title: General qPCR and Plate Reader Methods for Rapid Optimization of Membrane Protein Purification and Crystallization Using Thermostability Assays.
abstract: This unit describes rapid and generally applicable methods to identify conditions that stabilize membrane proteins using temperature-based denaturation measurements as a proxy for target time-dependent stability. Recent developments with thiol-reactive dyes sensitive to the unmasking of cysteine residues upon protein unfolding have allowed for routine application of thermostability assays to systematically evaluate the stability of membrane protein preparations after various purification procedures. Test conditions can include different lipid cocktails, lipid-detergent micelles, pH, salts, osmolytes, and potential active-site ligands. Identification and use of conditions that stabilize the structure have proven successful in enabling the structure determination of numerous families of membrane proteins that otherwise were intractable.
authors.affiliation: Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, California.
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Tomasiak
authors.affiliation: Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, California.
authors.forename: Bjørn P
authors.initials: BP
authors.lastname: Pedersen
authors.affiliation: Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, California.
authors.forename: Sarika
authors.initials: S
authors.lastname: Chaudhary
authors.affiliation: Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, California.
authors.forename: Andrew
authors.initials: A
authors.lastname: Rodriguez
authors.affiliation: Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, California.
authors.forename: Yaneth Robles
authors.initials: YR
authors.lastname: Colmanares
authors.affiliation: Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, California.
authors.forename: Zygy
authors.initials: Z
authors.lastname: Roe-Zurz
authors.affiliation: Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, California.
authors.forename: Sobha
authors.initials: S
authors.lastname: Thamminana
authors.affiliation: Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, California.
authors.forename: Meseret
authors.initials: M
authors.lastname: Tessema
authors.affiliation: Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, California.
authors.forename: Robert M
authors.initials: RM
authors.lastname: Stroud
mesh_terms: D018360:Crystallography, X-Ray / Q000379:methods; D006863:Hydrogen-Ion Concentration; D008055:Lipids / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D008823:Micelles
chemical_list: D008055:Lipids; D008565:Membrane Proteins; D008823:Micelles
keywords: dye-based assay; membrane proteins; thermostability
references.citation: Curr Opin Biotechnol. 1997 Oct;8(5):569-72
references.pmid: 9353228
references.citation: Nature. 2013 Apr 25;496(7446):533-6
references.pmid: 23542591
references.citation: J Biomol Screen. 2006 Dec;11(8):940-8
references.pmid: 17092916
references.citation: Nat Struct Mol Biol. 2013 Feb;20(2):135-8
references.pmid: 23381628
references.citation: Nat Protoc. 2007;2(9):2212-21
references.pmid: 17853878
references.citation: Annu Rev Biophys Biomol Struct. 2004;33:25-51
references.pmid: 15139803
references.citation: Methods. 2011 Dec;55(4):324-9
references.pmid: 21840396
references.citation: Structure. 2011 Jan 12;19(1):17-25
references.pmid: 21220112
references.citation: Curr Protein Pept Sci. 2008 Aug;9(4):338-69
references.pmid: 18691124
references.citation: Mol Cell Biochem. 2008 Jan;307(1-2):249-64
references.pmid: 17874175
references.citation: Anal Biochem. 1986 Apr;154(1):186-93
references.pmid: 3706721
references.citation: Biochemistry. 2005 Apr 5;44(13):5258-66
references.pmid: 15794662
references.citation: Nat Protoc. 2012 Feb 09;7(3):453-66
references.pmid: 22322218
references.citation: Nat Protoc. 2009;4(5):706-31
references.pmid: 19390528
references.citation: Structure. 2012 Aug 8;20(8):1293-9
references.pmid: 22884106
references.citation: Structure. 2008 Mar;16(3):351-9
references.pmid: 18334210
references.citation: Anal Biochem. 2006 Oct 15;357(2):289-98
references.pmid: 16962548
references.citation: Angew Chem Int Ed Engl. 2007;46(37):7023-5
references.pmid: 17691085
references.citation: Methods Enzymol. 2010;470:695-707
references.pmid: 20946832
references.citation: Acta Crystallogr D Biol Crystallogr. 2011 Nov;67(Pt 11):915-9
references.pmid: 22101817
references.citation: Science. 2007 Nov 23;318(5854):1258-65
references.pmid: 17962520
references.citation: Proc Natl Acad Sci U S A. 2008 Jan 22;105(3):877-82
references.pmid: 18192400
references.citation: Nature. 2013 Jul 4;499(7456):107-10
references.pmid: 23685453
references.citation: Biotechnol J. 2011 Jun;6(6):700-6
references.pmid: 21567964
references.citation: Methods. 2011 Dec;55(4):337-41
references.pmid: 21982781
references.citation: Proc Natl Acad Sci U S A. 2006 Oct 24;103(43):15835-40
references.pmid: 17035505