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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.
In this present volume, different approaches are detailed to produce membrane proteins, purify them, study their function, determine their structure, and model them in membrane. Since every membrane protein behaves mostly in a unique way /fashion, knowledge of guidelines and tricks may help to increase chances to express, purify and characterize a peculiar membrane protein. Production of correctly folded protein remains a challenge. Moreover, getting a functional and stable protein requires to optimize membrane mimicking environments that can be detergent or artificial membranes. In some cases, the finding of the correct ligand which will stabilize the desired conformation is needed. In other cases, stabilization can be obtained using specific antibodies. This volume also presents different techniques to analyze the functional status of membrane proteins. Written in the highly successful Methods in Molecular Biology series format, chapters in Membrane Protein Structure and Function Characterization: Methods and Protocols provide different techniques to analyze the functional and structural status of membrane proteins. 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 practical, Membrane Protein Structure and Function Characterization: Methods and Protocols aims to ensure successful results in the further study of this vital field.
The modern biosciences make many new proteins available. Nevertheless the handling of these proteins is quite difficult due to problems with their stability. This collection gives - in the form of tables - protein stability data for various temperatures and solvents. These data are most useful for the development of protein folding and the improvement of biotechnological stability for applications of proteins. The first supplement contains material covering 1997-1999. Some previous data have also been included into the present work. Previous papers on denaturant-induced protein unfolding have been reconsidered to include additional parameters. Furthermore, data on TFE-induced unfolding have been arranged in a new Table. Finally, some data have been added which slipped through during the preparation of the data collection.
Knowledge of the three-dimensional structure of a protein is absolutely required for the complete understanding of its function. The spatial orientation of amino acids in the active site of an enzyme demonstrates how substrate specificity is defined, and assists the medicinal chemist in the design of s- cific, tight-binding inhibitors. The shape and contour of a protein surface hints at its interaction with other proteins and with its environment. Structural ana- sis of multiprotein complexes helps to define the role and interaction of each individual component, and can predict the consequences of protein mutation or conditions that promote dissociation and rearrangement of the complex. Determining the three-dimensional structure of a protein requires milligram quantities of pure material. Such quantities are required to refine crystallization conditions for X-ray analysis, or to overcome the sensitivity limitations of NMR spectroscopy. Historically, structural determination of proteins was limited to those expressed naturally in large amounts, or derived from a tissue or cell source inexpensive enough to warrant the use of large quantities of cells. H- ever, with the advent of the techniques of modern gene expression, many p- teins that are constitutively expressed in minute amounts can become accessible to large-scale purification and structural analysis.
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.
The precise knowledge of the structure of biological macromolecules forms the basis of understanding their function and their mechanism of action. It also lays the foundation for rational protein and drug design. The only method to obtain this knowledge is still crystallography. At present, the structures of about 400 proteins are known at or nearly at atomic proteins. However, only two of them are membrane proteins or complexes of the membrane proteins. The reasons for the difference is not the crystals of membrane proteins resists forming special problems when being analysed. The reason is that the membrane proteins resist into forming into well-ordered crystals. The intention of this book is to help to produce well-ordered crystals proteins and to provide guidelines, it is aimed at both biochemists and protein crystallographer‘s.