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Evolutionary optimization of peptide substrates for proteases that exhibit rapid hydrolysis kinetics
Kevin T Boulware1, Abeer Jabaiah, Patrick S Daugherty
1Department of Chemical Engineering, University of California-Santa Barbara, 93106, USA.
Biotechnology and Bioengineering
|February 12, 2010
Summary
A new method called two-color cellular library of peptide substrates (CLiPS) optimizes protease substrate cleavage. This technique identified highly efficient tobacco etch virus (TEV) protease substrates, improving enzyme kinetics.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Protease cleavage site identification often yields substrates with suboptimal specificity and activity.
- Evolutionary optimization of substrate cleavage kinetics is crucial for understanding protease function.
Purpose of the Study:
- To develop and apply a novel methodology for evolutionary optimization of protease substrate cleavage kinetics.
- To identify and optimize peptide substrates for the tobacco etch virus (TEV) protease.
Main Methods:
- Development of the two-color cellular library of peptide substrates (CLiPS) methodology.
- Application of CLiPS to screen a random pentapeptide library for TEV protease substrates.
- Optimization of identified substrates using a focused, extended substrate library and quantitative screening.
Main Results:
- Identification of seven amino acid substrates with rapid hydrolysis by TEV protease.
- Discovered a strong consensus motif (EXLYPhiQG) for TEV protease substrates.
- Demonstrated co-evolution of the native TEV substrate with the protease for efficient hydrolysis.
- Identified related putative viral protease substrates using consensus motifs.
Conclusions:
- The CLiPS methodology enables evolutionary optimization of protease substrate selectivity and activity.
- Optimized substrates can be used to design improved protease activity probes, molecular imaging agents, and prodrugs.
- The study provides insights into the co-evolution of viral proteases and their substrates.
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