Key result
Yeast actin R177H mutation reduces thermal stability and accelerates nucleotide exchange versus wild-type.
Why the study?
The biochemical consequences of the zebrafish cardiofunk actin mutation R177H were unknown and needed assessment in a model system.
The R177H mutation disrupts the structural integrity of the actin monomer and filament, providing biochemical insights into its role in abnormal zebrafish cardiogenesis.
Does not alter clinical management of actin-related heart defects; leaves open translation of yeast biochemical defects to human cardiomyopathy.
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.
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Wen et al. (2003) studied Zebrafish cardiofunk actin mutation (R177H). R177H mutation in yeast actin vs. Wild type (WT) actin was evaluated on Biochemical consequences including thermal stability, nucleotide exchange rate, and polymerization. The R177H mutation in yeast actin exhibited reduced thermal stability, accelerated nucleotide exchange, and an extended nucleation phase with faster elongation compared to wild-type actin.