Early protein evolution: building domains from ligand-binding polypeptide segments.
It has been suggested that in the early evolution of proteins, segments of polypeptide, unable to fold in isolation, may have collapsed together to form folded proto-domains. We wondered whether the incorporation of segments with a pre-existing binding activity into a folded domain could, by fixing the ligand binding conformation and/or providing additional contacts, lead to large affinity improvements and provide an evolutionary advantage. As a model, we took a segment of polypeptide from hen egg lysozyme that in the native protein forms the binding interface with the monoclonal antibodies HyHEL5 and F10 (KD=60 pM). When expressed in bacteria the isolated segment was unfolded, readily proteolysed and only bound weakly to the antibodies (KD>1 microM). We then combined the segment with random genomic segments to create a repertoire of chimaeric polypeptides displayed on filamentous bacteriophage. By use of proteolysis (to select folded polypeptide) and anti-lysozyme antibodies (to select an active conformation) we isolated a folded dimeric protein with an enhanced antibody affinity (KD=400 pM). Unexpectedly the dimer also incorporated a single heme molecule (KD=33 nM) that stabilised the dimer (Tm=59 degrees C with heme, 35 degrees C without heme). These results show that the binding affinities of flexible polypeptide segments can be greatly enhanced on protein folding, and that the folding can be stabilised by prosthetic groups. This supports the hypothesis that sub-domain polypeptide segments with functional activities may have contributed to domain creation in early evolution.
Riechmann
Lutz
L
Division of Protein and Nucleic Acid Chemistry, MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK. lutz@mrc-lmb.cam.ac.uk
Winter
Greg
G
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D002645:Chickens; D019143:Evolution, Molecular; D006418:Heme / Q000378:metabolism; D008024:Ligands; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D000911:Antibodies, Monoclonal; D008024:Ligands; D010455:Peptides; D011993:Recombinant Fusion Proteins; D006418:Heme; D009113:Muramidase