The dynamics of the relay loop tryptophan residue in the Dictyostelium myosin motor domain and the origin of spectroscopic signals

Steady-state and time-resolved fluorescence measurements were performed on a Dictyostelium discoideum myosin II motor domain construct retaining a sin gle tryptophan residue at position 501, located on the relay loop. Other tr yptophan residues were mutated to phe nylalanine, The Trp-501 residue showe d a large enhancement in fluorescence in the presence of ATP and a small qu ench in the presence of ADP as a result of perturbing both the ground and e xcited state processes. Fluorescence lifetime and quantum yield measurement s indicated that at least three microstates of Trp-801 were present in all nucleotide states examined, and these could not be assigned to a particular gross conformation of the motor domain. Enhancement in emission intensity was associated with a reduction of the contribution from a statically quenc hed component and an increase in a component with a 5-ns lifetime, with lit tle change in the contribution from a 1-ns lifetime component, Anisotropy m easurements indicated that the Trp-501 side chain was relatively immobile i n all nucleotide states, and the fluorescence was effectively depolarized b y rotation of the whole motor domain with a correlation time on 50-70 ns. O verall these data suggest that the backbone of the relay loop remains struc tured throughout the myosin ATPase cycle but that the Trp-801 side chain ex periences a different weighting in local environments provided by surroundi ng residues as the adjacent converter domain rolls around the relay loop.