Thermal stability as a probe of S-1 structure.

Abstract

The stabilizing effect of nucleotides against thermal denaturation of subfragment-1(S-1) was studied quantitatively. We showed that ATP- (ADP.P1) and AMPPNP-bound species are very stable, while ADP- and PPi-bound species are much less so (after ligand affinities are accounted for); these two stability classes correspond to the two CD and UD spectral classes. We further showed that the result of PPi- and ADP-binding is a weakening of the affinity between S-1 heavy chain and light chains, resulting in turbidity due to formation of aggregates of naked S-1 heavy chain. We also showed that while PPi- and ADP-binding decrease the inactivation rate of S-1 ATPase by protecting the 50-kDa fragment, they actually increase the denaturation rate of the remaining moieties (27 and 20 kDa); the rate of decrease of the 27- and 20-kDa tryptic fragments is much slower than that of the 50-kDa fragment with ligand-free S-1; however, the rate of decrease of these two fragments is fast and equal to that of the 50-kDa fragment with ADP- and PPi-bound S-1. Therefore, nucleotide binding to S-1 strongly affects the structure of the light chain binding site on the heavy chain, and it seems that the domain structure of S-1 is maintained by light chain binding.