Biochemical consequences of the cardiofunk (R177H) mutation in yeast actin

Kuo-Kuang Wen; Peter A Rubenstein

doi:10.1074/jbc.M308980200

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Biochemical consequences of the cardiofunk (R177H) mutation in yeast actin

Journal article

Open access

Peer reviewed

Biochemical consequences of the cardiofunk (R177H) mutation in yeast actin

Kuo-Kuang Wen and Peter A Rubenstein

The Journal of biological chemistry, Vol.278(48), pp.48386-48394

11/28/2003

DOI: 10.1074/jbc.M308980200

PMID: 13129918

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Abstract

The zebrafish cardiofunk actin mutation, R177H, causes abnormal heart development. We have introduced this mutation into yeast actin to assess its biochemical consequences. R177H G-actin exhibited reduced thermal stability and an accelerated nucleotide exchange rate. R177H actin has an increased critical concentration and polymerizes with a greatly extended nucleation phase but a faster elongation process, suggesting that significant fragmentation accompanies filament formation. Pi release from R177H actin is tightly coupled to polymerization, as with wild type (WT) actin, suggesting that the R177H mutation does not affect ATPase activity and Pi release. R177H actin shows no polymerization-dependent decrease in intrinsic Trp fluorescence, and the fluorescence yield of a pyrene at Cys374 is decreased. An equivalent amount of WT actin significantly but not completely rescues the mutant's polymerization defect. Tropomyosin greatly exacerbates the elongation of the nucleation phase of R177H actin but slightly decreases its critical concentration. It has only a slight effect on a 1:1 WT/mutant mixture. The defects we observed with R177H actin in vitro indicate that Arg177 is crucial for the control of the structural integrity of the actin monomer and the actin filament and provide insight into the defects caused by this mutation in zebrafish cardiogenesis.

Oligonucleotides - chemistry

Temperature

Mutagenesis, Site-Directed

Models, Chemical

Phosphates - chemistry

Microscopy, Electron

Actins - genetics

Pyrenes - chemistry

Hydrogen Bonding

Time Factors

Myocardium - metabolism

Actins - chemistry

Heterozygote

Protein Conformation

Kinetics

Mutation

Tropomyosin - chemistry

Tropomyosin - metabolism

Hydrogen-Ion Concentration

Yeasts - metabolism

Details

Title: Subtitle: Biochemical consequences of the cardiofunk (R177H) mutation in yeast actin
Creators: Kuo-Kuang Wen - Department of Biochemistry, University of Iowa Carver College of Medicine, Iowa City, Iowa 52242, USA
Peter A Rubenstein
Resource Type: Journal article
Publication Details: The Journal of biological chemistry, Vol.278(48), pp.48386-48394
DOI: 10.1074/jbc.M308980200
PMID: 13129918
NLM abbreviation: J Biol Chem
ISSN: 0021-9258
eISSN: 1083-351X
Publisher: United States
Grant note: GM33689 / NIGMS NIH HHS AI45728 / NIAID NIH HHS
Language: English
Date published: 11/28/2003
Academic Unit: Stead Family Department of Pediatrics; Biochemistry and Molecular Biology; Internal Medicine
Record Identifier: 9984024404302771

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