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

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

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

GZP045F2: 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.

Mentions: Native, snake-derived fasciculin-II was purchased from Alomone Laboratories (Jerusalem, Israel). TcAChE was purified from electric organ tissue of T.californica (Sussman et al., 1988), and AChE activity essays were performed as previously described (Ellman et al., 1961). TcAChE at 0.04 nM concentration was pre-incubated for 20 min either alone or together with a Fas variant at the desired concentration in 50 mM phosphate, pH 8.0, containing 0.1 mg/ml BSA and 0.01% NaN3. The same assay mixture without the enzyme was used as a control to monitor non-specific substrate hydrolysis and subtracted from the sample readings. A range of concentrations were explored for each Fas mutant. The reaction was started by the addition of the substrate acetylthiocholine iodide (ATC) at 0.8 mM and 5,5′-Dithiobis-2-nitro-benzoic acid (DTNB) at 0.4 mM. The increase in absorption at 412 nm was monitored over 1 min at 10 s intervals, and the initial velocity of the reaction was calculated from the slope of the line thus obtained. The fraction of TcAChE activity for a particular concentration of a Fas variant was calculated by dividing the initial velocity of the reaction by the initial velocity of the reaction in the absence of Fas. The experiment was repeated for a range of Fas concentrations to obtain a full inhibitory profile (Fig. 2). Each curve was fitted to determine the Kd of binding. To fit the data, we assumed that Fas is a non-competitive inhibitor of TcAChE (Weiner et al., 2009) that forms a 1:1 complex with the enzyme, whose affinity is not affected by binding of substrate to the enzyme. In this assumption, the binding could be described by a reaction: and the Kd of binding is:(1)where [Fas], [AChE] and [Fas · AChE] are the concentrations of the unbound Fas, free TcAChE and the inactive complex, respectively, when the system had reached equilibrium. [Fas]tot and [AChE]tot are the total concentration of Fas and TcAChE, respectively. The fraction of the residual active enzyme measured in Fig. 2 then becomes:(2)


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)

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

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

GZP045F2: 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.
Mentions: Native, snake-derived fasciculin-II was purchased from Alomone Laboratories (Jerusalem, Israel). TcAChE was purified from electric organ tissue of T.californica (Sussman et al., 1988), and AChE activity essays were performed as previously described (Ellman et al., 1961). TcAChE at 0.04 nM concentration was pre-incubated for 20 min either alone or together with a Fas variant at the desired concentration in 50 mM phosphate, pH 8.0, containing 0.1 mg/ml BSA and 0.01% NaN3. The same assay mixture without the enzyme was used as a control to monitor non-specific substrate hydrolysis and subtracted from the sample readings. A range of concentrations were explored for each Fas mutant. The reaction was started by the addition of the substrate acetylthiocholine iodide (ATC) at 0.8 mM and 5,5′-Dithiobis-2-nitro-benzoic acid (DTNB) at 0.4 mM. The increase in absorption at 412 nm was monitored over 1 min at 10 s intervals, and the initial velocity of the reaction was calculated from the slope of the line thus obtained. The fraction of TcAChE activity for a particular concentration of a Fas variant was calculated by dividing the initial velocity of the reaction by the initial velocity of the reaction in the absence of Fas. The experiment was repeated for a range of Fas concentrations to obtain a full inhibitory profile (Fig. 2). Each curve was fitted to determine the Kd of binding. To fit the data, we assumed that Fas is a non-competitive inhibitor of TcAChE (Weiner et al., 2009) that forms a 1:1 complex with the enzyme, whose affinity is not affected by binding of substrate to the enzyme. In this assumption, the binding could be described by a reaction: and the Kd of binding is:(1)where [Fas], [AChE] and [Fas · AChE] are the concentrations of the unbound Fas, free TcAChE and the inactive complex, respectively, when the system had reached equilibrium. [Fas]tot and [AChE]tot are the total concentration of Fas and TcAChE, respectively. The fraction of the residual active enzyme measured in Fig. 2 then becomes:(2)

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