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 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.
Department of Biochemistry, University of Leicester, Leicester LE1 7RH, United Kingdom.
A
A
Malnasi-Csizmadia
M
M
Kovacs
R J
RJ
Woolley
S W
SW
Botchway
C R
CR
Bagshaw
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D016880:Anisotropy; D003001:Cloning, Molecular; D004023:Dictyostelium / Q000737:chemistry* / Q000502:physiology*; D007700:Kinetics; D008956:Models, Chemical; D009218:Myosins / Q000378:metabolism; D010649:Phenylalanine / Q000737:chemistry; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry*
D010649:Phenylalanine; D000244:Adenosine Diphosphate; D014364:Tryptophan; D000255:Adenosine Triphosphate; D009218:Myosins