The binding of mutant actins to profilin, ATP and DNase I.
Twenty-five mutations were created in the Drosophila melanogaster Act88F actin gene by in vitro mutagenesis and the mutant actins expressed in vitro. The affinity of the mutant actins for ATP, profilin and DNase I was determined. They were also tested for conformational changes by non-denaturing gel electrophoresis. Mutations at positions 364 (highly conserved) and 366 (invariant) caused changes in conformation, reduced ATP binding and increased profilin binding. At position 362 (invariant) only the conservative change from tyrosine to phenylalanine had no effect; other changes at this position affected conformation, ATP and profilin binding. Although only glycine or serine occur naturally at position 368, changes to threonine or glutamine had no effect on the actin. The mutant in which Asp363 was replaced by His and that in which Glu364 was replaced by Lys decreased DNase I binding, yet neither amino acid occurs in the DNase I binding site. Likewise several mutations affect ATP and profilin binding but are distant from the binding sites. We conclude that, although actin has a highly conserved amino acid sequence, individual amino acids can have variable tolerance for substitutions. Also amino acid changes can exert significant effects on the binding of ligands to distant parts of the actin structure.
Drummond
D R
DR
Department of Biology, University of York, England.
Hennessey
E S
ES
Sparrow
J C
JC
D000199:Actins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D003285:Contractile Proteins; D003850:Deoxyribonuclease I / Q000378:metabolism*; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008840:Microfilament Proteins / Q000378:metabolism*; D016296:Mutagenesis; D051304:Profilins; D011485:Protein Binding; D011487:Protein Conformation; D013329:Structure-Activity Relationship
D000199:Actins; D003285:Contractile Proteins; D029721:Drosophila Proteins; D008840:Microfilament Proteins; D051304:Profilins; C495737:chic protein, Drosophila; D000255:Adenosine Triphosphate; D003850:Deoxyribonuclease I