the only membrane integrated AAA+ proteases, which critically contribute to membrane protein degradation. To investigate the mechanisms of membrane protein degradation mediated by FtsH, I successfully reconstituted the degradation process using FtsH of E. coli in a lipid bilayer environment (Chapter 2). I also developed a six-helical bundle intramembrane protease GlpG of E. coli into a model membrane substrate to study the quantitative relationship between folding and degradation (Chapter 2). I found that FtsH has a substantial ability to accelerate unfolding of membrane substrates up to 800 fold using ATP hydrolysis, and the intrinsic folding properties of the substrates such as local stability, spontaneous unfolding rates, and hydrophobicity also impact degradation rates. Finally, I quantified the total ATP cost that FtsH consumes to degrade membrane proteins (Chapters 3 and 4). To degrade membrane proteins, FtsH needs to overcome large energetic costs for unfolding substrates in the membranes and extracting them towards its protease domain located outside the membrane. I found that FtsH utilizes ATP hydrolysis in degrading membrane proteins with similar efficiency to other AAA+ proteases in degrading water-soluble substrates. This efficiency is achieved by coupling multiple ATP hydrolysis events to degradation in a highly cooperative manner. These findings provide new insights into the physical principles of ATP-dependent degradation of membrane proteins, and the in vitro system developed will serve as a model for further refining the mechanisms of membrane protein degradation.
In Protein Structure, Stability, and Folding, Kenneth P. Murphy and a panel of internationally recognized investigators describe some of the newest experimental and theoretical methods for investigating these critical events and processes. Among the techniques discussed are the many methods for calculating many of protein stability and dynamics from knowledge of the structure, and for performing molecular dynamics simulations of protein unfolding. New experimental approaches presented include the use of co-solvents, novel applications of hydrogen exchange techniques, temperature-jump methods for looking at folding events, and new strategies for mutagenesis experiments. Unique in its powerful combination of theory and practice, Protein Structure, Stability, and Folding offers protein and biophysical chemists the means to gain a more comprehensive understanding of some of this complex area by detailing many of the major techniques in use today.
This volume is comprised of a collection of experimental protocols for common techniques and strategies used to study the biogenesis of b-barrel outer membrane proteins in Gram-negative bacteria. The BAM Complex: Methods and Protocols guides readers through methods on the function of the BAM complex, the roles played by each of the individual components, the expression and purification of the components, crystallization and structure determination of the components, and how the individual Bam components may assemble into a functional complex. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, The BAM Complex: Methods and Protocols will serve as an invaluable reference for those interested in studying the BAM complex.
Many individual aspects of the dynamics and assembly of biological membranes have been studied in great detail. Cell biological approaches, advanced genetics, biophysics and biochemistry have greatly contributed to an increase in our knowledge in this field.lt is obvious however, that the three major membrane constituents - lipids, proteins and carbohydrates- are studied, in most cases separately and that a coherent overview of the various aspects of membrane biogenesis is not readily available. The NATO Advanced Study Institute on "New Perspectives in the Dynamics of Assembly of Biomembranes" intended to provide such an overview: it was set up to teach students and specialists the achievements obtained in the various research areas and to try and integrate the numerous aspects of membrane assembly into a coherent framework. The articles in here reflect this. Statting with detailed contributions on phospholipid structure, dynamics, organization and biogenesis, an up to date overview of the basic, lipidic backbone of biomembranes is given. Extensive progress is made in the research on membrane protein biosynthesis. In particular the post- and co-translational modification processes of proteins, the mechanisms of protein translocation and the sorting mechanisms which are necessary to direct proteins to their final, intra - or extracellular destination have been characterized in detail. Modern genetic approaches were indispensable in this research area: gene cloning, hybrid protein construction, site directed mutagenesis and sequencing techniques elucidated many functional aspects of specific nucleic acid and amino acid sequences.
Sugar chains (glycans) are often attached to proteins and lipids and have multiple roles in the organization and function of all organisms. "Essentials of Glycobiology" describes their biogenesis and function and offers a useful gateway to the understanding of glycans.
This volume of Current Topics in Membranes focuses on Membrane Protein Crystallization, beginning with a review of past successes and general trends, then further discussing challenges of mebranes protein crystallization, cell free production of membrane proteins and novel lipids for membrane protein crystallization. This publication also includes tools to enchance membrane protein crystallization, technique advancements, and crystallization strategies used for photosystem I and its complexes, establishing Membrane Protein Crystallization as a needed, practical reference for researchers.