Investigation of the mechanism of domain closure in citrate synthase by molecular dynamics simulation.

Six, 2 ns molecular dynamics simulations have been performed on the homodimeric enzyme citrate synthase. In three, both monomers were started from the open, unliganded X-ray conformation. In the remaining three, both monomers started from a closed, liganded X-ray conformation, with the ligands removed. Projecting the motion from the simulations onto the experimental domain motion revealed that the free-energy profile is rather flat around the open conformation, with steep sides. The most closed conformations correspond to hinge-bending angles of 12-14 compared to the 20 degrees that occurs upon the binding of oxaloacetate. It is also found that the open, unliganded X-ray conformation is situated at the edge of the steep rise in free energy, although conformations that are about 5 degrees more open were sampled. A rigid-body essential dynamics analysis of the combined open trajectories has shown that domain motions in the direction of the closed X-ray conformation are compatible with the natural domain motion of the unliganded protein, which has just two main degrees of freedom. The simulations starting from the closed conformation suggest a free-energy profile with a small barrier in going from the closed to open conformation. A combined essential dynamics and hinge-bending analysis of a trajectory that spontaneously converts from the closed to open state shows an almost exact correspondence to the experimental transition that occurs upon ligand binding. The simulations support the conclusion from an earlier analysis of the experimental transition that the beta-hairpin acts as a mechanical hinge by attaching the small domain to the large domain through a conserved main-chain hydrogen bond and salt-bridges, and allowing rotation to occur via its two flexible termini. The results point to a mechanism of domain closure in citrate synthase that has analogy to the process of closing a door.

Sensitive assay for laboratory evolution of hydroxylases toward aromatic and heterocyclic compounds.

Powerful directed evolution methods have been developed for tailoring proteins to our needs in industrial applications. Here, the authors report a medium-throughput assay system designed for screening mutant libraries of oxygenases capable of inserting a hydroxyl group into a C-H bond of aromatic or O-heterocyclic compounds and for exploring the substrate profile of oxygenases. The assay system is based on 4-aminoantipyrine (4-AAP), a colorimetric phenol detection reagent. By using 2 detection wavelengths (509 nm and 600 nm), the authors achieved a linear response from 50 to 800 microM phenol and standard deviations below 11% in 96-well plate assays. The monooxygenase P450 BM-3 and its F87A mutant were used as a model system for medium-throughput assay development, identification of novel substrates (e.g., phenoxytoluene, phenylallyether, and coumarone), and discovery of P450 BM-3 F87A mutants with 8-fold improvement in 3-phenoxytoluene hydroxylation activity. This activity increase was achieved by screening a saturation mutagenesis library of amino acid position Y51 using the 4-AAP protocol in the 96-well format.

A molecular dynamics study of the 41-56 beta-hairpin from B1 domain of protein G.

The structural and dynamical behavior of the 41-56 beta-hairpin from the protein G B1 domain (GB1) has been studied at different temperatures using molecular dynamics (MD) simulations in an aqueous environment. The purpose of these simulations is to establish the stability of this hairpin in view of its possible role as a nucleation site for protein folding. The conformation of the peptide in the crystallographic structure of the protein GB1 (native conformation) was lost in all simulations. The new equilibrium conformations are stable for several nanoseconds at 300K (>10 ns), 350 K (>6.5 ns), and even at 450 K (up to 2.5 ns). The new structures have very similar hairpin-like conformations with properties in agreement with available experimental nuclear Overhauser effect (NOE) data. The stability of the structure in the hydrophobic core region during the simulations is consistent with the experimental data and provides further evidence for the role played by hydrophobic interactions in hairpin structures. Essential dynamics analysis shows that the dynamics of the peptide at different temperatures spans basically the same essential subspace. The main equilibrium motions in this subspace involve large fluctuations of the residues in the turn and ends regions. Of the six interchain hydrogen bonds, the inner four remain stable during the simulations. The space spanned by the first two eigenvectors, as sampled at 450 K, includes almost all of the 47 different hairpin structures found in the database. Finally, analysis of the hydration of the 300 K average conformations shows that the hydration sites observed in the native conformation are still well hydrated in the equilibrium MD ensemble.

Molecular dynamics simulation of the docking of substrates to proteins.

A simple method is described to perform docking of substrates to proteins or probes to receptor molecules by a modification of molecular dynamics simulations. The method consists of a separation of the center-of-mass motion of the substrate from its internal and rotational motions, and a separate coupling to different thermal baths for both types of motion of the substrate and for the motion of the receptor. Thus the temperatures and the time constants of coupling to the baths can be arbitrarily varied for these three types of motion, allowing either a frozen or a flexible receptor and allowing control of search rate without disturbance of internal structure. In addition, an extra repulsive term between substrate and protein was applied to smooth the interaction. The method was applied to a model substrate docking onto a model surface, and to the docking of phosphocholine onto immunoglobulin McPC603, in both cases with a frozen receptor. Using translational temperatures of the substrate in the range of 1300-1700 K and room temperature for the internal degrees of freedom of the substrate, an efficient nontrapping exploratory search ("helicopter view") is obtained which visits the correct binding sites. Low energy conformations can then be further investigated by separate search or by dynamic simulated annealing. In both cases the correct minima were identified. The possibility to work with flexible receptors is discussed.

Docking of flexible ligands to flexible receptors in solution by molecular dynamics simulation.

In this paper, a method of simulating the docking of small flexible ligands to flexible receptors in water is reported. The method is based on molecular dynamics simulations and is an extension of an algorithm previously reported by Di Nola et al. (Di Nola et al., Proteins 1994;19:174-182). The method allows a fast exploration of the receptor surface, using a high temperature of the center of mass translational motion, while the ligand internal motions, the solvent, and the receptor are simulated at room temperature. In addition, the method allows a fast center of mass motion of the ligand, even in solution. The dampening effect of the solvent can be overcome by applying different weights to the interactions between system subsets (solvent, receptor, and ligand). Specific ligand-receptor distances have been used to compare the results of the simulations with the crystal structure. The method is applied, as a test system, to the docking of the phosphocholine to the immunoglobulin McPC603. The results show the similarity of structure between the complex in solution and in the crystal.