CONSEQUENCES OF REACTING THE THIOLS OF MYOSIN SUBFRAGMENT-1

When subfragment 1 (S-1) of myosin is exposed (120 h at 0 °C) to a series of increasing concentrations of methyl methanethiosulfonate (MMTS) up to 25ËŸ [S-1], the accessible thiols of S-1 are progressively reacted to form -SSCH3. If “SHf”, the more readily reactive thiol of S-1, is first labeled with the fluorescent dye 1,5-IAEDANS [N-(iodoacetyl)-N-(5-sulfo-1-naphthyl)ethylenediamine], seven additional thiols can be reacted with MMTS. In 8 M urea 12 thiols of S-1 are titrated with PCMB [p-(chloromercuri)-benzoate]. Thus, four thiols are considered to be “inaccessible” to MMTS under the exposure conditions used. S-1 ATPase activities (Ca2+, Mg2+, and actin activated) decline as more MMTS is bound but do not fall to zero even at [MMTS]/ [S-1] ratios up to 100:1. With fluorescent-labeled S-1, the affinity of S-1 for F-actin is measured by following the time-resolved fluorescence anisotropy decay. As more MMTS is bound to S-1, the affinity constant decreases from about 10 μ M-1 to about 2 μ M-1 (25 °C; 0.284 M KCl, 1.35 mM MgCl2, and 0.45 mM EGTA, buffered at pH 7.4). In the presence of ATP there is no measurable affinity. Since reacting S-1 thiols with a small blocking group such as MMTS does not abolish S-1 ATPase activity or actin binding, it is concluded that thiols are not directly involved in these functions. The effects of reacting S-1 thiols with MMTS may be attributed to conformational changes in S-1. To test this hypothesis, we applied certain probes of structural changes at different stages of blockage with MMTS. The fluorescence of 8-anilino-1-naphthalenesulfonate equilibrated with the system increased with increased blockage but not hand in hand with the ATPase activity. However, signals (emanating from 1,5-IAEDANS placed at thiol SH1) from two other probes correlated very well with loss of ATPase activity and with loss of actin affinity, and their behavior can be taken to mean that certain structural changes are progressively caused by progressive blockage. The rotational correlation time of the S-1 particle increases, suggesting a progressive global deformation. The magnitude of the excited-state lifetime decreases upon ATP addition, suggesting a progressive accessibility of solvent to the region immediately near to SH1. © 1979, American Chemical Society. All rights reserved.