The Trp-501 residue in the Dictyostelium myosin motor domain showed enhanced fluorescence with ATP and quenching with ADP, indicating the relay loop backbone remains structured during the ATPase cycle.
This biophysical study demonstrates that the relay loop backbone of the Dictyostelium myosin motor domain remains structured during the ATPase cycle, while the local environment of the Trp-501 side chain changes as the converter domain moves.
Steady-state and time-resolved fluorescence measurements were performed on a Dictyostelium discoideum myosin II motor domain construct retaining a single tryptophan residue at position 501, located on the relay loop. Other tryptophan residues were mutated to phenylalanine. The Trp-501 residue showed a large enhancement in fluorescence in the presence of ATP and a small quench in the presence of ADP as a result of perturbing both the ground and excited state processes. Fluorescence lifetime and quantum yield measurements indicated that at least three microstates of Trp-501 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 quenched component and an increase in a component with a 5-ns lifetime, with little change in the contribution from a 1-ns lifetime component. Anisotropy measurements indicated that the Trp-501 side chain was relatively immobile in all nucleotide states, and the fluorescence was effectively depolarized by rotation of the whole motor domain with a correlation time on 50-70 ns. Overall these data suggest that the backbone of the relay loop remains structured throughout the myosin ATPase cycle but that the Trp-501 side chain experiences a different weighting in local environments provided by surrounding residues as the adjacent converter domain rolls around the relay loop.
Málnási‐Csizmadia et al. (2001) studied this question. ATP and ADP was evaluated on Fluorescence lifetime and quantum yield. The Trp-501 residue in the Dictyostelium myosin motor domain showed enhanced fluorescence with ATP and quenching with ADP, indicating the relay loop backbone remains structured during the ATPase cycle.