In vitro evolution of a dimeric variant of human pancreatic ribonuclease.
Site-directed mutagenesis of human pancreatic RNase (HP-RNase) was used as a model system for investigating the genetic events underlying the evolutionary origins of protein oligomers. HP-RNase is a monomeric enzyme with no natural tendency to oligomerize (K(d) for any dimers in solution of >280 mM). Nevertheless, deletion of five amino acid residues in the loop linking the N-terminal helix of HP-RNase to the rest of the protein was found to drive polypeptide chains to fold into dimers. These dimers could not be dissociated by heating at 70 degrees C, and small amounts of monomer were detected only in highly diluted samples. Measurement of dimer and monomer concentrations under equilibrium conditions yielded a K(d) of 1.5 microM. This implies that the deletion increases the protein propensity to dimerize at least 5.2 orders of magnitude. Moreover, the HP-RNase dimers were found to be over 4.6 orders of magnitude more stable than the dimers of bovine pancreatic RNase A obtained by lyophilization from acetic acid (K(d) > 73 mM). Cross-linking experiments with divinyl sulfone indicated that the HP-RNase dimers are stabilized by the exchange between subunits of their N-terminal helices. This generates composite active sites, i.e., each contributed by two subunit chains, that retain full enzymatic activity. Overall, these results show that a deletion of few residues in a key region of a monomeric protein can be the primary event irreversibly leading to oligomerization of the protein through the swap of a secondary structure element between protomers.
Russo
N
N
Department of Life Sciences, Second University of Naples, Caserta, Italy.
Antignani
A
A
D'Alessio
G
G
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D019281:Dimerization; D004795:Enzyme Stability / Q000235:genetics; D014644:Genetic Variation; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000138:chemical synthesis; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000235:genetics*; D017384:Sequence Deletion; D013329:Structure-Activity Relationship
D010446:Peptide Fragments; D011994:Recombinant Proteins; D012259:Ribonuclease, Pancreatic