LCP-Tm: an assay to measure and understand stability of membrane proteins in a membrane environment.
Structural and functional studies of membrane proteins are limited by their poor stability outside the native membrane environment. The development of novel methods to efficiently stabilize membrane proteins immediately after purification is important for biophysical studies, and is likely to be critical for studying the more challenging human targets. Lipidic cubic phase (LCP) provides a suitable stabilizing matrix for studying membrane proteins by spectroscopic and other biophysical techniques, including obtaining highly ordered membrane protein crystals for structural studies. We have developed a robust and accurate assay, LCP-Tm, for measuring the thermal stability of membrane proteins embedded in an LCP matrix. In its two implementations, protein denaturation is followed either by a change in the intrinsic protein fluorescence on ligand release, or by an increase in the fluorescence of a thiol-binding reporter dye that measures exposure of cysteines buried in the native structure. Application of the LCP-Tm assay to an engineered human beta2-adrenergic receptor and bacteriorhodopsin revealed a number of factors that increased protein stability in LCP. This assay has the potential to guide protein engineering efforts and identify stabilizing conditions that may improve the chances of obtaining high-resolution structures of intrinsically unstable membrane proteins.
Liu
Wei
W
Department of Molecular Biology, The Scripps Research Institute, La Jolla, California, USA.
Hanson
Michael A
MA
Stevens
Raymond C
RC
Cherezov
Vadim
V
D017122:Bacteriophage T4 / Q000201:enzymology; D001436:Bacteriorhodopsins / Q000378:metabolism*; D001681:Biological Assay / Q000379:methods*; D003902:Detergents / Q000494:pharmacology; D019617:Halobacterium salinarum / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D008024:Ligands; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism; D008055:Lipids / Q000494:pharmacology; D008823:Micelles; D009113:Muramidase / Q000378:metabolism; D011489:Protein Denaturation / Q000187:drug effects; D015202:Protein Engineering; D055550:Protein Stability / Q000187:drug effects; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism*; D044366:Transition Temperature / Q000187:drug effects
D003902:Detergents; D008024:Ligands; D008051:Lipid Bilayers; D008055:Lipids; D008823:Micelles; D018343:Receptors, Adrenergic, beta-2; D001436:Bacteriorhodopsins; D009113:Muramidase
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