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...

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Bibliographic Details
Published in:The Journal of biological chemistry 2001-06, Vol.276 (22), p.19483-19490
Main Authors: Malnasi-Csizmadia, A, Kovacs, M, Woolley, R J, Botchway, S W, Bagshaw, C R
Format: Article
Language:English
Subjects:
Adenosine Diphosphate - metabolism
Adenosine Triphosphate - metabolism
Animals
Anisotropy
Cloning, Molecular
Dictyostelium
Dictyostelium - chemistry
Dictyostelium - physiology
Kinetics
Models, Chemical
myosin II
Myosins - metabolism
Phenylalanine - chemistry
Protein Structure, Tertiary
Spectrometry, Fluorescence
Spectrophotometry
Time Factors
Tryptophan - chemistry
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Summary: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.
ISSN:0021-9258
DOI:10.1074/jbc.M010886200