Relative binding free energy calculations of inhibitors to two mutants (Glu46-- Ala/Gln) of ribonuclease T1 using molecular dynamics/free energy perturbation approaches |
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Authors: | Hirono Shuichi; Kollman Peter A |
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Affiliation: | Department of Pharmaceutical Chemistry, University of California San Francisco, CA 941430446, USA |
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Abstract: | We present free energy perturbation calculations on the complexesof Glu46 Ala46 (E46A) and Glu46 Gln46 (E46Q) mutantsof ribonuclease T1 (RNaseT1) with inhibitors 2-guanosinemonophosphate (GMP) and 2adenosine monophosphate (AMP)by a thermodynamic perturbation method implemented with moleculardynamics (MD). Using the available crystal structure of theRNaseT1GMP complex, the structures of E46A-GMP and E46Q-GMPwere model built and equilibrated with MD simulations. The structuresof E46A-AMP and E46Q-AMP were obtained as a final structureof the GMPAMP perturbation calculation respectively.The calculated difference in the free energy of binding (![{Delta}](http://peds.oxfordjournals.org/math/Delta.gif) Gbind)was 0.31 kcal/mol for the E46A system and 1.04 kcal/molfor the E46Q system. The resultant free energies are much smallerthan the experimental and calculated value of 3 kcal/mol forthe native RNase T1, which suggests that both mutants have greaterrelative adenine affinities than native RNaseT1. EspeciallyE46Q is calculated to have a larger affinity for adenine thanguanine, as we suggested previously from the calculation onthe native RNaseT1. Thus, the molecular dynamics/free energyperturbation method may be helpful in protein engineering, directedtoward increasing or changing the substrate specificity of enzymes. |
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Keywords: | adenine specificity/ dissociation constant/ guanine specificity/ electrostatic interaction/ site-directed mutagenesis |
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