Structural kinetics of myosin by transient time-resolved FRET.
For many proteins, especially for molecular motors and other enzymes, the functional mechanisms remain unsolved due to a gap between static structural data and kinetics. We have filled this gap by detecting structure and kinetics simultaneously. This structural kinetics experiment is made possible by a new technique, (TR)(2)FRET (transient time-resolved FRET), which resolves protein structural states on the submillisecond timescale during the transient phase of a biochemical reaction. (TR)(2)FRET is accomplished with a fluorescence instrument that uses a pulsed laser and direct waveform recording to acquire an accurate subnanosecond time-resolved fluorescence decay every 0.1 ms after stopped flow. To apply this method to myosin, we labeled the force-generating region site specifically with two probes, mixed rapidly with ATP to initiate the recovery stroke, and measured the interprobe distance by (TR)(2)FRET with high resolution in both space and time. We found that the relay helix bends during the recovery stroke, most of which occurs before ATP is hydrolyzed, and two structural states (relay helix straight and bent) are resolved in each nucleotide-bound biochemical state. Thus the structural transition of the force-generating region of myosin is only loosely coupled to the ATPase reaction, with conformational selection driving the motor mechanism.
Department of Biochemistry, University of Minnesota Medical School, Minneapolis, MN 55455, USA. yuri.nesmelov@uncc.edu
Yuri E
YE
Nesmelov
Roman V
RV
Agafonov
Igor V
IV
Negrashov
Sarah E
SE
Blakely
Margaret A
MA
Titus
David D
DD
Thomas
D000255:Adenosine Triphosphate / Q000737:chemistry; D004023:Dictyostelium / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D007700:Kinetics; D009218:Myosins / Q000737:chemistry*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
D000255:Adenosine Triphosphate; D009218:Myosins
J Biol Chem. 2008 Mar 28;283(13):8153-63
18211892
Rev Sci Instrum. 2010 Oct;81(10):103101
21034069
Biochemistry. 1982 Mar 16;21(6):1284-94
7074085
Biophys J. 2007 Jan 1;92(1):172-84
17028139
Cell. 2000 Sep 1;102(5):683-94
11007486
Biochemistry. 2000 Dec 26;39(51):16135-46
11123942
Biochemistry. 1978 Aug 22;17(17):3432-42
150856
J Biol Chem. 2002 Aug 9;277(32):28459-67
11971905
Proc Natl Acad Sci U S A. 2009 Apr 14;106(15):6140-5
19325123
Biophys J. 1995 Oct;69(4):1491-507
8534820
J Biol Chem. 2009 Jun 12;284(24):16432-16441
19369252
Biophys J. 1996 Nov;71(5):2329-45
8913574
Annu Rev Biochem. 1999;68:687-728
10872464
Biophys J. 1995 Apr;68(4 Suppl):135S-141S
7787056
Cell. 1999 May 14;97(4):459-70
10338210
Science. 2006 Sep 15;313(5793):1638-42
16973882
Nature. 2007 Dec 13;450(7172):964-72
18075575
Proc Natl Acad Sci U S A. 2008 Sep 2;105(35):12867-72
18725645
Biophys J. 2008 Jul;95(1):247-56
18339764
J Mol Biol. 2006 Mar 10;356(5):1107-17
16406406
Proc Natl Acad Sci U S A. 2008 Sep 9;105(36):13397-402
18765799
J Biol Chem. 2006 Mar 3;281(9):5711-7
16377637
Biochemistry. 1995 Jul 18;34(28):8960-72
7619795
Proc Natl Acad Sci U S A. 2009 Dec 22;106(51):21625-30
19966224
Anal Biochem. 1979 Nov 15;100(1):95-7
161695
Biochemistry. 1981 Mar 31;20(7):2004-12
6452904
Trends Biochem Sci. 2010 Dec;35(12):684-90
20801044
Proc Natl Acad Sci U S A. 2010 May 4;107(18):8207-12
20404208
Proc Natl Acad Sci U S A. 2008 Jun 24;105(25):8631-6
18552179
Nature. 1994 Mar 10;368(6467):113-9
8139653
Biochemistry. 2001 Oct 23;40(42):12727-37
11601998
Biophys J. 2008 Dec;95(11):5238-46
18805936
Annu Rev Biophys. 2009;38:347-69
19416073