Fluorescence resonance energy transfer within the regulatory light chain of myosin.
Rabbit skeletal muscle myosin regulatory light chain-2 (LC2) contains two reactive cysteine residues, Cys125 and Cys154, and one tryptophan at position 137. Using wild-type rabbit LC2 or its genetically engineered mutant with Cys125-->Arg (C125R), these residues can be selectively modified with fluorescent or chromophoric probes for spectroscopic studies. We have bound suitable donor/acceptor probe pairs to the two cysteine residues and Trp137 in LC2 or C125R, and measured the distance in solution between the probes by fluorescence resonance energy transfer spectroscopy. C125R was made to facilitate specific labelling of the less reactive Cys154, thus allowing the distance between Cys154 and Trp137 to be measured. Our measurements show that these residues are in close proximity to each other, the distance between them ranging from 1.7 nm (between Cys125 and Trp137) to 2.7 nm (Cys125 and Cys154). These results suggest that Cys125, Trp137 and Cys154, spanning up to 29 residues in the sequence of LC2, are spatially close, consistent with these residues residing within a C-terminal globular domain. The distances we obtained are in agreement with previous crosslinking studies [Huber, P. A., Brunner, U.T. & Schaub, M. C. (1989) Biochemistry 28, 9116-9123; Saraswat, L. & Lowey, S. (1991) J. Biol. Chem. 266, 19777-19785] and structure predictions of LC2. LC2 is located at the head-rod junction of the myosin crossbridge, and provides the primary regulatory mechanism in molluscan and smooth muscle. In skeletal muscle, its functional role is unclear, although it has been implicated in modulating actomyosin interaction [Metzger, J. M. & Moss, R. L. (1992) Biophys. J. 63, 460-468]. The incorporation of spectroscopic probes onto the light chains of myosin in solution or in fibres has become a valuable tool for evaluating the dynamic properties of the crossbridge during force generation.
Boey
W
W
Department of Anatomy and Histology, University of Sydney, Australia.
Huang
W
W
Bennetts
B
B
Sparrow
J
J
Dos Remedios
C
C
Hambly
B
B
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine; D003545:Cysteine; D017931:DNA Primers; D004735:Energy Transfer; D005456:Fluorescent Dyes; D009132:Muscles / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009218:Myosins / Q000737:chemistry* / Q000302:isolation & purification; D017354:Point Mutation; D011487:Protein Conformation; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence
D017931:DNA Primers; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D001120:Arginine; D009218:Myosins; D003545:Cysteine