title: Protein modification from mutational analysis of an autologous peptide fragment.
abstract: 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 dispensable 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 pre-existing 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.(ABSTRACT TRUNCATED AT 250 WORDS)
authors: CSIRO Division of Biomolecular Engineering, Laboratory for Molecular Biology, North Ryde, NSW, Australia.
authors: I S
authors: IS
authors: Dunn
authors: P A
authors: PA
authors: Jennings
mesh_terms: D000595:Amino Acid Sequence; D004795:Enzyme Stability; D008969:Molecular Sequence Data; D016296:Mutagenesis; D015202:Protein Engineering; D001616:beta-Galactosidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D001616:beta-Galactosidase