Rajni Verma

Computer-Aided Protein Directed Evolution: a Review of Web Servers, Databases and other Computational Tools for Protein Engineering

The combination of computational and directed evolution methods has proven a winning strategy for protein engineering. We refer to this approach as computer-aided protein directed evolution (CAPDE) and the review summarizes the recent developments in this rapidly growing field. We will restrict ourselves to overview the availability, usability and limitations of web servers, databases and other computational tools proposed in the last five years. The goal of this review is to provide concise information about currently available computational resources to assist the design of directed evolution based protein engineering experiment.

A Potential Antitumor Drug (Arginine Deiminase) Reengineered for Efficient Operation under Physiological Conditions

Arginine deiminase (ADI, EC 3.5.3.6) is a potential antitumor drug for the treatment of arginine‐auxotrophic tumors such as hepatocellular carcinomas (HCCs) and melanomas, and studies on human lymphatic leukemia cell lines have confirmed that ADI has antiangiogenic activity. Recent studies showed that a combination of taxane and ADI‐PEG20, which induces caspase‐independent apoptosis, is more effective than taxane monotherapy for prostate cancer. The main limitation of ADI from Pseudomonas plecoglossicida (PpADI) and of many other ADI enzymes lies in their pH‐dependent activity profile. PpADI has a pH optimum at 6.5 and a pH shift from 6.5 to 7.5 results in an ∼80 % activity drop (the pH of human plasma is 7.35 to 7.45). In 2010, we reported a proof of concept for ADI engineering by directed evolution that resulted in variant M2 (K5T/D44E/H404R). M2 has a pH optimum of pH 7.0, a fourfold higher kcat value than the wild‐type PpADI (pH 7.4, 0.5 M phosphate buffer), and an increased Km value for substrate arginine. In our latest work, variants M5 (K5T/D38H/D44E/A128T/H404R) and M6 (K5T/D38H/D44E/A128T/E296K/H404R) were generated by directed evolution by employing PBS buffer (pH 7.4), which mimics physiological conditions. The S0.5 value of parent M3 (K5T/D44E/A128T/H404R) decreased from 2.01 to 1.48 mM (M5) and 0.81 mM (M6). The S0.5 value of M6 (0.81 mM) is lower than that of wild‐type PpADI (1.30 mM); the kcat values improved from 0.18 s−1 (wild‐type PpADI) to 17.56 s−1 (M5, 97.6‐fold) and 11.64 s−1 (M6, 64.7‐fold).

MAP2.03D: A Sequence/Structure Based Server for Protein Engineering

The Mutagenesis Assistant Program (MAP) is a web-based tool to provide statistical analyses of the mutational biases of directed evolution experiments on amino acid substitution patterns. MAP analysis assists protein engineers in the benchmarking of random mutagenesis methods that generate single nucleotide mutations in a codon. Herein, we describe a completely renewed and improved version of the MAP server, the MAP(2.0)3D server, which correlates the generated amino acid substitution patterns to the structural information of the target protein. This correlation aids in the selection of a more suitable random mutagenesis method with specific biases on amino acid substitution patterns. In particular, the new server represents MAP indicators on secondary and tertiary structure and correlates them to specific structural components such as hydrogen bonds, hydrophobic contacts, salt bridges, solvent accessibility, and crystallographic B-factors. Three model proteins (D-amino oxidase, phytase, and N-acetylneuraminic acid aldolase) are used to illustrate the novel capability of the server. MAP(2.0)3D server is available publicly at http://map.jacobs-university.de/map3d.html.

Insight into the redox partner interaction mechanism in cytochrome P450BM-3 using molecular dynamics simulations.

Flavocytochrome P450BM-3 is a soluble bacterial reductase composed of two flavin (FAD/FMN) and one HEME domains. In this article, we have performed molecular dynamics simulations on both the isolated FMN and HEME domains and their crystallographic complex, with the aim to study their binding modes and to garner insight into the interdomain electron transfer (ET) mechanism. The results evidenced an interdomain conformational rearrangement that reduces the average distance between the FMN and HEME cofactors from 1.81 nm, in the crystal structure, to an average value of 1.41±0.09 nm along the simulation. This modification is in agreement with previously proposed hypotheses suggesting that the crystallographic FMN/HEME complex is not in the optimal arrangement for favorable ET rate under physiological conditions. The calculation of the transfer rate along the simulation, using the Pathways Path method, demonstrated the occurrence of seven ET pathways between the two redox centers, with three of them providing ET rates (KET ) comparable with the experimental one. The sampled ET pathways comprise the amino acids N319, L322, F390, K391, P392, F393, A399, C400, and Q403 of the HEME domain and M490 of the FMN domain. The values of KET closer to the experiment were found along the pathways FMN(C7)→F390→K391→P392→HEME(Fe) and FMN(C8)→M490→F393→HEME(Fe). Finally, the analysis of the collective modes of the protein complex evidences a clear correlation of the first two essential modes with the activation of the most effective ET pathways along the trajectory.

Conformational dynamics of the FMN-binding reductase domain of monooxygenase P450BM-3

In the cytochrome P450BM-3, the flavin mononucleotide (FMN) binding domain is an intermediate electron donor between the flavin adenine dinucleotide (FAD) binding domain and the HEME domain. Experimental evidence has shown that different redox states of FMN cofactor were found to induce conformational changes in the FMN domain. Herein, molecular dynamics (MD) simulation is used to gain insight into the latter phenomenon at the atomistic level. We have studied the effect of FMN cofactor and its redox states (oxidized and reduced) on the structure and dynamics of the FMN domain. The results of our study show significant differences in the atomic fluctuation amplitude of the FMN domain in both holo- and apoprotein. The change in the protonation state of FMN cofactor mostly affects its binding in holo-protein. In particular, the loops involved in the binding of the isoalloxazine ring (Lβ4) and ribityl side chain (Lβ1) adopt different conformations in both reduced and oxidized states. In addition, the reduced FMN cofactor mainly induces a conformational change in Trp574 residue (Lβ4) that is essential for controlling electron transfer (ET) within P450BM-3 domains. The structure of the apoprotein in solution remains mostly unchanged with respect to the crystal structure of the holo-protein. However, FMN binding loops were more flexible in apoprotein that might favor the rebinding of FMN cofactor. In the holo-protein simulation, the largest conformational changes in FMN cofactor are caused by the ribityl side chain. The isoalloxazine ring of FMN cofactor remains almost planar (∼177°) in the oxidized state and bends along the N5-N10 axis at an angle of ∼160° in the reduced state. The collective modes of the isoalloxazine ring were identical in both protonation states of FMN cofactor except the first eigenvector. In the reduced state, the isoalloxazine ring attains the butterfly motion as a dominant collective motion in the first eigenvector due to the bending along the N5-N10 axis.

Unraveling Binding Effects of Cobalt(II) Sepulchrate with the Monooxygenase P450 BM-3 Heme Domain Using Molecular Dynamics Simulations

One of the major limitations to exploit enzymes in industrial processes is their dependence on expensive reduction equivalents like NADPH to drive their catalytic cycle. Soluble electron-transfer (ET) mediators like cobalt(II) sepulchrate have been proposed as a cost-effective alternative to shuttle electrons between an inexpensive electron source and an enzymes redox center. The interactions of these molecules with enzymes have not yet been elucidated at the molecular level. Herein, molecular dynamics simulations are performed to understand the binding and ET mechanism of the cobalt(II) sepulchrate with the heme domain of cytochrome P450 BM-3. The study provides a detailed map of ET mediator binding sites on the protein surface that are prevalently composed of Asp and Glu amino acids. The cobalt(II) sepulchrate does not show a preferential binding to these sites. However, among the observed binding sites, only few of them provide efficient ET pathways to heme iron. The results of this study can be used to improve the ET mediator efficiency of the enzyme for possible biotechnological applications.

Molecular Modeling of Cetylpyridinium Bromide, a Cationic Surfactant, in Solutions and Micelle.

Cationic surfactants are widely used in biological and industrial processes. Notably, surfactants with pyridinium salts, such as cetylpyridinium bromide (CPB), have diverse applications. The cetylpyridium cation has a quaternary nitrogen in the aromatic heterocyclic ring of the headgroup and 16 carbons in the hydrocarbon tail. At present and in the past, it has been widely used in germicides. Recently, several interesting applications of CPB have been explored, including its use in protein folding, polymerization, enzyme studies, and gene delivery as well as in pharmaceuticals as a drug delivery tool. A molecular-level understanding of CPB and its micelle in solution can enhance its development in such applications. Herein, we have proposed the first united-atom force field for CPB that yields stable micellar aggregates in molecular dynamics (MD) simulations. The force field is validated through classical MD simulations of the CPB monomer in pure water and 1-octanol as well as in an aqueous CPB micelle. We have performed principal component analysis (PCA) and calculated the translational and rotational diffusion coefficients, spatial distribution of solvent, counterion distribution, and rotational correlation time of CPB molecule in solutions and in micelle, comparing these data to previous experimental and theoretical results for a strong validation of the force field. PCA confirms that the pyridinium ring remains planar, whereas the movement of the hydrophobic tail region leads to conformational changes during the simulations. The collective modes of the pyridinum ring were identical for CPB molecule in solution and micelle, but conformational dynamics of the CPB tail were restricted in the micelle relative to motions in water and 1-octanol. Using this force field, a spherical CPB micelle was shown to be stable throughout the course of simulation, and its solvation and structural properties are characterized.

To keep or not to keep? the question of crystallographic waters for enzyme simulations in organic solvent

Abstract The use of enzymes in non-aqueous solvents expands the use of biocatalysts to hydrophobic substrates, with the ability to tune selectivity of reactions through solvent selection. Non-aqueous enzymology also allows for fundamental studies on the role of water and other solvents in enzyme structure, dynamics, and function. Molecular dynamics simulations serve as a powerful tool in this area, providing detailed atomic information about the effect of solvents on enzyme properties. However, a common protocol for non-aqueous enzyme simulations does not exist. If you want to simulate enzymes in non-aqueous solutions, how many and which crystallographic waters do you keep? In the present work, this question is addressed by determining which crystallographic water molecules lead most quickly to an equilibrated protein structure. Five different methods of selecting and keeping crystallographic waters are used in order to discover which crystallographic waters lead the protein structure to reach an equilibrated structure more rapidly in organic solutions. It is found that buried waters contribute most to rapid equilibration in organic solvent, with slow-diffusing waters giving similar results.

In Silico Studies of Small Molecule Interactions with Enzymes Reveal Aspects of Catalytic Function

Small molecules, such as solvent, substrate, and cofactor molecules, are key players in enzyme catalysis. Computational methods are powerful tools for exploring the dynamics and thermodynamics of these small molecules as they participate in or contribute to enzymatic processes. In-depth knowledge of how small molecule interactions and dynamics influence protein conformational dynamics and function is critical for progress in the field of enzyme catalysis. Although numerous computational studies have focused on enzyme–substrate complexes to gain insight into catalytic mechanisms, transition states and reaction rates, the dynamics of solvents, substrates, and cofactors are generally less well studied. Also, solvent dynamics within the biomolecular solvation layer play an important part in enzyme catalysis, but a full understanding of its role is hampered by its complexity. Moreover, passive substrate transport has been identified in certain enzymes, and the underlying principles of molecular recognition are an area of active investigation. Enzymes are highly dynamic entities that undergo different conformational changes, which range from side chain rearrangement of a residue to larger-scale conformational dynamics involving domains. These events may happen nearby or far away from the catalytic site, and may occur on different time scales, yet many are related to biological and catalytic function. Computational studies, primarily molecular dynamics (MD) simulations, provide atomistic-level insight and site-specific information on small molecule interactions, and their role in conformational pre-reorganization and dynamics in enzyme catalysis. The review is focused on MD simulation studies of small molecule interactions and dynamics to characterize and comprehend protein dynamics and function in catalyzed reactions. Experimental and theoretical methods available to complement and expand insight from MD simulations are discussed briefly.