Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2009 Oct;22(10):641-8.
doi: 10.1093/protein/gzp045. Epub 2009 Jul 30.

Design, expression and characterization of mutants of fasciculin optimized for interaction with its target, acetylcholinesterase

Oz Sharabi  1 , Yoav Peleg, Efrat Mashiach, Eyal Vardy, Yacov Ashani, Israel Silman, Joel L Sussman, Julia M Shifman
Affiliations

Design, expression and characterization of mutants of fasciculin optimized for interaction with its target, acetylcholinesterase

Oz Sharabi et al. Protein Eng Des Sel. 2009 Oct.

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.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
(A) The amino acid sequence of Fas, showing the four intra-chain disulphide bridges and the interfacial residues chosen for redesign. (B) The Fas/TcAChE binding interface before redesign; (C) The Fas/TcAChE binding interface after redesign. Fas (pink) sits at the entrance of the narrow gorge leading to the active site of TcAChE (gray). The side-chains on Fas and TcAChE selected for computational optimization are displayed as pink and blue sticks, respectively. The numbers are shown for the Fas residues for which affinity-enhancing mutations had been predicted.
Fig. 2
Fig. 2
Comparison of recombinant wild-type Fas expressed in E.coli to native Fas purified from mamba venom. Activity profiles of TcAChE when inhibited by recombinant wild-type Fas (closed triangle) and by native Fas (closed circle). [Fas] are plotted on a log scale. TcAChE activity is measured with an accuracy of ±0.05. An insert shows an SDS–PAGE gel for equal amounts of recombinant Fas (lane 1) and native Fas (lane 2). Molecular weight markers are shown in the leftmost lane.
Fig. 3
Fig. 3
(A) Residual TcAChE activity in the presence of Fas variants (viz., T9N, K32R, Fasdes, R11K, T8V/T9N, FasWT, FasdesR32K, T8V and H29R) is displayed as a function of their concentrations. TcAChE was maintained at a concentration of 0.04 nM in all experiments. A wide range of Fas concentrations were explored in order to encompass the entire activity profile for each mutant. The curves were fitted to a 1:1 binding model to determine the Kd values for interaction of TcAChE with the Fas mutants [See Eq. (2)]. Each experiment was repeated two to four times, and the average Kd and the standard deviation were calculated; (B) ΔΔGbind values for interaction of the Fas mutants with TcAChE were calculated according to ΔΔGbind=0.59 kcal/mol ln [Kd(Fasmutant)/Kd(FasWT)]. The error bars were calculated from the standard deviations in the Kd values determined by repeating the experiments shown in (A).
Fig. 4
Fig. 4
(A) TcAChE activity in the presence of Fas mutants under conditions in which interaction between the two proteins approaches equilibrium. For clarity, the data for only three mutants are shown: K32R (closed square), Fasdes (closed circle) and H29R (closed diamond). TcAChE activity decreases with time until a plateau is reached. Slightly different concentrations were used for each Fas mutant so as to obtain optimal results. TcAChE at 0.025 nM was incubated with 0.75 nM Fasdes and 1.25 nM K32R; TcAChE at 0.006 nM was incubated with 0.15 nM H29R. The data were fitted to Eq. (3). (B) and (C) summarize the rates of association with TcAChE (B) and dissociation from the enzyme (C) of the various Fas mutants. Error bars represent the standard deviation obtained by repeating the experiments shown in (A). The experiment for K32R was performed only once.
Fig. 5
Fig. 5
Correlation between the experimentally observed change in free energy of binding, ΔΔGbind, and the change in the calculated energies for the Fas–TcAChE complex. (A) Correlation with the total energy change, ΔEtotal. (B) Correlation with the inter-molecular energy change, ΔEinter. (Closed circle) Fas mutants tested for binding to TcAChE in this study.(Closed inverted triangle) Fas mutants previously tested for binding to mAChE. We excluded Fas mutants with amino acid deletions or with mutations outside the Fas–mAChE interface, as well as mutants for which an exact value of Kd had not been reported. The lines represent the linear fitting of all the data points except for that for Fasdes.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Bolon D.N., Grant R.A., Baker T.A., Sauer R.T. Proc. Natl Acad. Sci. USA. 2005;102:12724–12729. - PMC - PubMed
    1. Bourne Y., Taylor P., Marchot P. Cell. 1995;83:503–512. - PubMed
    1. Cervenansky C., Engstrom A., Karlsson E. Biochim. Biophys. Acta. 1994;1199:1–5. - PubMed
    1. Cervenansky C., Engstrom A., Karlsson E. Eur. J. Biochem. 1995;229:270–275. - PubMed
    1. Chevalier B.S., Kortemme T., Chadsey M.S., Baker D., Monnat R.J., Stoddard B.L. Mol. Cell. 2002;10:895–905. - PubMed

Publication types

-