Fluorescence resonance energy transfer within the complex formed by actin and myosin subfragment 1. Comparison between weakly and strongly attached states.
Fluorescence resonance energy transfer measurements have been made between Cys-374 on actin and Cys-177 on the alkali light chain of myosin subfragment 1 (S1) using several pairs of donor-acceptor chromophores. The labeled light chain was exchanged into subfragment 1 and the resulting fluorescently labeled subfragment 1 isolated by ion-exchange chromatography on SP-Trisacryl. The efficiency of energy transfer was measured by steady-state fluorescence in a strong binding complex of acto-S1 and found to represent a spatial separation between the two probes of 5.6-6.3 nm. The same measurements were then made with weak binding acto-S1 complexes generated in two ways. First, actin was complexed with p-phenylenedimaleimide-S1, a stable analogue of S1-adenosine 5'-triphosphate (ATP), obtained by cross-linking the SH1 and SH2 heavy-chain thiols of subfragment 1 [Greene, L. E., Chalovich, J. M., & Eisenberg, E. (1986) Biochemistry 25, 704-709]. Large increases in transfer efficiency indicated that the two probes had moved closer together by some 3 nm. Second, weak binding complexes were formed between subfragment 1 and actin in the presence of the regulatory proteins troponin and tropomyosin, the absence of calcium, and the presence of ATP [Chalovich, J. M., & Eisenberg, E. (1982) J. Biol. Chem. 257, 2432-2437]. The measured efficiency of energy transfer again indicated that the distance between the two labeled sites had moved closer by about 3 nm. These data support the idea that there is a considerable difference in the structure of the acto-S1 complex between the weakly and strongly bound states.
Trayer
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Department of Biochemistry, University of Birmingham, Edgbaston, U.K.
Trayer
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D000199:Actins / Q000502:physiology*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D003545:Cysteine; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D015879:Myosin Subfragments; D009218:Myosins / Q000378:metabolism / Q000502:physiology*; D010446:Peptide Fragments / Q000032:analysis / Q000502:physiology*; D011485:Protein Binding; D011817:Rabbits; D013050:Spectrometry, Fluorescence
D000199:Actins; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D015879:Myosin Subfragments; D010446:Peptide Fragments; D000255:Adenosine Triphosphate; D009218:Myosins; D003545:Cysteine; D002118:Calcium