Location of a contact site between actin and myosin in the three-dimensional structure of the acto-S1 complex.
Using fluorescence resonance energy transfer (FRET), we measured distances from chromophores located at or near the actin-binding stretch of amino acids 633-642 of myosin subfragment 1 (S1), to five points in the acto-S1 complex. Specific labeling of this site was achieved by first attaching the desired chromophore to an "antipeptide" that by means of its charge complementarity specifically binds to this segment of S1 [Chaussepied & Morales (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7471] and then cross-linking the fluorescent peptide to the protein. According to this technique, antipeptides containing three different labels, viz., N-dansylaziridine, (iodoacetamido)fluorescein, and monobromobimane, were purified and covalently bound to S1. A second chromophoric group, required for FRET measurements, was selected in such a way as to provide a good spectral overlap with the corresponding peptide chromophore. Cys-707 (SH1) and Cys-697 (SH2) on S1 were modified by using iodoacetamido and maleimido derivatives of rhodamine, 1,N6-ethenoadenosine 5'-diphosphate was trapped at the S1 active site with orthovanadate, Cys-374 on actin was modified with either N-[4-[4-(dimethylamino)phenyl]azo]phenyl]maleimide or N-[(iodoacetyl)-amino]ethyl]-5-naphthylamine-1-sulfonate, and ADP bound to F-actin was exchanged with the fluorescent etheno analogue. By use of excited-state lifetime fluorometry, the following distances from the stretch 633-642 of S1 to other points on S1 or actin have been measured: Cys-707 (S1), 50.3 A; Cys-697 (S1), 49.4 A; active site of S1, greater than or equal to 44 A; nucleotide binding site (actin), 41.1 A; and Cys-374 (actin), approximately 53 A.(ABSTRACT TRUNCATED AT 250 WORDS)
Kasprzak
A A
AA
Cardiovascular Research Institute, University of California, San Francisco 94143-0524.
Chaussepied
P
P
Morales
M F
MF
D000199:Actins; D000251:Adenosine Triphosphatases; D000255:Adenosine Triphosphate; D000345:Affinity Labels; D055598:Chemical Phenomena; D002621:Chemistry; D004735:Energy Transfer; D005453:Fluorescence; D008274:Magnesium; D009218:Myosins; D010446:Peptide Fragments; D010449:Peptide Mapping; D011487:Protein Conformation
D000199:Actins; D000345:Affinity Labels; D010446:Peptide Fragments; C033025:actin subfragments; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D009218:Myosins; D008274:Magnesium