High affinity RNase S-peptide variants obtained by phage display have a novel "hot-spot" of binding energy.
Using phage display mutagenesis, high affinity variants of RNase S-peptide were produced that bind to RNase S-protein over 100-fold more tightly than the wild type S-peptide. The S-peptide: S-protein interface was further characterized using "biased" phage display libraries, where each targeted residue was constrained to be either polar or nonpolar. The use of these tailored libraries placed constraints on the type of interactions present during affinity maturation process and allowed more amino acids to be randomized simultaneously. These results, in conjunction with kinetic association and dissociation constants determined by surface plasmon resonance (SPR), highlight the role of a single mutation (A5W) in increasing S-peptide binding affinity. High affinity S-peptide variants were only identified when tryptophan was present in the phage display library at position 5, suggesting that this residue is a "hot-spot" of binding energy in the high affinity variants. Analysis of SPR data in the presence of denaturant suggests that the increased affinity is a result of increased hydrophobic interactions in the transition state rather than a stabilization of helical structure.
Dwyer
J J
JJ
Department of Biochemistry and Molecular Biology, Institute for Biophysical Dynamics, University of Chicago, 920 East 58th Street, Chicago, Illinois 60637, USA.
Dwyer
M A
MA
Kossiakoff
A A
AA
D001665:Binding Sites; D002942:Circular Dichroism; D016384:Consensus Sequence; D000939:Epitopes; D007700:Kinetics; D008958:Models, Molecular; D016296:Mutagenesis; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D019151:Peptide Library; D011897:Random Allocation; D012260:Ribonucleases / Q000235:genetics / Q000378:metabolism*; D013696:Temperature; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism*
D000939:Epitopes; D010446:Peptide Fragments; D019151:Peptide Library; C023560:ribonuclease S-peptide; D014364:Tryptophan; D012260:Ribonucleases