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Journal Article

Functional characterisation of Dictyostelium myosin II with conserved tryptophanyl residue 501 mutated to tyrosine

MPS-Authors
http://pubman.mpdl.mpg.de/cone/persons/resource/persons92120

Batra,  Renu
Emeritus Group Biophysics, Max Planck Institute for Medical Research, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons94215

Manstein,  Dietmar J.
Emeritus Group Biophysics, Max Planck Institute for Medical Research, Max Planck Society;

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Citation

Batra, R., & Manstein, D. J. (1999). Functional characterisation of Dictyostelium myosin II with conserved tryptophanyl residue 501 mutated to tyrosine. Biological Chemistry, 380(7), 1017-1023. doi:10.1515/BC.1999.126.


Cite as: http://hdl.handle.net/11858/00-001M-0000-0024-559A-6
Abstract
We created a Dictyostelium discoideum myosin II mutant in which the highly conserved residue Trp-501 was replaced by a tyrosine residue. The mutant myosin alone, when expressed in a Dictyostelium strain lacking the functional myosin II heavy chain gene, supported cytokinesis and multicellular development, processes which require a functional myosin in Dictyostelium. Additionally, we expressed the W501 Y mutant in the soluble myosin head fragment M761-2R (W501Y-2R) to characterise the kinetic properties of the mutant myosin motor domain. The affinity of the mutant myosin for actin was approximately 6-fold decreased, but other kinetic properties of the protein were changed less than 2-fold by the W501Y mutation. Based on spectroscopic studies and structural considerations, Trp-501, corresponding to Trp-510 in chicken fast skeletal muscle myosin, has been proposed to be the primary ATP-sensitive tryptophanyl residue. Our results confirm these conclusions. While the wild-type construct displayed a 10% fluorescence increase, addition of ATP to W501Y-2R was not followed by an increase in tryptophan fluorescence emission