Nucleotide-induced and actin-induced structural changes in SH1-SH2-modified myosin subfragment 1.
We compared the structural properties of myosin subfragment 1 (S1) modified at both reactive SH-groups, SH1 (Cys707) and SH2 (Cys697), with the properties of unmodified S1 and SH1-modified S1. It is shown using differential scanning calorimetry (DSC) that SH1 modification has no noticeable influence on the changes in S1 thermal unfolding induced by the formation of S1 ternary complexes with ADP and P(i) analogs (V(i), AlF(4)(-), and BeF(x)). These changes, however, normally expressed in a significant increase of S1 thermal stability, are almost fully prevented by modification of both SH1 and SH2. In contrast, SH2 modification had no effect on the changes induced by the formation of the ternary complexes S1-ADP-V(i), S1-ADP-AlF(4)(-), and S1-ADP-BeF(x) in EPR spectra of S1 spin-labeled at SH1 group. Interaction of S1 with F-actin substantially increased the thermal stability of S1; a similar effect was observed by DSC with both SH1- and SH1-SH2-modified S1. Overall, our results demonstrate that modification of both reactive SH-groups on S1 has no influence on the actin-induced changes of S1 and on the local nucleotide-induced conformational changes in the SH1 group region, but strongly prevents the global nucleotide-induced structural changes in the entire S1 molecule. The results suggest that modification of SH1 and SH2 impairs the spread of nucleotide-induced conformational changes from the ATPase site throughout the structure of the entire S1 molecule, thus disturbing a coupling between the motor and regulatory domains in the myosin head.
Shakirova
Lubov
L
A. N. Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky prosp. 33, 119071, Moscow, Russia.
Mikhailova
Valeria
V
Siletskaya
Elena
E
Timofeev
Vladimir P
VP
Levitsky
Dmitrii I
DI
D000199:Actins / Q000378:metabolism*; D000205:Actomyosin / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism*; D017607:Aluminum Compounds / Q000378:metabolism; D000818:Animals; D001608:Beryllium / Q000378:metabolism; D002152:Calorimetry, Differential Scanning; D004578:Electron Spin Resonance Spectroscopy; D005459:Fluorides / Q000378:metabolism; D015879:Myosin Subfragments / Q000737:chemistry / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011817:Rabbits; D014638:Vanadates / Q000378:metabolism
D000199:Actins; D017607:Aluminum Compounds; D015879:Myosin Subfragments; D014638:Vanadates; C035982:beryllium fluoride; D000244:Adenosine Diphosphate; D000205:Actomyosin; D009218:Myosins; D001608:Beryllium; D005459:Fluorides; C032311:aluminum fluoride