Amino acid substitution analysis of E. coli thymidylate synthase: the study of a highly conserved region at the N-terminus.
Amino acid substitution analysis within a highly conserved region of Escherichia coli thymidylate synthase (TS), using suppression of amber mutations by tRNA suppressors, has yielded a bank of 124 new mutationally altered TS proteins. These mutant proteins have been used to study the structure-function relationship of the Escherichia coli TS protein at the N-terminus corresponding to residues 20 through 35. This region contains a block of amino acids whose sequence has been well conserved among other known TS proteins from various organisms. Positions 20 through 25 contain a surface loop structure and positions 26 through 35 encompass a beta-strand. We find that residues surrounding a beta-bulge structure within the beta-strand are particularly sensitive to amino acid substitution, suggesting that this structure is maintained by a highly ordered packing arrangement. Three residues in the surface loop that are present at the base of the substrate binding pocket are also sensitive to amino acid substitution. The remainder of the conserved sites, including those at the dimer interface, are tolerant to most, if not all, of the substitutions tested.
Kim
C W
CW
Molecular Biology Institute, University of California, Los Angeles 90024.
Michaels
M L
ML
Miller
J H
JH
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D001483:Base Sequence; D015153:Blotting, Western; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013940:Thymidylate Synthase / Q000737:chemistry / Q000235:genetics*
D000596:Amino Acids; D001426:Bacterial Proteins; D013940:Thymidylate Synthase