STUDY OF EFFECTS OF PH ON THE STABILITY OF DOMAINS IN MYOSIN ROD BY HIGH-RESOLUTION DIFFERENTIAL SCANNING CALORIMETRY

Differential scanning calorimetry (DSC) has detected at least six quasi-independent structure domains in myosin rod [Potekhin, S. A., & Privalov, P. L. (1978) Biofizika 23, 219–223], These domains were found to be remarkably sensitive to pH in the physiological range, i.e., pH 6–8. We compared the thermodynamic characteristics, and studied effects of pH on the stability, of individual domains in rod, light meromyosin (LMM), and subfragment 2 (S-2). In rod, the lowest stability domain (approximately 400 amino acid residues per double strand), with a Tm of 42.4 °C, a ΔHcal of 190 kcal/mol, and a AG of 3.39 kcal/mol, at pH 7.02, destabilized by absorption of protons, is located at the LMM/S-2 junction and split into two parts, one associated with S-2 (approximately 100 residues per double strand) and the other with LMM (300 residues per double strand). The fragment with S-2 is likely a part of the “hinge” suggested by Swenson and Ritchie [(1980) Biochemistry 19, 5371–5375]. All other domains of rod released protons on melting. The domains located in S-2 were the most sensitive to pH and released a total of 0.9 proton on melting. The thermal meltings of all domains in myosin rod, LMM, and S-2 were independent of each other, and enthalpies of melting were additive in the whole pH range studied. Their sensitivities to pH and KCl were also unaffected by the presence or absence of other fragments. For example, domains in an isolated S-2 behaved similarly as they were in the rod, and so were domains in LMM. The three fragments studied here also exhibited nearly the same specific heat of unfolding, i.e., 4.24 ± 0.30 cal/g, 4.18 ± 0.35 cal/g, and 4.01 ± 0.60 cal/mol for rod, LMM, and S-2, respectively. At least two of the six domains (Tm's of 44.6 and 46.2 °C at pH 7.02) in the rod showed less than full cooperativity in melting (ΔHvH/cal< 1) at pH below 6.8. Deconvolution of these rod endotherms into more than six domains was attempted, but unique solutions were not attainable. However, the endotherms of LMM and S-2, at all pHs, could be fitted, respectively, into five and three two-state-like transitions. Thermodynamic parameters associated with each of these transitions were extracted and their meaning discussed. We have also found that the two most stable domains of the rod changed their relative sizes with pH, suggesting migration of structure from one domain to the other upon protonation of certain histidine residue(s). © 1990, American Chemical Society. All rights reserved.