Chapter 8 Methods for Measuring the Thermodynamic Stability of Membrane Proteins

Department of Chemistry and Biochemistry, UCLA-DOE Center for Genomics and Proteomics, Molecular Biology Institute, University of California, Los Angeles, California, USA.
Methods in enzymology (Impact Factor: 2.09). 02/2009; 455:213-36. DOI: 10.1016/S0076-6879(08)04208-0
Source: PubMed


Learning how amino acid sequences define protein structure has been a major challenge for molecular biology since the first protein structures were determined in the 1960s. In contrast to the staggering progress with soluble proteins, investigations of membrane protein folding have long been hampered by the lack of high-resolution structures and the technical challenges associated with studying the folding process in vitro. In the past decade, however, there has been an explosion of new membrane protein structures and a slower but notable increase in efforts to study the factors that define these structures. Here we review the methods that have been used to evaluate the thermodynamic stability of membrane proteins and provide some salient examples of how the methods have been used to begin to understand the energetics of membrane protein folding.

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    ABSTRACT: The stability of globular proteins is important in medicine, proteomics, and basic research. The conformational stability of the folded state can be determined experimentally by analyzing urea, guanidinium chloride, and thermal denaturation curves. Solvent denaturation curves in particular may give useful information about a protein such as the existence of domains or the presence of stable folding intermediates. The linear extrapolation method (LEM) for analyzing solvent denaturation curves gives the parameter m, which is a measure of the dependence of ΔG on denaturant concentration. There is much recent interest in the m value as it relates to the change in accessible surface area of a protein when it unfolds and what it may reveal about the denatured states of proteins.
    Methods in enzymology 01/2009; 466:549-65. DOI:10.1016/S0076-6879(09)66023-7 · 2.09 Impact Factor
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    ABSTRACT: Although the structures of many beta-barrel membrane proteins are available, our knowledge of the principles that govern their energetics and oligomerization states is incomplete. Here we describe a computational method to study the transmembrane (TM) domains of beta-barrel membrane proteins. Our method is based on a physical interaction model, a simplified conformational space for efficient enumeration, and an empirical potential function from a detailed combinatorial analysis. Using this method, we can identify weakly stable regions in the TM domain, which are found to be important structural determinants for beta-barrel membrane proteins. By calculating the melting temperatures of the TM strands, our method can also assess the stability of beta-barrel membrane proteins. Predictions on membrane enzyme PagP are consistent with recent experimental NMR and mutant studies. We have also discovered that out-clamps, in-plugs, and oligomerization are 3 general mechanisms for stabilizing weakly stable TM regions. In addition, we have found that extended and contiguous weakly stable regions often signal the existence of an oligomer and that strands located in the interfaces of protein-protein interactions are considerably less stable. Based on these observations, we can predict oligomerization states and can identify the interfaces of protein-protein interactions for beta-barrel membrane proteins by using either structure or sequence information. In a set of 25 nonhomologous proteins with known structures, our method successfully predicted whether a protein forms a monomer or an oligomer with 91% accuracy; in addition, our method identified with 82% accuracy the protein-protein interaction interfaces by using sequence information only when correct strands are given.
    Proceedings of the National Academy of Sciences 08/2009; 106(31):12735-40. DOI:10.1073/pnas.0902169106 · 9.67 Impact Factor
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    ABSTRACT: Over the years, membrane-soluble peptides have provided a convenient model system to investigate the folding and assembly of integral membrane proteins. Recent advances in experimental and computational methods are now being translated into the design of functional membrane proteins. Applications include artificial modulators of membrane protein function, inhibitors of protein-protein interactions, and redox membrane proteins.
    Current opinion in chemical biology 10/2009; 13(5-6):643-51. DOI:10.1016/j.cbpa.2009.09.017 · 6.81 Impact Factor
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