Nucleotide-induced states of myosin subfragment 1 cross-linked to actin.
Actomyosin interactions and the properties of weakly bound states in carbodiimide-cross-linked complexes of actin and myosin subfragment 1 (S-1) were probed in tryptic digestion, fluorescence, and thiol modification experiments. Limited proteolysis showed that the 50/20K junction on S-1 was protected in cross-linked acto-S-1 from trypsin even under high-salt conditions in the presence of MgADP, MgAMPPNP, and MgPPi (mu = 0.5 M). The same junction was exposed to trypsin by MgATP and MgATP gamma S but mainly on S-1 cross-linked via its 50K fragment to actin. p-Phenylenedimaleimide-bridged S-1, when cross-linked to actin, yielded similar tryptic cleavage patterns to those of cross-linked S-1 in the presence of MgATP. By using p-nitrophenylenemaleimide, it was found that the essential thiols of cross-linked S-1 were exposed to labeling in the presence of MgATP and MgATP gamma S in a state-specific manner. In contrast to this, the reactive thiols were protected from modification in the presence of MgADP, MgAMPPNP, and MgPPi at mu = 0.5 M. These modifications were compared with similar reactions on isolated S-1. Experiments with pyrene-actin cross-linked to S-1 showed enhancement of fluorescence intensity upon additions of MgATP and MgATP gamma S, indicating the release of the pyrene probe on actin from the sphere of S-1 influence. The results of this study contrast the "open" structure of weakly bound actomyosin states to the "tight" conformation of rigor complexes.
Duong
A M
AM
Department of Chemistry and Biochemistry, University of California, Los Angeles 90024.
Reisler
E
E
D000199:Actins; D000227:Adenine Nucleotides / Q000494:pharmacology*; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D003432:Cross-Linking Reagents; D066298:In Vitro Techniques; D015879:Myosin Subfragments; D009218:Myosins; D010446:Peptide Fragments; D011487:Protein Conformation / Q000187:drug effects; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014357:Trypsin
D000199:Actins; D000227:Adenine Nucleotides; D003432:Cross-Linking Reagents; D015879:Myosin Subfragments; D010446:Peptide Fragments; D000255:Adenosine Triphosphate; D014357:Trypsin; D009218:Myosins