Computational protein design requires methods to accurately estimate free energy changes in protein stability or binding upon an amino acid mutation. From the different approaches available, molecular dynamics-based alchemical free energy calculations are unique in their accuracy and solid theoretical basis. The challenge in using these methods lies in the need to generate hybrid structures and topologies representing two physical states of a system. A custom made hybrid topology may prove useful for a particular mutation of interest, however, a high throughput mutation analysis calls for a more general approach. In this work, we present an automated procedure to generate hybrid structures and topologies for the amino acid mutations in all commonly used force fields. The described software is compatible with the Gromacs simulation package. The mutation libraries are readily supported for five force fields, namely Amber99SB, Amber99SB*-ILDN, OPLS-AA/L, Charmm22*, and Charmm36.

Molecular dynamics simulations enable access to free energy differences governing the driving force underlying all biological processes. In the current chapter we describe alchemical methods allowing the calculation of relative free energy differences. We concentrate on the binding free energies that can be obtained using non-equilibrium approaches based on the Crooks Fluctuation Theorem. Together with the theoretical background, the chapter covers practical aspects of hybrid topology generation, simulation setup, and free energy estimation. An important aspect of the validation of a simulation setup is illustrated by means of calculating free energy differences along a full thermodynamic cycle. We provide a number of examples, including protein-ligand and protein-protein binding as well as ligand solvation free energy calculations.

The prediction of mutation‐induced free‐energy changes in protein thermostability or protein–protein binding is of particular interest in the fields of protein design, biotechnology, and bioengineering. Herein, we achieve remarkable accuracy in a scan of 762 mutations estimating changes in protein thermostability based on the first principles of statistical mechanics. The remaining error in the free‐energy estimates appears to be due to three sources in approximately equal parts, namely sampling, force‐field inaccuracies, and experimental uncertainty. We propose a consensus force‐field approach, which, together with an increased sampling time, leads to a free‐energy prediction accuracy that matches those reached in experiments. This versatile approach enables accurate free‐energy estimates for diverse proteins, including the prediction of changes in the melting temperature of the membrane protein neurotensin receptor 1.

The prediction of mutation-induced free-energy changes in protein thermostability or protein-protein binding is of particular interest in the fields of protein design, biotechnology,a nd bioengineering.H erein, we achieve remarkable accuracy in as can of 762 mutations estimating changes in protein thermostability based on the first principles of statistical mechanics.The remaining error in the free-energy estimates appears to be due to three sources in approximately equal parts,n amely sampling, force-field inaccuracies,a nd experimental uncertainty.W ep ropose ac onsensus force-field approach,w hich, together with an increased sampling time, leads to af ree-energy prediction accuracy that matches those reached in experiments.T his versatile approach enables accurate free-energy estimates for diverse proteins,i ncluding the prediction of changes in the melting temperature of the membrane protein neurotensin receptor 1.Evolution has optimized proteins to perform their specific functions in the environmental conditions native to the host organism. Altering certain thermodynamic properties of ap rotein is often sought after by the pharmaceutical and chemical industries, [1] for example,e nhancing the thermal stability of am olecule or altering the strength of as pecific protein-protein interaction. Such modifications may be achieved by means of amino acid mutations,a nd the prediction of free-energy changes upon mutation is thus of key interest.Foranideal free-energy prediction method, the predictive accuracy should be of the same range as that reached in experiments.Aperfect method ought to be system-independent, and hence not require fitting to experimental data. It should be able to robustly predict thermostabilities (or binding affinities) for different mutation types in the core of ap rotein as well as in the solvent-exposed regions,w hich requires that solute-solvent interactions are taken into account. Theability to change the environmental conditions, for example,t he temperature,p ressure,p H, or salt concentration, is another necessary requirement.Alchemical free-energy calculations have the potential to fulfill these requirements.T he approach relies on molecular dynamics (MD) simulations,w here both the solute and solvent are modeled atomistically.M Ds imulations are not restricted to any particular protein class and allow for precise control over the simulation conditions.T he estimation of the free-energy differences is based on rigorous theories.[2-4] The major bottlenecks to aroutine employment of these methods are high computational costs (subsequently leading to the related undersampling problem), the complex simulation setup,a nd the dependence of the results on the chosen molecular mechanics force field. Whereas the former two aspects are merely technical caveats,the force-field development is an active field requiring constant updates and benchmarks. [5,6] Herein, we utilized as tate-of-the-art setup [7] for alchemical free-energy calculations to carry out alarge-scale protein thermos...

We present a new replica exchange method, designed for optimal native state protein sampling in explicit solvent, called replica exchange with flexible tempering (REFT). The method was built upon the recently introduced replica exchange with solute tempering (REST). The potential function is adapted to direct the conformational search toward interdomain movements and the flexible portions of the protein. We demonstrate the improved sampling efficiency of REFT compared to the original REST for the bacteriophage T4 lysozyme.

In a conformational selection scenario, manipulating the populations of binding-competent states should be expected to affect protein binding. We demonstrate how in silico designed point mutations within the core of ubiquitin, remote from the binding interface, change the binding specificity by shifting the conformational equilibrium of the ground-state ensemble between open and closed substates that have a similar population in the wild-type protein. Binding affinities determined by NMR titration experiments agree with the predictions, thereby showing that, indeed, a shift in the conformational equilibrium enables us to alter ubiquitin’s binding specificity and hence its function. Thus, we present a novel route towards designing specific binding by a conformational shift through exploiting the fact that conformational selection depends on the concentration of binding-competent substates.

Nucleoside phosphoramidates (NPs) are a class of nucleotide analogues that has been developed as potential antiviral/antitumor prodrugs. Recently, we have shown that some amino acid nucleoside phosphoramidates (aaNPs) can act as substrates for viral polymerases like HIV-1 RT. Herein, we report the synthesis and hydrolysis of a series of new aaNPs, containing either natural or modified nucleobases to define the basis for their differential reactivity. Aqueous stability, kinetics, and hydrolysis pathways were studied by NMR spectroscopy at different solution pD values (5-7) and temperatures. It was observed that the kinetics and mechanism (P-N and/or P-O bond cleavage) of the hydrolysis reaction largely depend on the nature of the nucleobase and amino acid moieties. Aspartyl NPs were found to be more reactive than Gly or β-Ala NPs. For aspartyl NPs, the order of reactivity of the nucleobase was 1-deazaadenine>7-deazaadenine>adenine>thymine≥3-deazaadenine. Notably, neutral aqueous solutions of Asp-1-deaza-dAMP degraded spontaneously even at 4 °C through exclusive P-O bond hydrolysis (a 50-fold reactivity difference for Asp-1-deaza-dAMP vs. Asp-3-deaza-dAMP at pD 5 and 70 °C). Conformational studies by NMR spectroscopy and molecular modeling suggest the involvement of the protonated N3 atom in adenine and 1- and 7-deazaadenine in the intramolecular catalysis of the hydrolysis reaction through the rare syn conformation.

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