An Arginine to Histidine Change at Position 177 of Cfk Alters the Location of Positive Charges in the Nucleotide Binding Cleft of Actin

<div><p>(A) The position of R177 (red) in the structure of the yeast-actin monomer. The bound ATP is shown in green. The structure shown is based on a crystal structure of actin (PDB:1YAG).</p> <p>(B) Magnification of the cleft region showing the hydrogen bonding (green dashed lines) involving R177, which will be disrupted in H177.</p> <p>(C) The R177H mutation restricts yeast growth under hyperosmolar stress. Wild-type (wt) and mutant cells were grown to a density of 3 × 10⁶ per milliliter, and aliquots were plated either on YPD or YPD plus 0.9 M NaCl at different dilutions of the culture. The cells were then incubated at 30°C for 72 h. The normosmolar and hyperosmolar experiments were done at different times.</p> <p>(D) Purified R177H actin has a higher critical concentration of polymerization and a delayed nucleation phase. Wild-type or mutant actin was purified from yeast cultures and polymerization mea-sured by light diffraction. Symbols and abbreviations: circles, 5 μM wild-type actin; triangles, 5 μM R177H actin; diamonds, 7.5 μM R177H actin; squares, 10 μM R177H actin; ATP, adenine triphosphate; wt, wild-type.</p>