Is SH1-SH2-cross-linked myosin subfragment 1 a structural analog of the weakly-bound state of myosin?

Myosin subfragment 1 (S1) with SH1 (Cys(707)) and SH2 (Cys(697)) groups cro ss-linked by p-phenylenedimaleimide (pPDM-S1) is thought to be an analog of the weakly bound states of myosin bound to actin. The structural propertie s of pPDM-S1 were compared in this study to those of S1.ADP.BeFx and S1.ADP .AlF4-, i.e., the established structural analogs of the myosin weakly bound states. To distinguish between the conformational effects of SH1-SH2 cross -linking and those due to their monofunctional modification, we used S1 wit h the SH1 and SH2 groups labeled with N-phenylmaleimide (NPM-S1) as a contr ol in our experiments. The state of the nucleotide pocket was probed using a hydrophobic fluorescent dye, 3-[4-(3-phenyl-2-pyrazolin-1-yl)benzene-1-su lfonylamido]phenylboronic acid (PPBA). Differential scanning calorimetry (D SC) was used to study the thermal stability of S1. By both methods the conf ormational state of pPDM-S1 was different from that of unmodified S1 in the S1.ADP.BeFx and S1.ADPAlF(4)(-) complexes and closer to that of nucleotide -free S1. Moreover, BeFx and AlF4- binding failed to induce conformational changes in pPDM-S1 similar to those observed in unmodified S1. Surprisingly , when pPDM cross-linking was performed on S1 ADP BeF, complex, ADP BeF, pr otected to some extent the nucleotide pocket of S1 from the effects of pPDM modification. NPM-S1 behaved similarly to pPDM-S1 in our experiments. Over all, this work presents new evidence that the conformational state of pPDM- S1 is different from that of the weakly bound state analogs, S1.ADP.BeFx an d S1.ADP.AlFx. The similar structural effects of pPDM cross-linking of SH1 and SH2 groups and their monofunctional labeling with NPM are ascribed to t he inhibitory effects of these modifications on the flexibility/mobility of the SH1-SH2 helix.