CONSERVED MOTIFS AS THE BASIS FOR RECOGNITION OF HOMOLOGOUS PROTEINS ACROSS SPECIES BOUNDARIES USING PEPTIDE-MASS FINGERPRINTING

Two-dimensional gel electrophoresis of any biological system presently resolves a plethora of highly purified proteins for which no function or identity has been determined. Theoretical and experimental data we re used to demonstrate that peptide-mass fingerprinting (PMF) could ai d in the recognition of conserved motifs across species boundaries, an d thereby assist in attributing putative function to some of these mol ecules. Amino acids residue substitutions produced by biological diver sity and phylogenetic distance combine to highlight regions of functio nal significance within proteins. Using 10 prokaryotic and two eukaryo tic elongation factors (EF), up to 25 peptide fragments (>800 Da) per molecule were compared across species boundaries within a 12 x 12 cont ingency table (66 cross-species comparisons), based upon the degree of molecular mass and amino acid sequence identity. Total amino acid seq uence identity ranged from 29.4-80.9% for these molecules. Peptide fra gments with homologous sequence across three or more EF were defined a s containing, or being near to, conserved functional motifs. Twelve su ch fragments (>800 Da) were found in this group of proteins. In additi on, an 808.9 Da peptide of unknown functional significance was seen to occur in three of the 12 molecules studied and in another three EF-Tu molecules. At the 83% (five of six residues) identity level, this fra gment was found in a further 35 EF-Tu molecules and in 14 unrelated pr oteins. Further investigation should reveal a role for this fragment ( motif) in structural integrity or protein function. A FASTA search con ducted on a peptide fragment containing a conserved GTP-binding motif (GHVDHGK) of EF-Tu from Euglena gracilis was used as an example to put atively attribute partial function to three hypothetical proteins deri ved from DNA sequencing initiatives. (C) 1997 by John Wiley & Sons, Lt d.