The fluorescence properties of Dictyostelium discoideum (Dd) myosin II constructs containing a single tryptophan residue have revealed detailed information regarding nucleotide binding and hydrolysis steps. Here we extend these studies to investigate the influence of actin on nucleotide-induced fluorescence transients. The fluorescence from native actin tryptophan residues is not significantly perturbed on binding to myosin, although an apparent signal is detected as a consequence of a light scatter artifact. Actin has a minor effect on the response of W129, located at the entrance to the nucleotide-binding pocket, and reduces the forward rate constants for the isomerization(s) associated with binding of ATP, ATPgammaS, and ADP by 3-fold or less. The isomerization detected by W129 clearly precedes the dissociation of actin in the case of ADP and ATPgammaS binding. The fluorescence from the conserved W501 residue, located at the distal end of the relay helix, is very sensitive to the switch 2 and/or lever arm disposition. Consequently, the observed fluorescence emission intensity can be used to estimate the equilibrium constant between the pre- and post-power stroke conformations. Actin modulates this equilibrium by no more than 2-fold in the presence of nucleoside triphosphate. These data have implications for the mechanism of product release and suggest that actin activates another process in the mechanism, such as switch 1 movement and Pi release, rather than influencing the switch 2 equilibrium and lever arm position directly.
Trypsin-like serine proteases play essential roles in diverse physiological processes such as hemostasis, apoptosis, signal transduction, reproduction, immune response, matrix remodeling, development, and differentiation. All of these proteases share an intriguing activation mechanism that involves the transition of an unfolded domain (activation domain) of the zymogen to a folded one in the active enzyme. During this conformational change, activation domain segments move around highly conserved glycine hinges. In the present study, hinge glycines were replaced by alanine residues via site directed mutagenesis. The effects of these mutations on the interconversion of the zymogen-like and active conformations as well as on catalytic activity were studied. Mutant trypsins showed zymogen-like structures to varying extents characterized by increased flexibility of some activation domain segments, a more accessible N-terminus and a deformed substrate binding site. Our results suggest that the trypsinogen to trypsin transition is hindered by the mutations, which results in a shift of the equilibrium between the inactive zymogen-like and active enzyme conformations toward the inactive state. Our data also showed, however, that the inactive conformations of the various mutants differ from each other. Binding of substrate analogues shifted the conformational equilibrium toward the active enzyme since inhibited forms of the trypsin mutants showed similar structural features as the wild-type enzyme. The catalytic activity of the mutants correlated with the proper conformation of the active site, which could be supported by varying conformations of the N-terminus and the autolysis loop. Transient kinetic measurements confirmed the existence of an inactive to active conformational transition occurring prior to substrate binding.
Sodium channel inhibitor drugs can exert their effect by either blocking, or modulating the channel. The extent of modulation versus channel block is crucial regarding the therapeutic potential of drug candidates. Modulation can be selective for pathological hyperactivity, while channel block affects vital physiological function as much as pathological activity. Previous results indicated that riluzole, a drug with neuroprotective and antiepileptic effects, may have a unique mechanism of action, where modulation is predominant, and channel block is negligible. We studied the effects of riluzole on rNaV1.4 channels expressed in HEK cells. We observed that inhibition by riluzole disappeared and reappeared at a rate that could not be explained by association/dissociation dynamics. In order to verify the mechanism of non-blocking modulation, we synchronized photolabeling with the voltage clamp protocol of patch-clamp experiments. Using this method, we could bind a photoreactive riluzole analog covalently to specific conformations of the channel. Photolabeling was ineffective at resting conformation, but effective at inactivated conformation, as judged from persisting modulated gating after removal of unbound photoactive drug from the solution. Mutation of the key residue of the local anesthetic binding site (F1579A) did not fully prevent ligand binding and inhibition, however, it eliminated most of the modulation caused by ligand binding. Our results indicate that riluzole binds with highest affinity to the local anesthetic binding site, which transmits inhibition by the unique non-blocking modulation mechanism. Our results also suggest the existence of one or more additional binding sites, with lower affinity, and different inhibition mechanism.
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