title: LCP-Tm: an assay to measure and understand stability of membrane proteins in a membrane environment.
abstract: 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.
authors.affiliation: Department of Molecular Biology, The Scripps Research Institute, La Jolla, California, USA.
authors.forename: Wei
authors.initials: W
authors.lastname: Liu
authors.forename: Michael A
authors.initials: MA
authors.lastname: Hanson
authors.forename: Raymond C
authors.initials: RC
authors.lastname: Stevens
authors.forename: Vadim
authors.initials: V
authors.lastname: Cherezov
mesh_terms: 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
chemical_list: D003902:Detergents; D008024:Ligands; D008051:Lipid Bilayers; D008055:Lipids; D008823:Micelles; D018343:Receptors, Adrenergic, beta-2; D001436:Bacteriorhodopsins; D009113:Muramidase
references.citation: Biomaterials. 2000 Feb;21(3):223-34
references.pmid: 10646938
references.citation: Cryst Growth Des. 2011 Apr 6;11(4):1182-1192
references.pmid: 22933857
references.citation: Biophys J. 2003 Nov;85(5):3084-96
references.pmid: 14581209
references.citation: Biochemistry. 1997 Sep 9;36(36):10959-74
references.pmid: 9283088
references.citation: Biochemistry. 2006 Oct 3;45(39):11713-26
references.pmid: 17002272
references.citation: Science. 2007 Nov 23;318(5854):1258-65
references.pmid: 17962520
references.citation: Science. 2007 Nov 23;318(5854):1266-73
references.pmid: 17962519
references.citation: J Biol Chem. 2000 Feb 4;275(5):3121-7
references.pmid: 10652295
references.citation: J Mol Biol. 2008 Mar 7;376(5):1305-19
references.pmid: 18222471
references.citation: J Mol Biol. 2006 Apr 14;357(5):1605-18
references.pmid: 16490208
references.citation: J Struct Biol. 2005 Apr;150(1):23-40
references.pmid: 15797727
references.citation: EMBO J. 1989 Aug;8(8):2149-70
references.pmid: 2676514
references.citation: Proc Natl Acad Sci U S A. 1996 Dec 10;93(25):14532-5
references.pmid: 8962086
references.citation: J Biomol Screen. 2001 Dec;6(6):429-40
references.pmid: 11788061
references.citation: Chem Phys Lipids. 2004 Jan;127(1):103-11
references.pmid: 14706744
references.citation: Nat Biotechnol. 2009 Jan;27(1):51-7
references.pmid: 19079241
references.citation: J Struct Biol. 2006 Jun;154(3):223-31
references.pmid: 16600634
references.citation: Methods Enzymol. 1974;31:667-78
references.pmid: 4418026
references.citation: Biochim Biophys Acta. 1998 Mar 13;1370(2):299-309
references.pmid: 9545586
references.citation: Annu Rev Biophys. 2009;38:29-51
references.pmid: 19086821
references.citation: Nat Protoc. 2009;4(5):706-31
references.pmid: 19390528
references.citation: Biophys J. 2001 Jul;81(1):225-42
references.pmid: 11423409
references.citation: Structure. 2008 Mar;16(3):351-9
references.pmid: 18334210
references.citation: Phys Rev E Stat Nonlin Soft Matter Phys. 2005 Dec;72(6 Pt 1):061913
references.pmid: 16485980
references.citation: Anal Biochem. 2006 Oct 15;357(2):289-98
references.pmid: 16962548
references.citation: Cell Mol Life Sci. 2006 Jan;63(1):36-51
references.pmid: 16314922
references.citation: Science. 2008 Nov 21;322(5905):1211-7
references.pmid: 18832607
references.citation: Structure. 2008 Jun;16(6):897-905
references.pmid: 18547522
references.citation: Methods. 2004 Nov;34(3):348-53
references.pmid: 15325652
references.citation: Annu Rev Pharmacol Toxicol. 2006;46:481-519
references.pmid: 16402913
references.citation: Biochim Biophys Acta. 1998 Nov 10;1376(3):401-15
references.pmid: 9805000
references.citation: Proc Natl Acad Sci U S A. 2006 Oct 24;103(43):15835-40
references.pmid: 17035505
references.citation: Chem Phys Lipids. 1998 Sep;95(1):11-21
references.pmid: 9807807
references.citation: Methods Mol Biol. 2003;228:305-16
references.pmid: 12824562
references.citation: Biophys J. 2002 Dec;83(6):3393-407
references.pmid: 12496106
references.citation: Nat Biotechnol. 2007 Oct;25(10):1119-26
references.pmid: 17921997
references.citation: J Struct Biol. 2004 Nov;148(2):169-75
references.pmid: 15477097
references.citation: J R Soc Interface. 2009 Oct 6;6 Suppl 5:S587-97
references.pmid: 19535414
references.citation: Biochemistry. 2001 Apr 10;40(14):4484-92
references.pmid: 11284705