title: FRET characterisation for cross-bridge dynamics in single-skinned rigor muscle fibres.
abstract: 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.
authors.affiliation: Newton International Fellow, Royal Society, London, UK. v.caorsi@imperial.ac.uk
authors.forename: Valentina
authors.initials: V
authors.lastname: Caorsi
authors.forename: Dmtry S
authors.initials: DS
authors.lastname: Ushakov
authors.forename: Timothy G
authors.initials: TG
authors.lastname: West
authors.forename: Niovi
authors.initials: N
authors.lastname: Setta-Kaffetzi
authors.forename: Michael A
authors.initials: MA
authors.lastname: Ferenczi
mesh_terms: 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*
chemical_list: D000255:Adenosine Triphosphate; D000205:Actomyosin
references.citation: Biophys J. 1979 May;26(2):161-93
references.pmid: 262414
references.citation: Biochemistry. 2004 Nov 16;43(45):14463-71
references.pmid: 15533051
references.citation: J Mol Biol. 1993 Dec 5;234(3):826-36
references.pmid: 8254675
references.citation: Structure. 2005 Jan;13(1):131-41
references.pmid: 15642268
references.citation: Biophys J. 2008 Sep 15;95(6):2880-94
references.pmid: 18539638
references.citation: Biochemistry. 1983 Nov 8;22(23):5261-70
references.pmid: 6140026
references.citation: Biophys J. 1994 Dec;67(6):2436-47
references.pmid: 7696482
references.citation: Biochemistry. 2001 Apr 3;40(13):3796-803
references.pmid: 11300759
references.citation: J Cell Biol. 1983 Dec;97(6):1663-7
references.pmid: 6417144
references.citation: Adv Biochem Eng Biotechnol. 2005;95:245-65
references.pmid: 16080271
references.citation: Biophys J. 1997 Aug;73(2):895-904
references.pmid: 9251806
references.citation: Biophys J. 2003 Jun;84(6):3992-4010
references.pmid: 12770904
references.citation: Science. 1993 Jul 2;261(5117):50-8
references.pmid: 8316857
references.citation: Biophys J. 2007 Sep 15;93(6):2091-101
references.pmid: 17496049
references.citation: J Mol Biol. 1992 Jan 5;223(1):185-203
references.pmid: 1530978
references.citation: J Physiol. 1984 May;350:497-518
references.pmid: 6611404
references.citation: Proc Natl Acad Sci U S A. 1997 Aug 5;94(16):8533-8
references.pmid: 9238011
references.citation: Annu Rev Physiol. 1987;49:691-709
references.pmid: 3032079
references.citation: Biophys J. 1995 Sep;69(3):1000-10
references.pmid: 8519955
references.citation: Biophys J. 2001 Sep;81(3):1562-9
references.pmid: 11509369
references.citation: Eur J Biochem. 2000 Jul;267(14):4334-8
references.pmid: 10880955
references.citation: Structure. 2007 May;15(5):553-64
references.pmid: 17502101
references.citation: Adv Protein Chem. 2005;71:161-93
references.pmid: 16230112
references.citation: Biophys J. 2005 Oct;89(4):2736-49
references.pmid: 16040744
references.citation: Methods. 2001 Jul;24(3):289-96
references.pmid: 11403577
references.citation: Proc Natl Acad Sci U S A. 1984 Apr;81(7):2060-4
references.pmid: 6585786
references.citation: Science. 1969 Jun 20;164(3886):1356-65
references.pmid: 4181952
references.citation: J Biotechnol. 2002 Jan;82(3):177-9
references.pmid: 11999688
references.citation: Microsc Res Tech. 2006 Mar;69(3):186-95
references.pmid: 16538624
references.citation: Biophys J. 1998 May;74(5):2702-13
references.pmid: 9591694
references.citation: PLoS One. 2009 Nov 25;4(11):e8031
references.pmid: 19946626
references.citation: Biophys J. 2010 Oct 6;99(7):2163-9
references.pmid: 20923650
references.citation: Microsc Res Tech. 2004 Jun 1;64(2):185-95
references.pmid: 15352090
references.citation: Science. 1993 Jul 2;261(5117):58-65
references.pmid: 8316858
references.citation: J Struct Biol. 2004 Sep;147(3):268-82
references.pmid: 15450296
references.citation: Curr Opin Struct Biol. 1997 Apr;7(2):247-57
references.pmid: 9094325
references.citation: Proc Natl Acad Sci U S A. 2010 May 4;107(18):8207-12
references.pmid: 20404208
references.citation: Proc Natl Acad Sci U S A. 2008 Jun 24;105(25):8631-6
references.pmid: 18552179
references.citation: Biophys J. 1996 Dec;71(6):3330-43
references.pmid: 8968602
references.citation: Nature. 1998 Nov 26;396(6709):383-7
references.pmid: 9845077
references.citation: J Biol Chem. 2000 Jan 28;275(4):2404-9
references.pmid: 10644692
references.citation: Annu Rev Biochem. 1978;47:819-46
references.pmid: 354506
references.citation: Curr Opin Biotechnol. 2005 Feb;16(1):19-27
references.pmid: 15722011
references.citation: Biophys J. 1980 Dec;32(3):891-906
references.pmid: 6266539
references.citation: J Mol Biol. 1984 Sep 5;178(1):105-11
references.pmid: 6481807
references.citation: J Microsc. 2007 Nov;228(Pt 2):139-52
references.pmid: 17970914
references.citation: Trends Cell Biol. 2001 May;11(5):203-11
references.pmid: 11316609
references.citation: Cell. 1998 Sep 4;94(5):559-71
references.pmid: 9741621