Selection of High-Affinity Peptidic Serine Protease Inhibitors with Increased Binding Entropy from a Back-Flip Library of Peptide–Protease Fusions
We have developed a new concept for designing peptidic protein modulators, by recombinantly fusing the peptidic modulator, with randomized residues, directly to the target protein via a linker and screening for internal modulation of the activity of the protein. We tested the feasibility of the conc...
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Published in: | Journal of molecular biology 2015-09, Vol.427 (19), p.3110-3122 |
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Main Authors: | , , , , , , |
Format: | Article |
Language: | English |
Subjects: | |
Citations: | Items that this one cites Items that cite this one |
Online Access: | Get full text |
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Summary: | We have developed a new concept for designing peptidic protein modulators, by recombinantly fusing the peptidic modulator, with randomized residues, directly to the target protein via a linker and screening for internal modulation of the activity of the protein. We tested the feasibility of the concept by fusing a 10-residue-long, disulfide-bond-constrained inhibitory peptide, randomized in selected positions, to the catalytic domain of the serine protease murine urokinase-type plasminogen activator. High-affinity inhibitory peptide variants were identified as those that conferred to the fusion protease the lowest activity for substrate hydrolysis. The usefulness of the strategy was demonstrated by the selection of peptidic inhibitors of murine urokinase-type plasminogen activator with a low nanomolar affinity. The high affinity could not have been predicted by rational considerations, as the high affinity was associated with a loss of polar interactions and an increased binding entropy.
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•The back-flip strategy, as demonstrated here with a peptidic protease inhibitor, provides a practical approach to engineering peptidic protein modulators in general and to mapping peptide–protein interaction surfaces.•The usefulness of the back-flip strategy is demonstrated by a rationally unpredictable improvement of a peptidic protease inhibitor following the loss of a peptide–protein polar interaction and an entropy penalty.•The high affinity of the new peptidic inhibitors to their target enzyme appears to be caused by their flexibility, enabling them to adapt to variable enzyme surfaces. |
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ISSN: | 0022-2836 1089-8638 |
DOI: | 10.1016/j.jmb.2015.08.005 |