PEDS Advance Access originally published online on July 30, 2009
Protein Engineering Design and Selection 2009 22(10):641-648; doi:10.1093/protein/gzp045
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Design, expression and characterization of mutants of fasciculin optimized for interaction with its target, acetylcholinesterase
1Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel 2Israel Structural Proteomics Center 3Department of Structural Biology 4Department of Neurobiology, Weizmann Institute of Science, Rehovot 76100, Israel
7 To whom correspondence should be addressed. E-mail: jshifman{at}cc.huji.ac.il
Predicting mutations that enhance protein–protein affinity remains a challenging task, especially for high-affinity complexes. To test our capability to improve the affinity of such complexes, we studied interaction of acetylcholinesterase with the snake toxin, fasciculin. Using the program ORBIT, we redesigned fasciculin's sequence to enhance its interactions with Torpedo californica acetylcholinesterase. Mutations were predicted in 5 out of 13 interfacial residues on fasciculin, preserving most of the polar inter-molecular contacts seen in the wild-type toxin/enzyme complex. To experimentally characterize fasciculin mutants, we developed an efficient strategy to over-express the toxin in Escherichia coli, followed by refolding to the native conformation. Despite our predictions, a designed quintuple fasciculin mutant displayed reduced affinity for the enzyme. However, removal of a single mutation in the designed sequence produced a quadruple mutant with improved affinity. Moreover, one designed mutation produced 7-fold enhancement in affinity for acetylcholinesterase. This led us to reassess our criteria for enhancing affinity of the toxin for the enzyme. We observed that the change in the predicted inter-molecular energy, rather than in the total energy, correlates well with the change in the experimental free energy of binding, and hence may serve as a criterion for enhancement of affinity in protein–protein complexes.
Keywords: acetylcholinesterase/binding affinity/computational protein design/fasciculin/protein–protein interactions
Received June 26, 2009; revised June 26, 2009; accepted June 29, 2009.
5 Present address: Blavatnik School of Computer Science, Raymond and Beverly Sackler Faculty of Exact Sciences, Tel Aviv University, Israel
6 Present address: The Department of Biochemistry, Duke University Medical Center, Durham, NC 27710