title: Design, expression and characterization of mutants of fasciculin optimized for interaction with its target, acetylcholinesterase.
abstract: 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.
authors: Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel.
authors: Oz
authors: O
authors: Sharabi
authors: Yoav
authors: Y
authors: Peleg
authors: Efrat
authors: E
authors: Mashiach
authors: Eyal
authors: E
authors: Vardy
authors: Yacov
authors: Y
authors: Ashani
authors: Israel
authors: I
authors: Silman
authors: Joel L
authors: JL
authors: Sussman
authors: Julia M
authors: JM
authors: Shifman
mesh_terms: D000110:Acetylcholinesterase / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D004546:Elapid Venoms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods*; D054730:Protein Interaction Domains and Motifs / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: D004546:Elapid Venoms; D011994:Recombinant Proteins; C044937:fasciculin; D000110:Acetylcholinesterase