title: Nonuniform chain collapse during early stages of staphylococcal nuclease folding detected by fluorescence resonance energy transfer and ultrarapid mixing methods.
abstract: The development of tertiary structure during folding of staphylococcal nuclease (SNase) was studied by time-resolved fluorescence resonance energy transfer measured using continuous- and stopped-flow techniques. Variants of this two-domain protein containing intradomain and interdomain fluorescence donor/acceptor pairs (Trp and Cys-linked fluorophore or quencher) were prepared to probe the intradomain and interdomain structural evolution accompanying SNase folding. The intra-domain donor/acceptor pairs are within the β-barrel domain (Trp27/Cys64 and Trp27/Cys97) and the interdomain pair is between the α-helical domain and the β-barrel domain (Trp140/Cys64). Time-resolved energy transfer efficiency accompanying folding and unfolding at different urea concentrations was measured over a time range from 30 μs to ≈ 10 s. Information on average donor/acceptor distances at different stages of the folding process was obtained by using a quantitative kinetic modeling approach. The average distance for the donor/acceptor pairs in the β-barrel domain decreases to nearly native values whereas that of the interdomain donor/acceptor pairs remains unchanged in the earliest intermediate (<500 μs of refolding). This indicates a rapid nonuniform collapse resulting in an ensemble of heterogeneous conformations in which the central region of the β-barrel domain is well developed while the C-terminal α-helical domain remains disordered. The distance between Trp140 and Cys64 decreases to native values on the 100-ms time scale, indicating that the α-helical domain docks onto the preformed β-barrel at a late stage of the folding. In addition, the unfolded state is found to be more compact under native conditions, suggesting that changes in solvent conditions may induce a nonspecific hydrophobic collapse.
authors.affiliation: Graduate School of Science, Nagoya University, Nagoya, Aichi 464-8602, Japan.
authors.forename: Takuya
authors.initials: T
authors.lastname: Mizukami
authors.forename: Ming
authors.initials: M
authors.lastname: Xu
authors.forename: Hong
authors.initials: H
authors.lastname: Cheng
authors.forename: Heinrich
authors.initials: H
authors.lastname: Roder
authors.forename: Kosuke
authors.initials: K
authors.lastname: Maki
mesh_terms: D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D004735:Energy Transfer; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D008836:Micrococcal Nuclease / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D020125:Mutation, Missense; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics; D014508:Urea / Q000737:chemistry
chemical_list: D014364:Tryptophan; D014508:Urea; D008836:Micrococcal Nuclease; D003545:Cysteine
keywords: Förster energy transfer; continuous-flow; fluorescence; protein folding; stopped-flow
references.citation: Curr Opin Struct Biol. 2006 Feb;16(1):86-93
references.pmid: 16442277
references.citation: J Mol Biol. 2004 Jan 30;335(5):1309-23
references.pmid: 14729346
references.citation: Biochemistry. 1991 Mar 12;30(10):2698-706
references.pmid: 2001357
references.citation: Proc Natl Acad Sci U S A. 1992 Jan 15;89(2):748-52
references.pmid: 1731350
references.citation: Proteins. 1986 Sep;1(1):34-42
references.pmid: 2835760
references.citation: Chem Rev. 2006 May;106(5):1836-61
references.pmid: 16683757
references.citation: J Mol Biol. 2011 Jul 8;410(2):329-42
references.pmid: 21554889
references.citation: Biochemistry. 1997 May 27;36(21):6529-38
references.pmid: 9174370
references.citation: Anal Biochem. 1979 Mar;93(2):442-4
references.pmid: 464271
references.citation: Science. 1970 Feb 6;167(3919):886-7
references.pmid: 5410853
references.citation: Proc Natl Acad Sci U S A. 2002 Jul 23;99(15):9807-12
references.pmid: 12096190
references.citation: Proc Natl Acad Sci U S A. 2002 Feb 5;99(3):1329-34
references.pmid: 11773620
references.citation: Biophys J. 2010 Feb 17;98(4):678-86
references.pmid: 20159164
references.citation: J Mol Biol. 2000 Jun 23;299(5):1363-71
references.pmid: 10873459
references.citation: J Mol Biol. 2004 Apr 23;338(2):383-400
references.pmid: 15066439
references.citation: Proc Natl Acad Sci U S A. 1994 Jan 18;91(2):449-53
references.pmid: 8290547
references.citation: Biophys J. 1998 May;74(5):2714-21
references.pmid: 9591695
references.citation: J Mol Biol. 2005 Oct 28;353(3):704-18
references.pmid: 16188274
references.citation: Nature. 1988 Oct 20;335(6192):694-9
references.pmid: 2845278
references.citation: Nat Struct Biol. 1998 May;5(5):385-92
references.pmid: 9587001
references.citation: Anal Biochem. 1979 Apr 1;94(1):75-81
references.pmid: 37780
references.citation: Protein Sci. 1996 Sep;5(9):1907-16
references.pmid: 8880915
references.citation: Biochemistry. 2001 Jan 9;40(1):105-18
references.pmid: 11141061
references.citation: J Mol Biol. 2003 Mar 7;326(5):1615-33
references.pmid: 12595269
references.citation: Adv Protein Chem. 2000;53:209-82
references.pmid: 10751946
references.citation: Nature. 1988 Oct 20;335(6192):700-4
references.pmid: 2845279
references.citation: Biochemistry. 1994 Aug 30;33(34):10457-62
references.pmid: 8068683
references.citation: J Mol Biol. 2007 Apr 20;368(1):244-55
references.pmid: 17331534
references.citation: Trends Biochem Sci. 1999 Feb;24(2):77-83
references.pmid: 10098403
references.citation: Protein Sci. 2002 Jan;11(1):82-91
references.pmid: 11742125
references.citation: J Mol Biol. 2000 Mar 31;297(3):781-9
references.pmid: 10731428
references.citation: J Mol Biol. 2009 Jan 30;385(4):1230-42
references.pmid: 19013178
references.citation: Biochemistry. 1997 May 13;36(19):5795-805
references.pmid: 9153420
references.citation: Biochemistry. 1997 Sep 16;36(37):11273-81
references.pmid: 9287170
references.citation: Science. 1993 Nov 5;262(5135):896-900
references.pmid: 8235611
references.citation: Biochemistry. 1992 Oct 27;31(42):10217-25
references.pmid: 1420143
references.citation: J Mol Biol. 2006 Jul 28;360(5):1067-80
references.pmid: 16815441
references.citation: Proc Natl Acad Sci U S A. 2004 Feb 3;101(5):1171-6
references.pmid: 14711991
references.citation: Biochemistry. 1999 Feb 16;38(7):2213-23
references.pmid: 10026306
references.citation: Biochemistry. 1973 Oct 9;12(21):4154-61
references.pmid: 4745664