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Many enzymes have buried active sites. The properties of the tunnels connecting the active site with bulk solvent affect ligand binding and unbinding and also the catalytic properties. Here, we investigate ligand passage in the haloalkane dehalogenase enzyme LinB and the effect of replacing leucine by a bulky tryptophan at a tunnel-lining position. Transient kinetic experiments show that the mutation significantly slows down the rate of product release. Moreover, the mechanism of bromide ion release is changed from a one-step process in the wild type enzyme to a two-step process in the mutant. The rate constant of bromide ion release corresponds to the overall steady-state turnover rate constant, suggesting that product release became the rate-limiting step of catalysis in the mutant. We explain the experimental findings by investigating the molecular details of the process computationally. Analysis of trajectories from molecular dynamics simulations with a tunnel detection software reveals differences in the tunnels available for ligand egress. Corresponding differences are seen in simulations of product egress using a specialized enhanced sampling technique. The differences in the free energy barriers for egress of a bromide ion obtained using potential of mean force calculations are in good agreement with the differences in rates obtained from the transient kinetic experiments. Interactions of the bromide ion with the introduced tryptophan are shown to affect the free energy barrier for its passage. The study demonstrates how the mechanism of an enzymatic catalytic cycle and reaction kinetics can be engineered by modification of protein tunnels.

The factors that determine the extent to which diffusion and thermal activation processes govern electron transfer (ET) between proteins are debated. The process of ET between plastocyanin (PC) and cytochrome f (CytF) from the cyanobacterium Phormidium laminosum was initially thought to be diffusion-controlled but later was found to be under activation control (Schlarb-Ridley, B. G.; et al. Biochemistry 2005, 44, 6232). Here we describe Brownian dynamics simulations of the diffusional association of PC and CytF, from which ET rates were computed using a detailed model of ET events that was applied to all of the generated protein configurations. The proteins were modeled as rigid bodies represented in atomic detail. In addition to electrostatic forces, which were modeled as in our previous simulations of protein-protein association, the proteins interacted by a nonpolar desolvation (hydrophobic) force whose derivation is described here. The simulations yielded close to realistic residence times of transient protein-protein encounter complexes of up to tens of microseconds. The activation barrier for individual ET events derived from the simulations was positive. Whereas the electrostatic interactions between P. laminosum PC and CytF are weak, simulations for a second cyanobacterial PC-CytF pair, that from Nostoc sp. PCC 7119, revealed ET rates influenced by stronger electrostatic interactions. In both cases, the simulations imply significant contributions to ET from both diffusion and thermal activation processes.

Recently we have developed a new approach to study protein-protein interactions using Fourier transform infrared spectroscopy in combination with titration experiments and principal component analysis (FTIR-TPCA). In the present paper we review the FTIR-TPCA results obtained for the interaction between cytochrome P450 and the redox partner protein in two P450 systems, the Pseudomonas putida P450cam (CYP101) with putidaredoxin (P450cam-Pdx), and the Bacillus megaterium P450BM-3 (CYP102) heme domain with the FMN domain (P450BMP-FMND). Both P450 systems reveal similarities in the structural changes that occur upon redox partner complex formation. These involve an increase in beta-sheets and alpha-helix content, a decrease in the population of random coil/3(10)-helix structure, a redistribution of turn structures within the interacting proteins and changes in the protonation states or hydrogen-bonding of amino acid carboxylic side chains. We discuss in detail the P450cam-Pdx interaction in comparison with literature data and conclusions drawn from experiments obtained by other spectroscopic techniques. The results are also interpreted in the context of a 3D structural model of the Pdx-P450cam complex.

We present an analysis of trajectories from Brownian dynamics simulations of diffusional protein-protein encounter for the well-studied system of barnase and barstar. This analysis reveals details about the optimal association pathways, the regions of the encounter complex, possible differences of the pathways for dissociation and association, the coupling of translational and rotation motion, and the effect of mutations on the trajectories. We found that a small free-energy barrier divides the energetically most favorable region into a region of the encounter complex above the barnase binding interface and a region around a second energy minimum near the RNA binding loop. When entering the region of the encounter complex from the region near the RNA binding loop, barstar has to change its orientation to increase the electrostatic attraction between the proteins. By concentrating the analysis on the successful binding trajectories, we found that the region of the second minimum is not essential for the binding of barstar to barnase. Nevertheless, this region may be helpful to steer barstar into the region of the encounter complex. When applying the same analysis to several barnase mutants, we found that single mutations may drastically change the free-energy landscape and may significantly alter the population of the two minima. Therefore, certain protein-protein pairs may require careful adaptation of the positions of encounter and transition states when interpreting mutation effects on kinetic rates of association and/or dissociation.

The extracellular ribonuclease barnase and its intracellular inhibitor barstar bind fast and with high affinity. Although extensive experimental and theoretical studies have been carried out on this system, it is unclear what the relative importance of different contributions to the high affinity is and whether binding can be improved through point mutations. In this work, we first applied Poisson-Boltzmann electrostatic calculations to 65 barnase-barstar complexes with mutations in both barnase and barstar. The continuum electrostatic calculations with a van der Waals surface dielectric boundary definition result in the electrostatic interaction free energy providing the dominant contribution favoring barnase-barstar binding. The results show that the computed electrostatic binding free energy can be improved through mutations at W44/barstar and E73/barnase. Furthermore, the determinants of binding affinity were quantified by applying COMparative BINding Energy (COMBINE) analysis to derive quantitative structure-activity relationships (QSARs) for the 65 complexes. The COMBINE QSAR model highlights approximately 20 interfacial residue pairs as responsible for most of the differences in binding affinity between the mutant complexes, mainly due to electrostatic interactions. Based on the COMBINE model, together with Brownian dynamics simulations to compute diffusional association rate constants, several mutants were designed to have higher binding affinities than the wild-type proteins.

The periplasmic oligopeptide binding component (OppA) of the oligopeptide permease found in Gram-negative bacteria acts as a receptor for peptide transport across the cell membrane and is a potential target for antibacterial drug design. OppA exhibits broad specificity, binding to diverse peptides of 2-5 amino acid residues length. Crystallographic and calorimetric measurements have been carried out by Tame et al. of the binding of 28 peptides of sequence K-X-K to OppA, where X is a natural or nonnatural amino acid. Despite this extensive experimental characterization, a clear relationship between structural and thermodynamic parameters could not be readily identified, with a complicating factor being the observation of varying numbers of water molecules at the binding interface in the different complexes. Consequently, we have applied COMparative BINding Energy (COMBINE) analysis to derive quantitative structure-activity relationships (QSARs) for these 28 OppA-tripeptide complexes. This is the first application of COMBINE analysis to predict binding enthalpies and entropies, and predictive QSAR models were obtained for these quantities as well as for binding free energies. These QSAR models highlight several protein residues and bound water molecules in the binding site, as well as the electrostatic desolvation energies of the protein and the peptides, as responsible for most of the differences in binding thermodynamics between the peptides studied. The QSAR models aid rationalization of the determinants of binding affinity of the OppA:peptide complexes and provide guides for further ligand design. This study also points to the general applicability of COMBINE analysis to estimating thermodynamic parameters for protein-peptide complexes.

Understanding the mechanism and specificity of substrate binding in the cytochrome P450 (P450) superfamily is an important step toward explaining its key role in drug metabolism, toxicity, xenobiotic degradation, and several biosynthetic pathways. Here we investigate the ligand exit pathways and mechanisms of P450cam (CYP101), P450BM-3 (CYP102), and P450eryF (CYP107A1) by using random expulsion molecular dynamics and classical molecular dynamics simulations. Although several different pathways are found for each protein, one pathway is common to all three. The mechanism of ligand exit along this pathway is, however, quite different in the three different proteins. For P450cam, small backbone conformational changes, in combination with aromatic side chain rotation, allow for the passage of the rather rigid, compact, and hydrophobic substrate, camphor. In P450BM-3, larger transient backbone changes are observed on ligand exit. R47, situated at the entrance to the channel, appears important in guiding negatively charged fatty acid substrates in and out of the active site. In P450eryF, an isolated buried arginine, R185, stabilized by four hydrogen bonds to backbone carbonyl oxygen atoms, is located in the exit channel and is identified as having a particularly unusual functionality, dynamically gating channel opening. The results for these three P450s suggest that the channel opening mechanisms are adjusted to the physico-chemical properties of the substrate and can kinetically modulate protein-substrate specificity.

The rate of protein-protein association limits the response time due to protein-protein interactions. The bimolecular association rate may be diffusion-controlled or influenced, and in such cases, Brownian dynamics simulations of protein-protein diffusional association may be used to compute association rates. Here, we report Brownian dynamics simulations of the diffusional association of five different protein-protein pairs: barnase and barstar, acetylcholinesterase and fasciculin-2, cytochrome c peroxidase and cytochrome c, the HyHEL-5 antibody and hen egg lysozyme (HEL), and the HyHEL-10 antibody and HEL. The same protocol was used to compute the diffusional association rates for all the protein pairs in order to assess, by comparison to experimentally measured rates, whether the association of these proteins can be explained solely on the basis of diffusional encounter. The simulation protocol is similar to those previously derived for simulation of the association of barnase and barstar, and of acetylcholinesterase and fasciculin-2; these produced results in excellent agreement with experimental data for these protein pairs, with changes in association rate due to mutations reproduced within the limits of expected computational and modeling errors. Here, we find that for all protein pairs, the effects of mutations can be well reproduced by the simulations, even though the degree of the electrostatic translational and orientational steering varies widely between the cases. However, the absolute values of association rates for the acetylcholinesterase: fasciculin-2 and HyHEL-10 antibody: HEL pairs are overestimated. Comparison of bound and unbound protein structures shows that this may be due to gating resulting from protein flexibility in some of the proteins. This may lower the association rates compared to their bimolecular diffusional encounter rates.

Cytochrome P450s form a ubiquitous protein family with functions including the synthesis and degradation of many physiologically important compounds and the degradation of xenobiotics. Cytochrome P450cam from Pseudomonas putida has provided a paradigm for the structural understanding of cytochrome P450s. However, the mechanism by which camphor, the natural substrate of cytochrome P450cam, accesses the buried active site is a long-standing puzzle. While there is recent crystallographic and simulation evidence for opening of a substrate-access channel in cytochrome P450BM-3, for cytochrome P450cam, no such conformational changes have been observed either in different crystal structures or by standard molecular dynamics simulations. Here, a novel simulation method, random expulsion molecular dynamics, is presented, in which substrate-exit channels from the buried active site are found by imposing an artificial randomly oriented force on the substrate, in addition to the standard molecular dynamics force field. The random expulsion molecular dynamics method was tested in simulations of the substrate-bound structure of cytochrome P450BM-3, and then applied to complexes of cytochrome P450cam with different substrates and with product. Three pathways were identified, one of which corresponds to a channel proposed earlier on the basis of crystallographic and site-directed mutagenesis data. Exit via the water-filled channel, which was previously suggested to be a product exit channel, was not observed. The pathways obtained by the random expulsion molecular dynamics method match well with thermal motion pathways obtained by an analysis of crystallographic B-factors. In contrast to large backbone motions (up to 4 A) observed in cytochrome P450BM-3 for the exit of palmitoleic acid, passage of camphor through cytochrome P450cam only requires small backbone motions (less than 2.4 A) in conjunction with side-chain rotations. Concomitantly, in almost all the exit trajectories, salt-links that have been proposed to act as ionic tethers between secondary structure elements of the protein, are perturbed., (Copyright 2000 Academic Press.)

Three possible channels by which substrates and products can exit from the buried active site of cytochrome P450cam have been identified by means of random expulsion molecular dynamics simulations. In the investigation described here, we computed estimates of the relative probabilities of ligand passage through the three channels using steered molecular dynamics and adiabatic mapping. For comparison, the same techniques are also applied to investigate substrate egress from cytochrome P450-BM3. The channel in cytochrome P450cam, for which there is the most supporting evidence from experiments (which we name pathway 2a), is computed to be the most probable ligand exit channel. It has the smallest computed unbinding work and force. For this channel, the ligand exits between the F/G loop and the B' helix. Two mechanistically distinct, but energetically similar routes through this channel were observed, showing that multiple pathways along one channel are possible. The probability of ligand exit via the next most probable channel (pathway 3), which is located between the I helix and the F and G helices, is estimated to be less than 1/10 of the probability of exit along pathway 2a. Low-frequency modes of the protein extracted from an essential dynamics analysis of a 1 ns duration molecular dynamics simulation of cytochrome P450cam with camphor bound, support the opening of pathway 2a on a longer timescale. On longer timescales, it is therefore expected that this pathway becomes more dominant than estimated from the present computations., (Copyright 2000 Academic Press.)

Blue copper proteins are type-I copper-containing redox proteins whose role is to shuttle electrons from an electron donor to an electron acceptor in bacteria and plants. A large amount of experimental data is available on blue copper proteins; however, their functional characterization is hindered by the complexity of redox processes in biological systems. We describe here the application of a semiquantitative method based on a comparative analysis of molecular interaction fields to gain insights into the recognition properties of blue copper proteins. Molecular electrostatic and hydrophobic potentials were computed and compared for a set of 33 experimentally-determined structures of proteins from seven blue copper subfamilies, and the results were quantified by means of similarity indices. The analysis provides a classification of the blue copper proteins and shows that (I) comparison of the molecular electrostatic potentials provides useful information complementary to that highlighted by sequence analysis; (2) similarities in recognition properties can be detected for proteins belonging to different subfamilies, such as amicyanins and pseudoazurins, that may be isofunctional proteins; (3) dissimilarities in interaction properties, consistent with experimentally different binding specificities, may be observed between proteins belonging to the same subfamily, such as cyanobacterial and eukaryotic plastocyanins; (4) proteins with low sequence identity, such as azurins and pseudoazurins, can have sufficient similarity to bind to similar electron donors and acceptors while having different binding specificity profiles.

Triose phosphate isomerase (TIM) is a diffusion-controlled enzyme whose rate is limited by the diffusional encounter of the negatively charged substrate glyceraldehyde 3-phosphate (GAP) with the homodimeric enzyme's active sites. Translational and orientational steering of GAP toward the active sites by the electrostatic field of chicken muscle TIM has been observed in previous Brownian dynamics (BD) simulations. Here we report simulations of the association of GAP with TIMs from four species with net charges at pH 7 varying from -12e to +12e. Computed second-order rate constants are in good agreement with experimental data. The BD simulations and computation of average Boltzmann factors of substrate-protein interaction energies show that the protein electrostatic potential enhances the rates for all the enzymes. There is much less variation in the computed rates than might be expected on the basis of the net charges. Comparison of the electrostatic potentials by means of similarity indices shows that this is due to conservation of the local electrostatic potentials around the active sites which are the primary determinants of electrostatic steering of the substrate.

Protein association events are ubiquitous in biological systems. Some protein associations and subsequent responses are diffusion controlled in vivo. Hence, it is important to be able to compute bimolecular diffusional association rates for proteins. The Brownian dynamics simulation methodology may be used to simulate protein-protein encounter, compute association rates, and examine their dependence on protein mutation and the nature of the physical environment (e.g., as a function of ionic strength or viscosity). Here, the theory for Brownian dynamics simulations is described, and important methodological aspects, particularly pertaining to the correct modeling of electrostatic forces and definition of encounter complex formation, are highlighted. To illustrate application of the method, simulations of the diffusional encounter of the extracellular ribonuclease, barnase, and its intracellular inhibitor, barstar, are described. This shows how experimental rates for a series of mutants and the dependence of rates on ionic strength can be reproduced well by Brownian dynamics simulations. Potential future uses of the Brownian dynamics method for investigating protein-protein association are discussed.

The rate of protein association places an upper limit on the response time due to protein interactions, which, under certain circumstances, can be diffusion-controlled. Simulations of model proteins show that diffusion-limited association rates are approximately 10(6)-10(7) M-1 s-1 in the absence of long-range forces (Northrup, S. H., and H. P. Erickson. 1992. Kinetics of protein-protein association explained by Brownian dynamics computer simulations. Proc. Natl. Acad. Sci. U.S.A. 89:3338-3342). The measured association rates of barnase and barstar are 10(8)-10(9) M-1 s-1 at 50 mM ionic strength, and depend on ionic strength (Schreiber, G., and A. R. Fersht. 1996. Rapid, electrostatically assisted association of proteins. Nat. Struct. Biol. 3:427-431), implying that their association is electrostatically facilitated. We report Brownian dynamics simulations of the diffusional association of barnase and barstar to compute association rates and their dependence on ionic strength and protein mutation. Crucial to the ability to reproduce experimental rates is the definition of encounter complex formation at the endpoint of diffusional motion. Simple definitions, such as a required root mean square (RMS) distance to the fully bound position, fail to explain the large influence of some mutations on association rates. Good agreement with experiments could be obtained if satisfaction of two intermolecular residue contacts was required for encounter complex formation. In the encounter complexes, barstar tends to be shifted from its position in the bound complex toward the guanine-binding loop on barnase.