Synthetic peptide model of an essential region of an aminoacyl-tRNA synthetase.
A 40 amino acid sequence of the unsolved structure of Escherichia coli alanine-tRNA synthetase is essential for tRNA binding and encodes an immunological determinant that cross-reacts with antibodies raised against a eukaryote (insect Bombyx mori) alanine enzyme. The secondary structure of this sequence is predicted to be an amphiphilic alpha-helix that includes one aspartyl and eight glutamyl side chain carboxyl groups. The antibody reactivity and the conformation of a synthetic peptide model of this region (Glu346 to Ser385) were investigated. In addition, double Arg----Gln and Leu----Ala substitutions were separately placed in the enzyme on the hydrophilic and hydrophobic face, respectively, of the predicted helix. These mutations conserve the polar/nonpolar character of each face and retain the potential for helix formation. Circular dichroism spectra of the synthetic peptide model demonstrate the potential for amphiphilic helix formation for the segment from Glu346 to Ser385. The behavior of the mutations in the enzyme, together with earlier data and immunological assays presented here, suggests that one face of the putative helix is an antigenic region of the surface of the enzyme where it contributes to the interaction with alanine tRNA and that the specific sequence of the helix is an important determinant of enzyme stability.
Park
S J
SJ
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Miller
W T
WT
Schimmel
P
P
D000412:Alanine-tRNA Ligase / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D001483:Base Sequence; D012831:Bombyx / Q000201:enzymology; D003429:Cross Reactions; D004795:Enzyme Stability; D007158:Immunologic Techniques; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D012344:RNA, Transfer, Ala / Q000737:chemistry; D012995:Solubility
D000906:Antibodies; D012344:RNA, Transfer, Ala; D000412:Alanine-tRNA Ligase