Actin and temperature effects on the cross-linking of the SH1-SH2 helix in myosin subfragment 1.
Past biochemical work on myosin subfragment 1 (S1) has shown that the bent alpha-helix containing the reactive thiols SH1 (Cys(707)) and SH2 (Cys(697)) changes upon nucleotide and actin binding. In this study, we investigated the conformational dynamics of the SH1-SH2 helix in two actin-bound states of myosin and examined the effect of temperature on this helix, using five cross-linking reagents that are 5-15 A in length. Actin inhibited the cross-linking of SH1 to SH2 on both S1 and S1.MgADP for all of the reagents. Because the rate of SH2 modification was not altered by actin, the inhibition of cross-linking must result from a strong stabilization of the SH1-SH2 helix in the actin-bound states of S1. The dynamics of the helix is also influenced by temperature. At 25 degrees C, the rate constants for cross-linking in S1 alone are low, with values of approximately 0.010 min(-1) for all of the reagents. At 4 degrees C, the rate constants, except for the shortest reagent, range between 0.030 and 0.070 min(-1). The rate constants for SH2 modification in SH1-modified S1 show the opposite trend; they increase with the increases in temperature. The greater cross-linking at the lower temperature indicates destabilization of the SH1-SH2 helix at 4 degrees C. These results are discussed in terms of conformational dynamics of the SH1-SH2 helix.
Nitao
L K
LK
Department of Chemistry and Biochemistry and the Molecular Biology Institute, University of California, Los Angeles 90095, USA.
Reisler
E
E
D000199:Actins / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D003432:Cross-Linking Reagents; D003545:Cysteine; D007700:Kinetics; D018482:Muscle, Skeletal / Q000378:metabolism; D015879:Myosin Subfragments / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011817:Rabbits; D013816:Thermodynamics
D000199:Actins; D003432:Cross-Linking Reagents; D015879:Myosin Subfragments; D000244:Adenosine Diphosphate; D003545:Cysteine