PROTEIN MODIFICATION FROM MUTATIONAL ANALYSIS OF AN AUTOLOGOUS PEPTIDE FRAGMENT

In the alpha-complementation of beta-galactosidase an N-terminal peptide fragment (alpha-peptide) of the wild-type enzyme interacts with a defective beta-galactosidase enzyme to restore capacity for subunit assembly and activity. We have used previously a random mutagenesis and screening approach to identify a pentapeptide residue tract in the alpha-peptide that was highly tolerant of residue substitution, with some mutations conferring improved function. This tract is of clear importance for alpha-peptide function but is apparently dispensible in the intact parental enzyme. To investigate this further, we selected tract mutations and placed them into intact beta-galactosidase, at the corresponding N-terminal position as in the alpha-peptide. We then tested whether such specific tract sequences conferred properties to the whole enzyme which could be predicted from the behaviour of the defective enzyme complemented with the corresponding mutant alpha-peptide. This was shown for mutations which positively or negatively affected enzyme stability. Additionally, a subset of mutations which affected complementation efficiency in vivo were predicted to affect the formation of higher-order structures in the intact protein, and this was observed experimentally. Mutations which decreased peptide complementation dramatically decreased the level of formation of multimers in the intact protein and a mutation which increased peptide complementation produced marked enhancement of multimer formation in a protein with a preexisting impairment in higher-order structure formation. Such subtle effects are difficult to detect directly in the whole protein by randomization/selection approaches, but in the complementing peptide the role of the residues within the pentapeptide tract is effectively amplified. Identification of residue tracts exhibiting functional tolerance to amino acid substitution in an active peptide fragment can thus be combined with transferral of potentially useful mutant peptide sequences back into the intact protein. Manipulation of a complementation system in this manner affords a sensitive approach towards targeted improvement of proteins.