Hierarchical folding mechanism of apomyoglobin revealed by ultra-fast H/D exchange coupled with 2D NMR.
The earliest steps in the folding of proteins are complete on an extremely rapid time scale that is difficult to access experimentally. We have used rapid-mixing quench-flow methods to extend the time resolution of folding studies on apomyoglobin and elucidate the structural and dynamic features of members of the ensemble of intermediate states that are populated on a submillisecond time scale during this process. The picture that emerges is of a continuum of rapidly interconverting states. Even after only 0.4 ms of refolding time a compact state is formed that contains major parts of the A, G, and H helices, which are sufficiently well folded to protect amides from exchange. The B, C, and E helix regions fold more slowly and fluctuate rapidly between open and closed states as they search docking sites on this core; the secondary structure in these regions becomes stabilized as the refolding time is increased from 0.4 to 6 ms. No further stabilization occurs in the A, G, H core at 6 ms of folding time. These studies begin to time-resolve a progression of compact states between the fully unfolded and native folded states and confirm the presence an ensemble of intermediates that interconvert in a hierarchical sequence as the protein searches conformational space on its folding trajectory.
Uzawa
Takanori
T
Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan.
Nishimura
Chiaki
C
Akiyama
Shuji
S
Ishimori
Koichiro
K
Takahashi
Satoshi
S
Dyson
H Jane
HJ
Wright
Peter E
PE
D000577:Amides / Q000737:chemistry; D000818:Animals; D001059:Apoproteins / Q000737:chemistry* / Q000145:classification / Q000378:metabolism*; D041961:Deuterium Exchange Measurement / Q000379:methods*; D008297:Male; D008958:Models, Molecular; D009211:Myoglobin / Q000737:chemistry* / Q000145:classification / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013997:Time Factors; D014907:Whales
D000577:Amides; D001059:Apoproteins; D009211:Myoglobin; C010857:apomyoglobin
J Mol Biol. 1998 Feb 20;276(2):491-504
9512718
Biophys J. 2007 Jul 1;93(1):218-24
17416618
Proc Natl Acad Sci U S A. 2008 Apr 29;105(17):6278-83
18434548
J Mol Biol. 2003 Nov 21;334(2):293-307
14607120
Proc Natl Acad Sci U S A. 2005 Mar 29;102(13):4765-70
15769860
Biochemistry. 2000 Mar 21;39(11):2894-901
10715109
Proteins. 1993 Sep;17(1):87-92
8234247
C R Trav Lab Carlsberg. 1964;34:299-317
14214225
J Mol Biol. 1986 Nov 5;192(1):133-54
3820301
Biochemistry. 2001 Mar 27;40(12):3561-71
11297422
Science. 1993 Nov 5;262(5135):892-6
8235610
J Mol Biol. 1996 Nov 8;263(4):531-8
8918936
Biochemistry. 2005 May 10;44(18):7013-23
15865446
J Mol Biol. 2002 Sep 20;322(3):483-9
12225742
J Mol Biol. 1999 Jan 8;285(1):269-82
9878405
Biochemistry. 1993 Jun 29;32(25):6337-47
8518279
Biochemistry. 1993 Jun 29;32(25):6356-64
8518281
Methods. 2004 Sep;34(1):51-64
15283915
Science. 1990 Sep 28;249(4976):1544-8
2218495
J Mol Graph. 1996 Feb;14(1):51-5, 29-32
8744573
J Mol Biol. 2007 Apr 20;368(1):219-29
17331539
J Mol Biol. 2006 Jan 6;355(1):139-56
16300787
Adv Protein Chem. 1966;21:287-386
5333290
J Mol Biol. 2002 Sep 27;322(4):655-62
12270702
J Mol Biol. 2006 Mar 31;357(3):997-1008
16460755
Proteins. 1993 Sep;17(1):75-86
8234246
Annu Rev Biophys Biomol Struct. 2000;29:213-38
10940248
Nat Struct Biol. 1997 Jan;4(1):44-50
8989323
Nat Struct Biol. 2000 Jun;7(6):514-20
10881201
Chem Rev. 2006 May;106(5):1757-68
16683753
J Mol Biol. 2007 Oct 19;373(2):491-502
17850819
Protein Sci. 1999 Jan;8(1):45-9
10210182
J Biomol NMR. 1995 Nov;6(3):271-6
8520219
Proc Natl Acad Sci U S A. 2004 Feb 3;101(5):1171-6
14711991
Nat Struct Biol. 1998 Feb;5(2):148-55
9461081
Protein Sci. 1999 Jul;8(7):1484-91
10422837
Protein Sci. 1997 Mar;6(3):706-16
9070453