A d. functional study of the hydrolysis reaction of phosphodiesters with a series of attacking nucleophiles in the gas phase and in soln. is presented. The nucleophiles HOH, HO-​, CH3OH, and CH3O-​ were studied in reactions with ethylene phosphate, 2',​3'-​ribose cyclic phosphate and in their neutral (protonated) and monoanionic forms. Stationary-​point geometries for the reactions were detd. at the d. functional B3LYP​/6-​31++G(d,​p) level followed by energy refinement at the B3LYP​/6-​311++G(3df,​2p) level. Solvation effects were estd. by using a dielec. approxn. with the polarizable continuum model (PCM) at the gas-​phase optimized geometries. This series of reactions characterizes factors that influence the intrinsic reactivity of the model phosphate compds., including the effect of nucleophile, protonation state, cyclic structure, and solvent. The present study of the in-​line mechanism for phosphodiester hydrolysis, a reaction of considerable biol. importance, has implications for enzymic mechanisms. The anal. generally supports the associative mechanism for phosphate ester hydrolysis. The results highlight the importance for the reaction barrier of charge neutralization resulting from the protonation of the nonbridging phosphoryl oxygens and the role of internal hydrogen transfer in the gas-​phase mechanism. It also shows that solvent stabilization has a profound influence on the relative barrier heights for the dianionic, monoanionic, and neutral reactions. The calcns. provide a comprehensive data set for the in-​line hydrolysis mechanisms that can be used for the development of improved semiempirical quantum models for phosphate hydrolysis reactions.

The improper activation of the Abl tyrosine kinase results in chronic myeloid leukemia (CML)​. The recognition of an inactive conformation of Abl, in which a catalytically important Asp-​Phe-​Gly (DFG) motif is flipped by approx. 180° with respect to the active conformation, underlies the specificity of the cancer drug imatinib, which is used to treat CML. The DFG motif is not flipped in crystal structures of inactive forms of the closely related Src kinases, and imatinib does not inhibit c-​Src. We present a structure of the kinase domain of Abl, detd. in complex with an ATP-​peptide conjugate, in which the protein adopts an inactive conformation that resembles closely that of the Src kinases. An interesting aspect of the Src-​like inactive structure, suggested by mol. dynamics simulations and addnl. crystal structures, is the presence of features that might facilitate the flip of the DFG motif by providing room for the phenylalanine to move and by coordinating the aspartate side chain as it leaves the active site. One class of mutations in BCR-​Abl that confers resistance to imatinib appears more likely to destabilize the inactive Src-​like conformation than the active or imatinib-​bound conformations. Our results suggest that interconversion between distinctly different inactive conformations is a characteristic feature of the Abl kinase domain.

An autobiog. of Martin Karplus, who was born in Vienna and came to the United States as a refugee in Oct. 1938, is presented. This experience played an important role in his view of the world and my approach to science: It contributed to his realization that it was safe to stop working in fields that he felt he understood and to focus on different areas of research by asking questions that would teach him and others something new. He describe the experiences that led him from chem. and physics back to his first love, biol., and outline some of the contributions he has made as part of his ongoing learning experience.

We show that the balanced min.-​cut procedure introduced in PNAS 2004, 101, 14766 can be reinterpreted as a method for solving the constrained optimization problem of finding the min. cut among the cuts with a particular value of an additive function of the nodes on either side of the cut. Such an additive function (e.g., the partition function of the reactant region) can be used as a progress coordinate to det. a one-​dimensional profile (FEP) of the free-​energy surface of the protein-​folding reaction as well as other complex reactions. The algorithm is based on the network (obtained from an equil. mol. dynamics simulation) that represents the calcd. reaction behavior. The resulting FEP gives the exact values of the free energy as a function of the progress coordinate; i.e., at each value of the progress coordinate, the profile is obtained from the surface with the minimal partition function among the surfaces that divide the full free-​energy surface between two chosen end points. In many cases, the balanced min.-​cut procedure gives results for only a limited set of points. An approx. method based on pfold is shown to provide the profile for a more complete set of values of the progress coordinate. Applications of the approach to model problems and to realistic systems (β-​hairpin of protein G, LJ38 cluster) are presented.

A theor. study showed that the base excision repair enzyme uracil-​DNA glycosylase (UDG) exploits electrostatic interactions with backbone phosphate groups in the substrate for catalysis. Although expts. performed to test the calcd. results confirmed the predicted importance of the -​2, -​1, and +1 phosphate groups, there was an apparent disagreement with regard to the +2 phosphate group. The calcns. indicated that it made an important contribution, while exptl., the effect of its deletion or neutralization was small. The +2 phosphate group interacts directly with an active site histidine (H148 in humans) in the crystal structure of UDG in complex with double-​stranded (ds) DNA. We previously calcd. that H148 has a strong anticatalytic effect due to its protonation, and here we use alchem. free energy simulations to est. its site-​specific pKa. We find that it is pos. charged over the entire exptl. pH range (4-​10)​, so its deprotonation cannot compensate for deletion or neutralization of the +2 phosphate group. The free energy simulations are facilitated by an efficient charge-​scaling procedure that allows quant. correction for the implicit treatment of solvent far from the active site; improvements are made to that method to account carefully for differences in the truncation of electrostatic interactions in the contributing mol.-​mech. and continuum-​electrostatic approaches. Addnl. simulations are used to demonstrate that the +2 phosphate group is fully solvent exposed in complexes with single-​stranded DNA substrates like those used in the expts. In contrast, it is well-​structured and buried in the dsDNA complex used in the original simulations. Differences in solvent shielding thus account for the apparent lack of an effect obsd. exptl. upon neutralization or deletion of this group.

Nicotinic acetylcholine receptors (nAChR) are pentameric ligand gated ion channels composed of subunits that consist of an extracellular domain that carries the ligand-​binding site and a distinct ion-​pore domain. Signal transduction results from the allosteric coupling between the two domains: the distance from the binding site to the gate of the pore domain is 50 Å. Normal mode anal. with a Cα Gaussian network of a new structural model of the neuronal α7 nAChR showed that the lowest mode involves a global quaternary twist motion that opens the ion pore. A mol. probe anal., in which the network is modified at each individual amino acid residue, demonstrated that the major effect is to change the frequency, but not the form, of the twist mode. The largest effects were obsd. for the ligand-​binding site and the Cys-​loop. Most (24​/27) of spontaneous mutations known to cause congenital myasthenia and autosomal dominant nocturnal frontal lobe epilepsy are located either at the interface between subunits or, within a given subunit, at the interface between rigid blocks. These interfaces are modified significantly by the twist mode. The present anal., thus, supports the quaternary twist model of the nAChR allosteric transition and provides a qual. interpretation of the effect of the mutations responsible for several receptor pathologies.

A review on major aspects of the multi-​copy simultaneous search methodol. used for calcg. functionality maps.

A 27-residue lattice heteropolymer subject to Monte Carlo dynamics on a simple cubic lattice is studied over a range of temps.  Folding time distributions are used to obtain information concerning the details of folding kinetics.  The results are compared with those from methods based on mean force surfaces expressed in terms of a reduced set of variables and on a disconnectivity graph for the same system.  A detailed anal. of the folding trajectories is given, and the importance of dead-end traps in detg. the folding time is demonstrated.  We show that the calcd. folding kinetics can be modeled by a system of kinetic equations, with the essential rate consts. detd. from the Monte Carlo simulations and the resulting folding time distributions.  The kinetic equations make possible an anal. of the variation of the importance of different channels with temp.  In particular, we show that the presence of intermediates may be masked in the folding time distributions, with the mean folding time being independent of the height of the barrier between the intermediates and collapsed globule state of the system.  This and other results demonstrate that care has to be used in interpreting exptl. folding data in terms of the underlying kinetics.  Correspondingly, simulations are shown to have to satisfy certain requirements to obtain proper sampling of the dead-end traps.

A review.  Living cells are a collection of mol. machines which carry out many of the functions essential for the cell's existence, differentiation, and reprodn.  Most, although not all, of these machines are made up of proteins.  Because of their complexity, an understanding of how they work requires a synergistic combination of exptl. and theor. studies.  Here, the authors outline and discuss their studies of 2 such protein machines.  One is GroEL, the mol. chaperone from Escherichia coli, which aids in protein folding, and the other is F1-ATPase, a motor protein which synthesizes and hydrolyzes ATP.

How DNA repair proteins distinguish between the rare sites of damage and the vast expanse of normal DNA is poorly understood.  Recognizing the mutagenic lesion 8-oxoguanine (oxoG) represents an esp. formidable challenge, because this oxidized nucleobase differs by only two atoms from its normal counterpart, guanine (G).  Here we report the use of a covalent trapping strategy to capture a human oxoG repair protein, 8-oxoguanine DNA glycosylase I (hOGG1), in the act of interrogating normal DNA.  The x-ray structure of the trapped complex features a target G nucleobase extruded from the DNA helix but denied insertion into the lesion recognition pocket of the enzyme.  Free energy difference calcns. show that both attractive and repulsive interactions have an important role in the preferential binding of oxoG compared with G to the active site.  The structure reveals a remarkably effective gate-keeping strategy for lesion discrimination and suggests a mechanism for oxoG insertion into the hOGG1 active site.

A method for the simulation of conformational transitions is presented.  The method, based on targeted mol. dynamics (TMD), limits the conformational change at each mol. dynamics step to a fixed size, that minimizes the root mean square deviation from the target.  The method is more efficient than std. TMD and yields lower energy pathways, but, like the TMD method, requires only a single mol. dynamics simulation.  Test calcns. and comparisons with std. TMD calcns. for the alanine dipeptide with the analytic continuum electrostatics implicit solvent model are presented.

The mol. chaperone HSP90 is an attractive target for chemotherapy because its activity is required for the functional maturation of a no. of oncogenes.  Among the know inhibitors, radicicol, a 14-member macrolide, stands out as the most potent.  A mol. dynamics/minimization of radicicol showed that there were three low energy conformers of the macrocycle.  The lowest of these is the bioactive conformation obsd. in the cocrystal structure of radicicol with HSP90.  Corresponding conformational analyses of several known analogs gave a good correlation between the bioactivity and the energy of the bioactive conformer, relative to other conformers.  Based on this observation, a no. of proposed analogs were analyzed for their propensity to adopt the bioactive conformation prior to synthesis.  This led to the identification of pochonin D, a recently isolated secondary metabolite of Pochonia chlamydosporia, as a potential inhibitor of HSP90.  Pochonin D was synthesized using polymer-bound reagents and shown to be nearly as potent an HSP90 inhibitor as radicicol.

Mol. dynamics simulations of the structurally homologous proteins TNfn3 and FNfn10 have been used to investigate the contributions to side-chain dynamics measured by NMR relaxation expts.  The results reproduce the variation in core side-chain dynamics obsd. by NMR and highlight the relevance of anharmonic motion and transitions between local min. for explaining NMR side-chain order parameters.  A method is described for calcg. converged order parameters by use of replica exchange mol. dynamics in conjunction with an implicit solvent model.  These simulations allow the influence of various factors, such as the flexibility of side-chains and their free vol., on the mobility to be tested by perturbing the system.  Deletion mutations are found to have the largest effect on the more densely packed FNfn10.  Some counterintuitive effects are seen, such as an increase in order parameters close to deletion mutation sites, but these can be rationalized in terms of direct interactions with the modified side-chains.  A statistical anal. of published order parameters supports the conclusions drawn from the simulations.

A rapid method for the calcn. of the electrostatic energy of a system without a cutoff is described in which the computational time grows linearly with the no. of particles or charges.  The inverse of the distance is approximated as a polynomial, which is then transformed into a function whose terms involve individual particles, instead of particle pairs, by a partitioning of the double sum.  In this way, the electrostatic energy that is detd. by the interparticle interactions is obtained without explicit calcn. of these interactions.  For systems of pos. charges positioned on a face-centered cubic lattice, the calcn. of the energy by the new method is shown to be faster than the calcn. of the exact energy, in many cases by an order of magnitude, and to be accurate to within 1-2%.  The application of this method to increase the accuracy of conventional truncation-based calcns. in condensed-phase systems is also demonstrated by combining the approximated long-range electrostatic interactions with the exact short-range interactions in a "hybrid" calcn.  For a 20-Å sphere of water mols., the forces are shown to be six times as accurate using this hybrid method as those calcd. with conventional truncation of the electrostatic energy function at 12 Å.  This is accomplished with a slight increase in speed, and with a sevenfold increase in speed relative to the exact all-pair calcn.  Structures minimized with the hybrid function are shown to be closer to structures minimized with an exact all-pair electrostatic energy function than are those minimized with a conventional 13-Å cutoff-based electrostatic energy function.  Comparison of the energies and forces calcd. with the exact method illustrate that the abs. errors obtained with std. truncation can be very large.  The extension of the current method to other pairwise functions as well as to multibody functions, is described.

The study of the dynamics and thermodn. of small icosahedral virus capsids is an active field of research.  Normal mode anal. is one of the computational tools that can provide important insights into the conformational changes of the virus assocd. with cell entry or caused by changing of the physicochem. environment.  Normal mode anal. of virus capsids has been limited due to the size of these systems, which often exceed 50,000 residues.  Here we present the first normal mode calcn. with full dihedral flexibility of several virus capsids, including poliovirus, rhinovirus, and cowpea chlorotic mottle virus.  The calcns. were made possible by applying group theor. methods, which greatly simplified the calcns. without any approxn. beyond the all-atom force field representations in general use for smaller protein systems.  Since a full Cartesian basis set was too large to be handled by the available computer memory, we used a basis set that includes all internal dihedral angles of the system with the exception of the peptide bonds, which were assumed rigid.  The fluctuations of the normal modes are shown to correlate well with crystallog. temp. factors.  The motions of the first several normal modes of each symmetry type are described.  A hinge bending motion in poliovirus was found that may be involved in the mechanism by which bound small mols. inhibit conformational changes of the capsid.  Fully flexible normal mode calcns. of virus capsids are expected to increase our understanding of virus dynamics and thermodn., and can be useful in the refinement of cryo-electron microscopy structures of viruses.

Mol. dynamics simulations of the allosteric enzyme chorismate mutase in yeasts are presented.  QM/MM mol. dynamics simulations confirm the instability of the CHAIR conformation relative to that of other isomers in the gas phase, and confirm its relative instability in soln.  Two nonreactive conformers which are more stable in soln. than CHAIR undergo rapid transformation to the (active) CHAIR conformation by interactions with the enzyme active site.  The QM/MM potential generates transient substrate structures that are closer to the transition state than is the CHAIR conformer.

Cks proteins are adapter mols. that coordinate the assembly of multiprotein complexes.  They share the ability to domain swap by exchanging a β-strand, β4.  Here we use NMR spectroscopy and mol. dynamics simulations to investigate the dynamic properties of human Cks1 and its response on assembly with components of the SCFSkp2 ubiquitin ligation machinery.  In the NMR expt. with the free form of Cks1, a subset of residues displayed elevated R2 values and the cross-peaks of neighboring residues were missing from the spectrum, indicating a substantial conformational exchange contribution on the microsecond to millisecond time scale.  Strikingly the region of greatest conformational variability was the β4-strand that domain swaps to form the dimer.  Binding of the ligand common to all Cks proteins, Cdk2, suppressed the conformational heterogeneity.  This response was specific to Cdk2 binding; in contrast, binding of Skp2, a ligand unique to human Cks1, did not alter the dynamic behavior.  Short time (<5 ns) mol. dynamics simulations indicate that residues of Cks1 that form the binding site for phosphorylated ligands are considerably more flexible in the free form of Cks1 than they are in the Cdk2-Cks1 complex.  A cooperative interaction between Cdk2 and Cks1 is suggested, which reduces the configurational entropy of Cks1 and therefore facilitates phosphoprotein binding.  Indications of an unusual dynamic behavior of strand β4 in the free form of Cks1 were obtained from longer time scale (50 ns) dynamics simulations.  A spontaneous reversible unzipping of hydrogen bonds between β4 and β2 was obsd., suggesting an early intermediate structure for unfolding and/or domain swapping.  We propose that the dynamic properties of the β-sheet and its modification upon ligand binding underlie the domain swapping ability and the adapter function of Cks proteins.

We have used mol. dynamics simulations restrained by exptl. ϕ values derived from protein engineering expts. to det. the structures of the transition state ensembles of ten proteins that fold with two-state kinetics.  For each of these proteins we then calcd. the av. contact order in the transition state ensemble and compared it with the corresponding exptl. folding rate.  The resulting correlation coeff. is similar to that computed for the contact orders of the native structures, supporting the use of native state contact orders for predicting folding rates.  The native contacts in the transition state also correlate with those of the native state but are found to be about 30% lower.  These results show that, despite the high levels of heterogeneity in the transition state ensemble, the large majority of contributing structures have native-like topologies and that the native state contact order captures this phenomenon.

A calcn. of the binding free energy for the dimerization of insulin has been performed using the mol. mechanics-generalized Born surface area approach.  The calcd. abs. binding free energy is -11.9 kcal/mol, in approx. agreement with the exptl. value of -7.2 kcal/mol.  The results show that the dimerization is mainly due to nonpolar interactions.  The role of the hydrogen bonds between the 2 monomers appears to give the direction of the interactions.  A per-atom decompn. of the binding free energy has been performed to identify the residues contributing most to the self assocn. free energy.  Residues B24-B26 are found to make the largest favorable contributions to the dimerization.  Other residues situated at the interface between the 2 monomers were found to make favorable but smaller contributions to the dimerization: Tyr B16, Val B12, and Pro B28, and to an even lesser extent, Gly B23.  The energy decompn. on a per-residue basis is in agreement with exptl. alanine scanning data.  The results obtained from a single trajectory (i.e., the dimer trajectory is also used for the monomer anal.) and 2 trajectories (i.e., sep. trajectories are used for the monomer and dimer) are similar.

The plastic network model (PNM) is used to generate a conformational change pathway for Escherichia coli adenylate kinase based on two crystal structures, namely that of an open and a closed conformer.  In this model, the energy basins corresponding to known conformers are connected at their lowest common energies.  The results are used to evaluate and analyze the minimal energy pathways between these basins.  The open to closed transition anal. provides an identification of hinges that is in agreement with the existing definitions based on the available X-ray structures.  The elastic energy distribution and the Cα pseudo-dihedral variation provide similar information on these hinges.  The ensemble of the 45 published structures for this protein and closely related proteins is shown to always be within 3.0 Å of the pathway, which corresponds to a conformational change between two end structures that differ by a Cα-atom root-mean-squared deviation of 7.1 Å.