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

被引:47
作者
Batra, R [1 ]
Manstein, DJ [1 ]
机构
[1] Max Planck Inst Med Res, D-69120 Heidelberg, Germany
关键词
fluorescence; molecular motor; myosin ATPase; protein engineering;
D O I
10.1515/BC.1999.126
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 Dictyosteliom 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 W501Y 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 e-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.
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页码:1017 / 1023
页数:7
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