Internal movement in myosin subfragment 1 detected by fluorescence resonance energy transfer.
We have determined intersite distances from Cys374 of actin to Cys707 (SH1) and Cys697 (SH2) of myosin subfragment 1 (S1) in actosubfragment 1 (A.S1) by fluorescence resonance energy transfer for rigor complex A.S1 and complexes containing bound ADP and ADP plus orthovanadate (Vi), A.S1.ADP, and A.S1.ADP.Vi. A single energy acceptor (4-dimethylaminophenylazophenyl-4'-maleimide, DABMI) was attached to Cys374, and two different energy donors [(5-(iodoacetamideothyl)aminonaphthalene-1-sulfonic acid (IAEDANS) and 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS)] were each attached to SH1 and SH2 for the distance determination. The two sites SH1 and SH2 of S1 were approximately equidistant (ca. 45 A) from actin Cys374 in rigor A.S1 when MIANS was the energy donor attached to the two thiols. The Cys374-SH1 distance decreased by 7-8 A in the presence of ADP plus Vi, but the distance Cys374-SH2 was essentially unaltered under identical conditions. Slightly different but similar distance results were obtained with AEDANS as energy donor. If the structure of actin monomer in A.S1 is assumed to be rigid [Miki, M. (1991) Biochemistry 30, 10878-10884], the present results indicate that MgADP plus Vi induced a movement of SH1 toward the actin site and that SH2 was insensitive to saturation of the active site pocket of S1 and relatively immobile. These results suggest that during the steady-state hydrolysis of ATP or in the weak-binding state of actomyosin, the short helical segment of S1 heavy chain containing SH1 moves closer to the COOH-terminal end of actin, while the adjacent helical segment containing SH2 remains stationary. The emission spectrum of MIANS attached to SH2 experienced a large red spectral shift (6-10 nm) in the presence of MgADP, MgADP + Vi, MgADP + beryllium fluoride, and ATP. A crude model of S1 based on the C alpha coordinates suggests that SH2 is located in a hydrophobic cage surrounded by three hydrophobic residues. Reorientation of one of these side chains could expose SH2 to the solvent. The observed red spectral shift of MIANS attached to SH2 could be explained by such a nucleotide-induced exposure, and this explanation would be consistent with the interpretation that SH2 is stationary.
Xing
J
J
Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham 35294-2041, USA.
Cheung
H C
HC
D000199:Actins / Q000737:chemistry; D000205:Actomyosin / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D003545:Cysteine / Q000737:chemistry; D004735:Energy Transfer; D005454:Fluorescence Polarization; D066298:In Vitro Techniques; D007700:Kinetics; D009038:Motion; D015879:Myosin Subfragments / Q000737:chemistry* / Q000378:metabolism; D009218:Myosins / Q000737:chemistry* / Q000378:metabolism; D011817:Rabbits; D013050:Spectrometry, Fluorescence
D000199:Actins; D015879:Myosin Subfragments; D000255:Adenosine Triphosphate; D000205:Actomyosin; D009218:Myosins; D003545:Cysteine