FRET characterisation for cross-bridge dynamics in single-skinned rigor muscle fibres.
In this work we demonstrate for the first time the use of Förster resonance energy transfer (FRET) as an assay to monitor the dynamics of cross-bridge conformational changes directly in single muscle fibres. The advantage of FRET imaging is its ability to measure distances in the nanometre range, relevant for structural changes in actomyosin cross-bridges. To reach this goal we have used several FRET couples to investigate different locations in the actomyosin complex. We exchanged the native essential light chain of myosin with a recombinant essential light chain labelled with various thiol-reactive chromophores. The second fluorophore of the FRET couple was introduced by three approaches: labelling actin, labelling SH1 cysteine and binding an adenosine triphosphate (ATP) analogue. We characterise FRET in rigor cross-bridges: in this condition muscle fibres are well described by a single FRET population model which allows us to evaluate the true FRET efficiency for a single couple and the consequent donor-acceptor distance. The results obtained are in good agreement with the distances expected from crystallographic data. The FRET characterisation presented herein is essential before moving onto dynamic measurements, as the FRET efficiency differences to be detected in an active muscle fibre are on the order of 10-15% of the FRET efficiencies evaluated here. This means that, to obtain reliable results to monitor the dynamics of cross-bridge conformational changes, we had to fully characterise the system in a steady-state condition, demonstrating firstly the possibility to detect FRET and secondly the viability of the present approach to distinguish small FRET variations.
Caorsi
Valentina
V
Newton International Fellow, Royal Society, London, UK. v.caorsi@imperial.ac.uk
Ushakov
Dmtry S
DS
West
Timothy G
TG
Setta-Kaffetzi
Niovi
N
Ferenczi
Michael A
MA
D000205:Actomyosin / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D001696:Biomechanical Phenomena; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D056004:Molecular Dynamics Simulation; D009119:Muscle Contraction; D018485:Muscle Fibers, Skeletal / Q000737:chemistry* / Q000378:metabolism; D009126:Muscle Relaxation; D009127:Muscle Rigidity / Q000378:metabolism / Q000473:pathology*; D011487:Protein Conformation; D012867:Skin / Q000378:metabolism / Q000473:pathology*
D000255:Adenosine Triphosphate; D000205:Actomyosin
Biophys J. 1979 May;26(2):161-93
262414
Biochemistry. 2004 Nov 16;43(45):14463-71
15533051
J Mol Biol. 1993 Dec 5;234(3):826-36
8254675
Structure. 2005 Jan;13(1):131-41
15642268
Biophys J. 2008 Sep 15;95(6):2880-94
18539638
Biochemistry. 1983 Nov 8;22(23):5261-70
6140026
Biophys J. 1994 Dec;67(6):2436-47
7696482
Biochemistry. 2001 Apr 3;40(13):3796-803
11300759
J Cell Biol. 1983 Dec;97(6):1663-7
6417144
Adv Biochem Eng Biotechnol. 2005;95:245-65
16080271
Biophys J. 1997 Aug;73(2):895-904
9251806
Biophys J. 2003 Jun;84(6):3992-4010
12770904
Science. 1993 Jul 2;261(5117):50-8
8316857
Biophys J. 2007 Sep 15;93(6):2091-101
17496049
J Mol Biol. 1992 Jan 5;223(1):185-203
1530978
J Physiol. 1984 May;350:497-518
6611404
Proc Natl Acad Sci U S A. 1997 Aug 5;94(16):8533-8
9238011
Annu Rev Physiol. 1987;49:691-709
3032079
Biophys J. 1995 Sep;69(3):1000-10
8519955
Biophys J. 2001 Sep;81(3):1562-9
11509369
Eur J Biochem. 2000 Jul;267(14):4334-8
10880955
Structure. 2007 May;15(5):553-64
17502101
Adv Protein Chem. 2005;71:161-93
16230112
Biophys J. 2005 Oct;89(4):2736-49
16040744
Methods. 2001 Jul;24(3):289-96
11403577
Proc Natl Acad Sci U S A. 1984 Apr;81(7):2060-4
6585786
Science. 1969 Jun 20;164(3886):1356-65
4181952
J Biotechnol. 2002 Jan;82(3):177-9
11999688
Microsc Res Tech. 2006 Mar;69(3):186-95
16538624
Biophys J. 1998 May;74(5):2702-13
9591694
PLoS One. 2009 Nov 25;4(11):e8031
19946626
Biophys J. 2010 Oct 6;99(7):2163-9
20923650
Microsc Res Tech. 2004 Jun 1;64(2):185-95
15352090
Science. 1993 Jul 2;261(5117):58-65
8316858
J Struct Biol. 2004 Sep;147(3):268-82
15450296
Curr Opin Struct Biol. 1997 Apr;7(2):247-57
9094325
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
Biophys J. 1996 Dec;71(6):3330-43
8968602
Nature. 1998 Nov 26;396(6709):383-7
9845077
J Biol Chem. 2000 Jan 28;275(4):2404-9
10644692
Annu Rev Biochem. 1978;47:819-46
354506
Curr Opin Biotechnol. 2005 Feb;16(1):19-27
15722011
Biophys J. 1980 Dec;32(3):891-906
6266539
J Mol Biol. 1984 Sep 5;178(1):105-11
6481807
J Microsc. 2007 Nov;228(Pt 2):139-52
17970914
Trends Cell Biol. 2001 May;11(5):203-11
11316609
Cell. 1998 Sep 4;94(5):559-71
9741621