title: Determining serpin conformational distributions with single molecule fluorescence.
abstract: Conformational plasticity is key to inhibitory serpin function, and this plasticity gives serpins relatively easy access to alternative, dysfunctional conformations. Thus, a given serpin population may contain both functional and dysfunctional proteins. Single molecule fluorescence (SMF), with its ability to interrogate one fluorescently labeled protein at a time, is a powerful method for elucidating conformational distributions and monitoring how these distributions change over time. SMF and related methods have been particularly valuable for characterizing serpin polymerization. Fluorescence correlation spectroscopy experiments have revealed a second lag phase during in vitro α(1)-antitrypsin polymerization associated with the formation of smaller oligomers that then condense to form longer polymers [Purkayastha, P., Klemke, J. W., Lavender, S., Oyola, R., Cooperman, B. S., and Gai, F. (2005). Alpha 1-antitrypsin polymerization: A fluorescence correlation spectroscopic study. Biochemistry44, 2642-2649.]. SMF studies of in vitro neuroserpin polymerization have confirmed that a monomeric intermediate is required for polymer formation while providing a test of proposed polymerization mechanisms [Chiou, A., Hägglöf, P., Orte, A., Chen, A. Y., Dunne, P. D., Belorgey, D., Karlsson-Li, S., Lomas, D., and Klenerman, D. (2009). Probing neuroserpin polymerization and interaction with amyloid-beta peptides using single molecule fluorescence. Biophys. J.97, 2306-2315.]. SMF has also been used to monitor protease-serpin interactions. Single pair Förster resonance energy transfer studies of covalent protease-serpin complexes suggest that the extent of protease structural disruption in the complex is protease dependent [Liu, L., Mushero, N., Hedstrom, L., and Gershenson, A. (2006). Conformational distributions of protease-serpin complexes: A partially translocated complex. Biochemistry45, 10865-10872.]. SMF techniques are still evolving and the combination of SMF with encapsulation methods has the potential to provide more detailed information on the conformational changes associated with serpin polymerization, protease-serpin complex formation, and serpin folding.
authors.affiliation: School of Medicine, University of Massachusetts, Worcester, Massachusetts, USA.
authors.forename: Nicole
authors.initials: N
authors.lastname: Mushero
authors.forename: Anne
authors.initials: A
authors.lastname: Gershenson
mesh_terms: D000818:Animals; D001665:Binding Sites; D002417:Cattle; D057056:Cysteine Proteases / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008301:Maleimides / Q000737:chemistry; D008958:Models, Molecular; D009479:Neuropeptides / Q000737:chemistry* / Q000378:metabolism; D058105:Polymerization; D011485:Protein Binding; D017433:Protein Structure, Secondary; D015843:Serpins / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods*; D000515:alpha 1-Antitrypsin / Q000737:chemistry* / Q000378:metabolism; D000097522:Neuroserpin
chemical_list: D057056:Cysteine Proteases; D005456:Fluorescent Dyes; D008301:Maleimides; D009479:Neuropeptides; D015843:Serpins; D000515:alpha 1-Antitrypsin; D000097522:Neuroserpin; C043592:maleimide
references.citation: Proc Natl Acad Sci U S A. 2007 Jan 2;104(1):105-10
references.pmid: 17185422
references.citation: Biochemistry. 1994 Jul 26;33(29):8757-63
references.pmid: 8038165
references.citation: Curr Opin Struct Biol. 2008 Feb;18(1):16-26
references.pmid: 18221865
references.citation: Annu Rev Phys Chem. 2004;55:79-96
references.pmid: 15117248
references.citation: Bioconjug Chem. 2003 Jan-Feb;14(1):195-204
references.pmid: 12526709
references.citation: Anal Chem. 2003 Apr 1;75(7):1664-70
references.pmid: 12705600
references.citation: J Biol Chem. 2002 May 10;277(19):17367-73
references.pmid: 11880376
references.citation: Nat Methods. 2008 Jun;5(6):507-16
references.pmid: 18511918
references.citation: J Phys Chem B. 2007 Aug 30;111(34):10253-62
references.pmid: 17676789
references.citation: J Phys Chem B. 2010 Nov 25;114(46):15221-6
references.pmid: 21028764
references.citation: J Biol Chem. 2007 Jan 26;282(4):2305-13
references.pmid: 17142451
references.citation: J Biol Chem. 1994 Nov 4;269(44):27657-62
references.pmid: 7961684
references.citation: J Phys Chem B. 2006 Apr 6;110(13):6970-8
references.pmid: 16571010
references.citation: Biophys J. 2005 Nov;89(5):3508-22
references.pmid: 16113120
references.citation: J Phys Chem B. 2010 Dec 9;114(48):15766-72
references.pmid: 21069980
references.citation: Proc Natl Acad Sci U S A. 2010 Aug 17;107(33):14609-14
references.pmid: 20639465
references.citation: Biochemistry. 2001 May 29;40(21):6275-83
references.pmid: 11371189
references.citation: Biophys J. 2009 Oct 21;97(8):2306-15
references.pmid: 19843463
references.citation: Annu Rev Biochem. 1971;40:83-114
references.pmid: 4331120
references.citation: J Mol Biol. 2007 Jan 19;365(3):856-69
references.pmid: 17084857
references.citation: Bioconjug Chem. 2008 Mar;19(3):786-91
references.pmid: 18275130
references.citation: Biophys J. 2004 Apr;86(4):2530-7
references.pmid: 15041689
references.citation: Acc Chem Res. 2005 Jul;38(7):523-33
references.pmid: 16028886
references.citation: Lab Chip. 2009 Mar 7;9(5):692-8
references.pmid: 19224019
references.citation: Anal Chem. 2006 Nov 15;78(22):7707-15
references.pmid: 17105162
references.citation: Biochemistry. 2006 Sep 12;45(36):10865-72
references.pmid: 16953572
references.citation: Annu Rev Phys Chem. 2010;61:345-67
references.pmid: 20055680
references.citation: Nature. 1992 Jun 18;357(6379):605-7
references.pmid: 1608473
references.citation: Nat Struct Mol Biol. 2005 Jan;12(1):46-53
references.pmid: 15580276
references.citation: Proc Natl Acad Sci U S A. 2007 Jun 19;104(25):10459-64
references.pmid: 17556539
references.citation: Proc Natl Acad Sci U S A. 2010 Jun 29;107(26):11793-8
references.pmid: 20547872
references.citation: FEBS Lett. 2001 Apr 6;494(1-2):30-3
references.pmid: 11297729
references.citation: Biochim Biophys Acta. 2010 Mar;1803(3):396-404
references.pmid: 19895852
references.citation: J Biol Chem. 2002 Oct 11;277(41):38901-14
references.pmid: 12077135
references.citation: Biophys J. 2010 Apr 7;98(7):1302-11
references.pmid: 20371330
references.citation: Biophys J. 2008 Jun;94(12):4819-27
references.pmid: 18326651
references.citation: J Biol Chem. 2002 Apr 5;277(14):11629-35
references.pmid: 11786547
references.citation: J Biol Chem. 2001 Jul 20;276(29):27541-7
references.pmid: 11325972
references.citation: Proc Natl Acad Sci U S A. 2007 Feb 20;104(8):2649-54
references.pmid: 17299036
references.citation: Methods Enzymol. 1986;130:454-84
references.pmid: 3773744
references.citation: Methods. 2011 Mar;53(3):255-66
references.pmid: 21115126
references.citation: J Mol Biol. 2007 Jun 1;369(2):462-73
references.pmid: 17442346
references.citation: Proc Natl Acad Sci U S A. 2008 Sep 23;105(38):14424-9
references.pmid: 18796612
references.citation: J Biol Chem. 2002 Aug 16;277(33):29927-35
references.pmid: 12055188
references.citation: J Biol Chem. 1995 Mar 10;270(10):5395-8
references.pmid: 7890653
references.citation: Methods Mol Biol. 2009;490:311-37
references.pmid: 19157089
references.citation: J Biol Chem. 2006 Feb 10;281(6):3452-7
references.pmid: 16321984
references.citation: Nature. 2000 Oct 19;407(6806):923-6
references.pmid: 11057674
references.citation: J Phys Chem B. 2007 Nov 8;111(44):12925-32
references.pmid: 17929964
references.citation: Curr Opin Struct Biol. 2010 Feb;20(1):121-7
references.pmid: 20061135
references.citation: Biochemistry. 2005 Feb 22;44(7):2642-9
references.pmid: 15709777
references.citation: Annu Rev Biochem. 1978;47:819-46
references.pmid: 354506
references.citation: J Biol Chem. 2006 Sep 29;281(39):29268-77
references.pmid: 16849336
references.citation: Anal Chem. 2008 Nov 15;80(22):8389-97
references.pmid: 18855410
references.citation: J Mol Biol. 2006 Jun 2;359(2):378-89
references.pmid: 16626735
references.citation: Biopolymers. 1974 Jan;13(1):29-61
references.pmid: 4818131
references.citation: Nature. 1999 Sep 23;401(6751):376-9
references.pmid: 10517635
references.citation: Biophys J. 2005 Feb;88(2):1413-22
references.pmid: 15556973
references.citation: Protein Sci. 2007 Nov;16(11):2403-11
references.pmid: 17962402
references.citation: Anal Chem. 2007 Apr 1;79(7):2771-7
references.pmid: 17343363
references.citation: Methods Enzymol. 2010;475:455-514
references.pmid: 20627168
references.citation: Cell. 2008 Apr 4;133(1):142-53
references.pmid: 18394994
references.citation: J Mol Biol. 2010 Oct 29;403(3):459-67
references.pmid: 20837024