STRUCTURAL COMPARISON OF TWO BINDING PROTEINS FROM GRAM-NEGATIVE BACTERIA. APPLICATION OF NEW PROCEDURES FOR TRYPTOPHANYL PEPTIDE BOND CLEAVAGE AND REVERSED-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY OF LARGE DENATURED PEPTIDES
Abstract
The complete primary structure of the Escherichia coli B/rD-galactose-binding protein was determined by the automated sequencing of fragments produced by cleavage with cyanogen bromide, limited trypsin digestion, mild acid hydrolysis, digestion with Staphylococcus aureus strain V8 protease, and o-iodosobenzoic acid. The protein, which has 309 amino acids, is notable in the extent to which it differs from the L-arabinose-binding protein. Comparison of these two proteins indicates only about 18% homology despite the close structural resemblance of the molecules which they bind. Secondary structural predictions suggest that chain folding for the two proteins could be very similar. Comparison of the galactose-binding protein with the Escherichia coli K12 ribose-binding protein, of which approximately 70% of the amino acid sequence is known, again indicates a low level of sequence homology over most of the molecule with the exception of residues 207 to 253 of the galactose-receptor. Forty-five percent of the residues in this area are identical in the two proteins with an additional 30% of the residues related by a single nucleotide base change. This region is probably important for the protein-protein association between these receptors and the chemotaxis signal generating protein, probably methyl-accepting chemotaxis protein III, the trg-gene product. The development of a new and quantitative cleavage procedure for tryptophanyl residues was critical for these studies as was the development of a new procedure for the reversed-phase high-performance liquid chromatography of large denatured protein fragments on 300(DEGREES)A pore size reversed-phase supports in solvents containing trifluoroacetic acid.
Degree
Ph.D.
Subject Area
Biochemistry
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