title: Collapse of a long axis: single-molecule Förster resonance energy transfer and serpin equilibrium unfolding.
abstract: The energy required for mechanical inhibition of target proteases is stored in the native structure of inhibitory serpins and accessed by serpin structural remodeling. The overall serpin fold is ellipsoidal with one long and two short axes. Most of the structural remodeling required for function occurs along the long axis, while expansion of the short axes is associated with misfolded, inactive forms. This suggests that ellipticity, as typified by the long axis, may be important for both function and folding. Placement of donor and acceptor fluorophores approximately along the long axis or one of the short axes allows single-pair Förster resonance energy transfer (spFRET) to report on both unfolding transitions and the time-averaged shape of different conformations. Equilibrium unfolding and refolding studies of the well-characterized inhibitory serpin α1-antitrypsin reveal that the long axis collapses in the folding intermediates while the monitored short axis expands. These energetically distinct intermediates are thus more spherical than the native state. Our spFRET studies agree with other equilibrium unfolding studies that found that the region around one of the β strands, s5A, which helps define the long axis and must move for functionally required loop insertion, unfolds at low denaturant concentrations. This supports a connection between functionally important structural lability and unfolding in the inhibitory serpins.
authors.affiliation: Department of Chemistry, Brandeis University , Waltham, Massachusetts 02453, United States.
authors.forename: Lu
authors.initials: L
authors.lastname: Liu
authors.forename: Michael
authors.initials: M
authors.lastname: Werner
authors.forename: Anne
authors.initials: A
authors.lastname: Gershenson
mesh_terms: D002942:Circular Dichroism; D005454:Fluorescence Polarization; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D008958:Models, Molecular; D011489:Protein Denaturation; D017510:Protein Folding; D055550:Protein Stability; D015843:Serpins; D000515:alpha 1-Antitrypsin / Q000737:chemistry*
chemical_list: D015843:Serpins; D000515:alpha 1-Antitrypsin
references.citation: Nat Struct Biol. 2003 Sep;10(9):731-7
references.pmid: 12923573
references.citation: Adv Protein Chem. 2000;53:209-82
references.pmid: 10751946
references.citation: Chem Rev. 2002 Dec;102(12):4751-804
references.pmid: 12475206
references.citation: J Mol Biol. 2007 Aug 3;371(1):245-55
references.pmid: 17568610
references.citation: J Biol Chem. 2012 Apr 6;287(15):12425-32
references.pmid: 22334651
references.citation: J Biol Chem. 2002 Nov 29;277(48):46347-54
references.pmid: 12244055
references.citation: Proc Natl Acad Sci U S A. 2007 Jan 2;104(1):105-10
references.pmid: 17185422
references.citation: Proc Natl Acad Sci U S A. 2002 Oct 29;99(22):14171-6
references.pmid: 12381795
references.citation: Nature. 2008 Oct 30;455(7217):1255-8
references.pmid: 18923394
references.citation: J Biol Chem. 1999 Apr 2;274(14):9482-8
references.pmid: 10092631
references.citation: Nat Struct Mol Biol. 2011 Feb;18(2):222-6
references.pmid: 21258324
references.citation: Biophys J. 2001 Oct;81(4):2344-56
references.pmid: 11566804
references.citation: Biochem Biophys Res Commun. 2004 Nov 12;324(2):729-35
references.pmid: 15474488
references.citation: Biochemistry. 1996 Dec 24;35(51):16443-8
references.pmid: 8987976
references.citation: Curr Opin Struct Biol. 2008 Feb;18(1):16-26
references.pmid: 18221865
references.citation: J Biol Chem. 2000 Oct 27;275(43):33663-8
references.pmid: 10924508
references.citation: Protein Sci. 2000 May;9(5):934-41
references.pmid: 10850803
references.citation: Proc Natl Acad Sci U S A. 2007 Dec 26;104(52):20799-804
references.pmid: 18093935
references.citation: Proc Natl Acad Sci U S A. 2004 Aug 24;101(34):12491-6
references.pmid: 15314214
references.citation: J Chem Theory Comput. 2013 Jan 8;9(1):119-25
references.pmid: 26589015
references.citation: J Biol Chem. 2000 Sep 15;275(37):28513-8
references.pmid: 10878020
references.citation: Proc Natl Acad Sci U S A. 2005 Feb 22;102(8):2754-9
references.pmid: 15699337
references.citation: J Biol Chem. 1999 Apr 16;274(16):11072-7
references.pmid: 10196190
references.citation: Proc Natl Acad Sci U S A. 2006 Aug 22;103(34):12724-8
references.pmid: 16908850
references.citation: Biochem Biophys Res Commun. 1996 Sep 13;226(2):378-84
references.pmid: 8806643
references.citation: J Comput Chem. 2004 Oct;25(13):1605-12
references.pmid: 15264254
references.citation: J Mol Biol. 2000 Mar 31;297(3):683-95
references.pmid: 10731421
references.citation: J Phys Chem B. 2007 Jun 28;111(25):7392-400
references.pmid: 17547447
references.citation: Nature. 1992 Jan 16;355(6357):270-3
references.pmid: 1731226
references.citation: J Mol Biol. 2001 Nov 9;313(5):1161-9
references.pmid: 11700071
references.citation: Biochemistry. 1990 Feb 13;29(6):1377-80
references.pmid: 2334702
references.citation: Nat Struct Biol. 1995 Jun;2(6):442-5
references.pmid: 7664104
references.citation: Biochemistry. 2006 Sep 12;45(36):10865-72
references.pmid: 16953572
references.citation: Biochemistry. 1993 Jan 19;32(2):500-8
references.pmid: 8422359
references.citation: Structure. 2011 Apr 13;19(4):566-76
references.pmid: 21481779
references.citation: Biochem Biophys Res Commun. 2001 Sep 28;287(3):636-41
references.pmid: 11563842
references.citation: J Biomol NMR. 2006 Dec;36(4):215-24
references.pmid: 17061025
references.citation: Protein Sci. 2000 Jul;9(7):1274-81
references.pmid: 10933492
references.citation: J Mol Biol. 2002 Sep 27;322(4):841-9
references.pmid: 12270718
references.citation: J Chem Phys. 2005 Jan 1;122(1):14707
references.pmid: 15638691
references.citation: Nature. 1992 Jun 18;357(6379):605-7
references.pmid: 1608473
references.citation: J Biol Chem. 2003 Sep 26;278(39):37881-7
references.pmid: 12860985
references.citation: Nat Struct Mol Biol. 2005 Jan;12(1):46-53
references.pmid: 15580276
references.citation: Proc Natl Acad Sci U S A. 2003 Mar 18;100(6):3197-202
references.pmid: 12612345
references.citation: Nucleic Acids Res. 2013 Jul;41(Web Server issue):W427-31
references.pmid: 23658222
references.citation: Protein Sci. 2008 Oct;17(10):1844-9
references.pmid: 18725454
references.citation: Biophys J. 2008 Jun;94(12):4819-27
references.pmid: 18326651
references.citation: Biochemistry. 2006 May 30;45(21):6561-9
references.pmid: 16716066
references.citation: J Biol Chem. 1999 Apr 2;274(14):9548-55
references.pmid: 10092640
references.citation: PLoS One. 2013;8(1):e54766
references.pmid: 23382962
references.citation: Proc Natl Acad Sci U S A. 2007 Feb 20;104(8):2649-54
references.pmid: 17299036
references.citation: J Biol Chem. 1994 Apr 1;269(13):9627-31
references.pmid: 8144550
references.citation: J Mol Biol. 2012 Nov 9;423(5):789-99
references.pmid: 22940366
references.citation: Proc Natl Acad Sci U S A. 2012 Oct 2;109(40):16155-60
references.pmid: 22984159
references.citation: Biochem Soc Trans. 2007 Dec;35(Pt 6):1564-8
references.pmid: 18031267
references.citation: J Mol Biol. 2006 Mar 17;357(1):313-24
references.pmid: 16426636
references.citation: Proc Natl Acad Sci U S A. 2000 Jun 20;97(13):7220-5
references.pmid: 10860987
references.citation: Nature. 2002 Oct 17;419(6908):743-7
references.pmid: 12384704
references.citation: Biochemistry. 2002 Apr 9;41(14):4575-81
references.pmid: 11926819
references.citation: J Biol Chem. 2008 Jun 20;283(25):17568-78
references.pmid: 18390904
references.citation: Proc Natl Acad Sci U S A. 2006 Aug 1;103(31):11539-43
references.pmid: 16857738
references.citation: Structure. 1999 Feb 15;7(2):111-8
references.pmid: 10368279
references.citation: Biochemistry. 1995 Feb 7;34(5):1702-13
references.pmid: 7849030
references.citation: Proc Natl Acad Sci U S A. 2012 Mar 20;109(12):4467-72
references.pmid: 22392975
references.citation: Biophys J. 2002 Jul;83(1):458-72
references.pmid: 12080133
references.citation: J Mol Biol. 2008 Feb 15;376(2):597-605
references.pmid: 18164723
references.citation: Nat Struct Biol. 2003 Sep;10(9):738-43
references.pmid: 12923571
references.citation: Biochemistry. 2000 Jul 25;39(29):8460-9
references.pmid: 10913251
references.citation: EMBO Rep. 2011 Sep 30;12(10):1011-7
references.pmid: 21909074
references.citation: Nat Commun. 2011 Oct 11;2:493
references.pmid: 21988909
references.citation: J Biol Chem. 2006 Feb 10;281(6):3452-7
references.pmid: 16321984
references.citation: J Biol Chem. 2009 Jun 12;284(24):16099-16107
references.pmid: 19336401
references.citation: Nat Struct Biol. 2001 Nov;8(11):979-83
references.pmid: 11685246
references.citation: Nature. 2000 Oct 19;407(6806):923-6
references.pmid: 11057674
references.citation: J Biol Chem. 1988 Nov 15;263(32):16626-30
references.pmid: 3263371
references.citation: J Biol Chem. 2000 Jun 2;275(22):16904-9
references.pmid: 10747976
references.citation: Methods Enzymol. 1972;26:43-50
references.pmid: 4680720
references.citation: EMBO Rep. 2007 Jul;8(7):658-63
references.pmid: 17557112
references.citation: Biopolymers. 1974 Jan;13(1):29-61
references.pmid: 4818131
references.citation: Proc Natl Acad Sci U S A. 2007 Jan 30;104(5):1528-33
references.pmid: 17251351
references.citation: Genome Res. 2000 Dec;10(12):1845-64
references.pmid: 11116082
references.citation: J Mol Biol. 1992 Mar 5;224(1):241-52
references.pmid: 1548702
references.citation: Mol Cell Proteomics. 2009 May;8(5):1072-81
references.pmid: 19136720
references.citation: Phys Chem Chem Phys. 2009 Jan 7;11(1):83-93
references.pmid: 19081910