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

Sharabi O, Peleg Y, Mashiach E, Vardy E, Ashani Y, Silman I, Sussman JL, Shifman JM - Protein Eng. Des. Sel. (2009)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel.

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.

Show MeSH

Related in: MedlinePlus

(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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2742391&req=5

GZP045F4: (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.

Mentions: cThese values were calculated by fitting the kinetic data presented in Fig. 4A to Eq. (3).


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

Sharabi O, Peleg Y, Mashiach E, Vardy E, Ashani Y, Silman I, Sussman JL, Shifman JM - Protein Eng. Des. Sel. (2009)

(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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2742391&req=5

GZP045F4: (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.
Mentions: cThese values were calculated by fitting the kinetic data presented in Fig. 4A to Eq. (3).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel.

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.

Show MeSH
Related in: MedlinePlus