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Chromophore Protonation States and the Proton Shuttle Mechanism in Green Fluorescent Protein: Inferences Drawn from ab Initio Theoretical Studies of Structures and Infrared Absorption Spectra
Hi-Young Yoo, J.A. Boatz, Volkhard Helms, J. Andrew McCammon and Peter W. Langhoff
Assignments are provided of prominent features in the recently measured Fourier transform infrared (FTIR) difference spectra of green fluorscent and photoactive yellow proteins (GFP, PYP) employing ab inito calculations of the ground electronic state structures and vibrational spectra of their chromophores in selected protonation states. Particular attention is addressed to inferring the protonation states of wild-type GFP chromophore and to reconciling the measured FTIR difference spectrum with a proposed proton shuttle mechanism in which protonated and deprotonated forms of the chromophore are paired with corresponding charge states of a Glu222 residue shuttle terminus. The calculated GFP IR difference spectrum obtained from the neutral-anionic pair of chromophores is found to be in general accord with the FTIR measurements on wild-type GFP in its protonated and deprotonated forms, whereas the spectrum obtained from the zwitterionic-cationic pair of chromophores provides a less satisfactory simulation of the data. The apparent absence of a carbonyl band in the measured GFP FTIR difference spectrum, a feature expected upon protonation of the carboxylic Glu222 residue, is reconciled by the presence of a carbonyl mode in the imidazole ring of the neutral chromophore which partially obscures the anticipated R-COOH Glu222 feature in the calculated spectrum. By contract, the corresponding carbonyl mode in the PYP chromophore is predicted to be significantly weaker and at lower frequency than in GFP, accounting in part for the presence of an unobscured prominent R-COOH Glu46 residue carbonyl mode in the measured PYP FTIR difference spectrum. Accordingly, the present ab initio theoretical study supports the predominance of neutral and anionic forms of wild-type GFP chromophore, and it argueably reconciles the available FTIR data with a probable Glu222 terminus of the proposed proton shuttle mechanism in the protein. Additional experimental studies of IR and Raman difference spectra in GFP and PYP, including particularly isotopic substitutions, are suggested to complement additional theoretical studies in progress.
The Adaptive Multilevel Finite Element Solution of the Poisson-Boltzmann Equation on Massively Parallel Computers
N.A. Baker, D. Sept, M.J. Holst and J.A. McCammon
Using new methods for the parallel solution of elliptic partial differential equations, the teraflops computing power of massively parallel computers can be leveraged to perform electrostatic calculations on large biological systems. This paper describes the adaptive multilevel finite element solution of the Poisson-Boltzmann equation for a microtubule on the NPACI IBM Blue Horizon supercomputer. The microtubule system is 40 nm in length, 24 nm in diameter, consists of roughly 600000 atoms, and has a net charge of -1800 e. Poisson-Boltzmann calculations are performed for several processor configurations and the algorithm shows excellent parallel scaling.
Load Balancing of Molecular Dynamics Simulation with NWChem
T.P. Straatsma and J.A. McCammon
NWChem is a computational chemistry software suite developed for massively parallel computers in the W. R. Wiley Environmental Molecular Sciences Laboratory at the U.S. Department of Energy's Pacific Northwest National Laboratory software computational chemistry applications classical molecular dynamics quantum mechanical calculations architecture memory domain decomposition intermolecular interactions periodic atomic reassignmentsoratory. This software integrates a range of modules for computational chemistry applications, including classical molecular dynamics simulations and quantum mechanical calculations. This contribution provides details of the classical molecular dynamics module and focuses on issues related to load balancing on massively parallel computers, in particular the IBM SP and the Cray T3E as examples of distributed and shared memory massively parallel architectures. The implementation of the molecular dynamics module of NWChem is based on a domain decomposition of the chemical system, taking advantage of the distribution of data to reduce the memory requirements and the locality of intermolecular interactions to reduce the communication requirements. This approach results in a more complex implementation because of the requirement of periodic atomic reassignments and the need for sophisticated load-balancing techniques.
A Model for Enzyme-Substrate Interactions in Alanine Racemase
Mary Jo Ondrechen, James M. Briggs and J. Andrew McCammon
We report on a theoretical model for the complex of the enzyme alanine racemase with its natural substrate (L-alanine) and cofactor (pyridoxal 5'-phosphate). Electrostatic potentials were calculated and ionization states were predicted for all the ionizable groups in alanine racemase. Some rather unusual charge states were predicted for certain residues. Tyr265' has an unusually low predicted pKa of 7.9 and at pH 7.0 has a predicted average charge of -0.37, meaning that 37% of the Tyr265' residues in an ensemble of enzyme molecules are in the phenolate form. At pH 8-9, the majority Tyr265' side groups will be in the phenolate form. This lends support to the experimental evidence that Tyr265' is the catalytic base involved in the conversion of L-alanine to D-alanine. Residues Lys39 and Lys129 have predicted average charges of +0.91 and +0.14 respectively at pH 7.0. Lys39 is believed to be the catalytic base for the conversion of D-alanine to L-alanine and the present results show that, at least some of the time, it is in the unprotonated amine form and thus able to act as a base. Cys311', which is located very close to the active site, has an unusally low predicted pKa of 5.8 and at pH 7.0 has a predicted average charge of -0.72. It appears that the enzyme has the ability to stabilize negative charge in the region of the active site. Implications for selective inhibitor design are discussed.
Calculation of Weak Protein-Protein Interactions: The pH Dependence of the Second Virial Coefficient
Adrian H. Elcock and J. Andrew McCammon
Interactions between proteins are often sufficiently weak that their study through the use of conventional structural techniques becomes highly problematic. Of the few techniques capable of providing experimental measures of weak protein-protein interactions perhaps the most useful is the second virial coefficient B22, which quantifies a protein solution's deviations from ideal behaviour. It has long been known that B22 can in principle be computed, but only very recently has it been demonstrated that such calculations can be performed using protein models of true atomic detail (Neal et al., Biophys. J. 1998, 75:2469-2477). The work reported here extends these previous efforts in an attempt to develop a transferable energetic model that is capable of reproducing the experimental trends obtained for two different proteins over a range of pH and ionic strength. We describe protein-protein interaction energies by a combination of three separate terms: (i) an electrostatic interaction term based on the use of effective charges, (ii) a term describing the electrostatic desolvation that occurs when charged groups are buried by an approaching protein partner, and (iii) a solvent accessible surface area (SASA) term that is used to describe contributions from van der Waals and hydrophobic interactions. The magnitude of the third term is governed by an adjustable parameter, gamma, that is altered to optimize agreement between calculated and experimental values of B22. The model is applied separately to the proteins lysozyme and chymotrypsinogen, yielding optimal values of gamma that are almost idential. However, there are clear difficulties in reproducing B22 values at extremes of pH. Rigorous calculations of the protonation states of charged groups in the 200 most favourable structures suggest that these difficulties are due to a neglect of the ionization state changes that often accompany complexation. Despite this problem, the fact that identical gamma values are obtained from two different proteins suggests that the energetic model developed here may well be transferable to other protein systems. Since the model proposed is extremely rapid to evaluate, it can be used in dynamical simulations of weak protein-protein interactions.
Statistical Analysis of the Fractal Gating Motions of the Enzyme Acetylcholinesterase
T.Y. Shen, Kaihsu Tai and J. Andrew McCammon
The enzyme acetylcholinesterase has an active site that is accessible only by a "gorge" or main channel from surface, and perhaps by secondary channels such as the "back door". Molecular dynamics simulations show that these channels are too narrow most of the time to admit substrate or other small molecules. Binding of substrate is therefore "gated" by structural fluctuations of the enzyme. Here, we analyze the fluctuations of these possible channels, as observed in the 10.8 ns trajectory of the simulation. The probability density function of the gorge proper radius (defined in text) was calculated. A double-peak feature of the function was discovered and therefore two states with a threshold were identified. The relaxation (transition probability) functions of these two states were also calculated. The results revealed a power-law decay trend and an oscillation around it, which show properties of fractal dynamics with a "complex exponent". The cross-correlation of potential energy versus proper radius was also investigated. We discuss possible physical models behind the fractal protein dynamics; the dynamic hierarchical model for glassy systems is evaluated in detail.
Historical Overview and Future Challenges
J. Andrew McCammon
The selective binding of molecules to form productive complexes is of central importance to pharmacology and medicinal chemistry. Although kinetic factors can influence the yields of different molecular complexes in cellular and other non-equilibrium environments, the primary factors that one must consider in the analysis of molecular recognition are thermodynamic. In particular, the equilibrium constant for the binding of molecules A and B to form the complex AB depends exponentially on the standard free energy change associated with complexation. Here, I provide a brief review of the history and new directions of free energy calculations.
The X-ray Crystal Structure of an SR Protein Kinase in Yeast (Sky1p) Reveals a Novel Mechanism for Constitutive Activity
Brad Nolen, Chi Y. Yun, Chung F. Wong, J. Andrew McCammon, Xiang-Dong Fu and Gourisankar Ghosh
The SR proteins and RS domain-containing proteins are a class of splicing factors rich in arginine-serine (RS) dipeptides that are phosphorylated by the SR protein kinases (SRPHs). SRPKs are constitutively active and display remarkable substrate specificity. Recently, the sole Saccharomyces cerevisiae SRPH family member, Sky1p, was shown to regulate nuclear import of the shuttling RNA binding protein Npl3p, which has been implicated in the mRNA export. Here we present the three-dimensional structure of a fully active truncated Sky1p. Analysis of the structure and structure-based functional studies reveal that the carboxyl-terminal tail, an unusual glutamine residue located in the P+1 loop, and a unique mechanism for the positioning of helix αC act together to render Sky1p constitutively active. We have modeled a Npl3p-derived substrate peptide bound to Sky1p. The model complex combined with mutagenesis studies illustrate the molecular basis for substrate recognition by this kinase, and suggest a mechanism for SRPKs to catalyze a novel sequential phosphorylation reaction on the RS dipeptide repeats characteristic of mammalian SR proteins.
Computer Simulation of Protein-Protein Interactions
Adrian H. Elcock, David Sept and J. Andrew McCammon
The use of computer simulations in investigations of protein-protein interactions is discussed. First, crystallographic analyses of known protein-protein complexes are summarized with particular emphasis being placed on the atomic nature of the interactions. Models available for describing macromolecular association energetics are then discussed, with special reference to the treatment of electrostatic and nonpolar interactions. The use of these models in combination with efficient search methods is discussed in context of the so-called protein docking problem and in the description of weaker (i.e., noncrystallisable) protein-protein interactions. Finally, simulations of the dynamics of protein-protein association events are outlined. In all cases, differences are stressed between the atomically detailed view of protein-protein interactions and the view implicit in the use of simpler colloidal models.
Identification of Protein Oligomerisation States by Analysis of Interface Conservation
Adrian H. Elcock and J. Andrew McCammon
The discrimination of true oligomeric protein-protein contacts from non-specific crystal contacts remains problematic. Criteria that have been used previously base the assignment of oligomeric state on consideration of the size of the interface area and/or the results of a scoring function based on statistical potentials. Both techniques have a high success rate, but fail in more than 10% of cases. More importantly, the oligomeric states of several proteins are incorrectly assigned by both methods. Here we test the hypothesis that true oligomeric contacts should be identifiable based on an increased degree of conservation of the residues involved in the interface. By quantifying the degree of conservation of the interface and comparing it with that of the remainder of the protein surface, we develop a new criterion that provides a highly effective complement to existing methods.
Computational Analysis of PKA-Balanol Interactions
Chung F. Wong, Philippe H. Hünenberger, Pearl Akamine, Narendra Narayana, Tom Diller, J. Andrew McCammon, Susan Taylor and Nguyen-Huu Xuong
Protein kinases are important targets for designing therapeutic drugs. This paper illustrates a computational approach to extend the usefulness of a single protein-inhibitor structure in aiding the design of protein kinase inhibitors. Using the complex structure of the catalytic subunit of PKA (cPKA) and balanol as a guide, we have analyzed and compared the distribution of amino acid types near the protein-ligand interface for nearly 400 kinases. This analysis has identified a number of sites that are more variable in amino acid types among the kinases analyzed, and these are useful sites to consider in designing specific protein kinase inhibitors. On the other hand, we have found kinases whose protein-ligand interfaces are similar to that of the cPKA-balanol complex and balanol can be a useful lead compound for developing effective inhibitors for these kinases. Generally, this approach can help us discover new drug targets for an existing class of compounds that have already been well characterized pharmacologically. The relative significance of the charge/polarity of residues at the protein-ligand interface has been quantified by carrying out computational sensitivity analysis in which the charge/polarity of an atom or functional group was turned off/on, and the resulting effects on binding affinity have been examined. The binding affinity was estimated by using an implicit-solvent model in which the electrostatic contributions were obtained by solving the Poisson equation and the hydrophobic effects were accounted for by using surface-area dependent terms. The same sensitivity analysis approach was applied to the ligand balanol to develop a pharmacophoric model for searching new drug leads from small-molecule libraries. To help evaluate the binding affinity of designed inhibitors before they are made, we have developed a semiempirical approach to improve the predictive reliability of the implicit-solvent binding model.
Analysis of a Ten-nanosecond Molecular Dynamics Simulation of Mouse Acetylcholinesterase
Kaihsu Tai, Tongye Shen, Ulf Börjesson, Marios Philippopoulos and J. Andrew McCammon
A 10 ns molecular dynamics simulation of mouse acetylcholinesterase was analysed, with special attention paid to the fluctuation in the width of the gorge, and opening events of the back door. The trajectory was first verified to ensure its stability. We defined the gorge proper radius as the measure for the extent of the gorge opening. We developed an expression of an inter-atom distance representative of the gorge proper radius in terms of projections on the principal components. This revealed the fact that collective motions of many scales contribute to the opening behavior of the gorge. Covariance and correlation results identified the motions of the protein backbone as the gorge opens. In the back door region, sidechain dihedral angles that define the opening were identified. Additional data from this molecular dynamics simulation can be found at http://mccammon.ucsd.edu/.
Thermodynamics and Kinetics of Actin Filament Nucleation
David Sept and J. Andrew McCammon
We have performed computer simulations and free energy calculations to determine the thermodynamics and kinetics of actin nucleation and thus identify a probable nucleation pathway and critical nucleus size. The binding free energies of structures along the nucleation pathway are found through a combination of electrostatic calculations and estimates of the entropic and surface area contributions. The association kinetics for the formation of each structure are determined through a series of Brownian dynamics simulations. The combination of the binding free energies and the association rate constants determines the dissociation rate constants, allowing for a complete characterization of the nucleation and polymerization kinetics. The results indicate that the trimer is the size of the critical nucleus, and the rate constants produce polymerization plots that agree very well with experimental results over a range of actin monomer concentrations.
Electrostatics of Nanosystems: Application to Microtubules and the Ribosome
Nathan A. Baker, David Sept, Simpson Joseph, Michael J. Holst and J. Andrew McCammon
Evaluation of the electrostatic properties of biomolecules has become a standard practice in molecular biophysics. Foremost among the models used to elucidate the electrostatic potential is the Poisson-Boltzmann equation; however, existing methods for solving this equation have limited the scope of accurate electrostatic calculations to relatively small biomolecular systems. Here we present the application of numerical methods to enable the trivially parallel solution of the Poisson-Boltzmann equation for supramolecular structures that are orders of magnitude larger in size. As a demonstration of this methodology, electrostatic potentials have been calculated for large microtubule and ribosome structures. The results point to the likely role of electrostatics in the variety of activities of these structures.
Ordered Water and Ligand Mobility in the HIV-1 Integrase-5CITEP Complex: A Molecular Dynamics Study
Haihong Ni, Christoph A. Sotriffer and J. Andrew McCammon
A 2 ns molecular dynamics simulation has been carried out for the HIV-1 integrase-5CITEP complex in order to understand the role of water in defining the ligand's binding mode and to address issues of binding site flexibility and ligand motion. Although the ligand retains considerable mobility within the active site, a structural water molecule bridging 5CITEP with Asp 64 and Asn 155 is identified in the simulation. Consideration of this water molecule could open a route to new HIV-1 integrase inhibitors.
Atomistic Brownian Dynamics Simulation of Peptide Phosphorylation
Tongye Shen, Chung F. Wong and J. Andrew McCammon
We report the implementation of an all-atom Brownian dynamics simulation model of peptides using the constraint algorithm LINCS. The algorithm has been added as a part of UHBD. It uses adaptive time steps to achieve a balance between computational speed and stability. The algorithm was applied to study the effect of phosphorylation on the conformational preference of the peptide Gly-Ser-Ser-Ser. We find that the middle serine residue experiences considerable conformational change from C7eq to the αR structure upon phosphorylation. NMR 3J coupling constants were also computed from the Brownian trajectories using the Karplus equation. The calculated 3J results agree reasonably well with experimental data for the phosphorylated peptide but less so for doubly charged phosphorylated one.
Native State Conformational Dynamics of GART: A Regulatory pH-Dependent Coil-Helix Transition Examined by Electrostatic Calculations
Dimitrios Morikis, Adrian H. Elcock, Patricia A. Jennings and J. Andrew McCammon
Glycinamide ribonucleotide transformylase (GART) undergoes a pH-dependent coil-helix transition with pKa = 7. An α-helix is formed at high pH spanning eight residues of a twenty-one residue long loop, comprising the segment Thr120-His121-Arg122-Gln123-Ala124-Leu125-Glu126-Asn127. To understand the electrostatic nature of this loop-helix, pKa values of all ionizable residues of GART have been calculated,using Poisson-Boltzmann electrostatic calculations and crystallogrphic data. Crystallographic structures of high and low pH E70A GART have been used in our analysis. Low pKa values of 5.3, 5.3, 3.9, 1.7, and 4.7, have been calculated for five functionally important histidines, His108, His119, His121, His132, and His137, respectively, using the high pH E70A GART structure. Ten theoretical single and double mutants of the high pH E70A structure have been constructed to identify pair-wise interactions of ionizable residues, which have aided in elucidating the multiplicity of electrostatic interactions of the activation loop-helix, and the impact of the activation helix on the catalytic site. Based on our pKa calculations and structural data, we propose that: (1) His121 forms a molecular switch for the coil-helix transition of the activation helix, depending on its protonation state, (2) a strong electrostatic interaction between His132 and His121 is observed, which can be of stabilizing or destabilizing nature for the activation helix, depending on the relative orientation and protonation states of the rings of His121 and His132, (3) electrostatic interactions involving His119 and Arg122 play a role in the stability of the activation helix, and (4) the activation helix contains the helix promoting sequence Arg122-Gln123-Ala125-Leu125-Glu126, but its alignment relative to the amino and carboxy termini of the helix is not optimal, and possibly of destablizing nature. Finally, we provide electrostatic evidence that the formation and closure of the activation helix creates a hydrophobic environment for catalytic site residue His108, to facilitate catalysis.
Proton Transfer Dynamics of GART: The pH-Dependent Catalytic Mechanism Examined by Electrostatic Calculations
Dimitrios Morikis, Adrian H. Elcock, Patricia A. Jennings and J. Andrew McCammon
The enzyme glycinamide ribonucleotide transformylase (GART) catalyzes the transfer of a formyl group from formyl tetrahydrofolate (fTHF) to glycinamide ribonucleotide (GAR), a process that is pH-dependent with pKa of = 8. Experimental studies of pH-rate profiles of wild type and site directed mutants of GART, have led to the proposal that HIS108, Asp144 and GAR are involved in catalysis, with His108 being an acid catalyst, while forming a salt bridge with Asp144, and GAR being a nucleophile to attack the formyl group of fTHF. This model implied a protonated histidine with pKa of 9.7 with a neutral GAR with pKa of 6.8 (Shim J.H. and Benkovic, S.J., 1999, Biochemistry, 38, 10024-10031). These proposed unusual pKa's have led us to investigate the electrostatic environment of the active site of GART. We have used Poisson-Boltzmann based electrostatic methods to calculate the pKa's of all ionizable groups, using the crystallographic structure of a ternary complex of GART involving the pseudo-substrate 5-deaza-5, 6, 7, 8-THF (5dTHF) and substrate GAR. Theoretical mutation and deletion analogs have been constructed to elucidate pair-wise electrostatic interations between key ionizable sites within the catalytic site. Also, a construct of a more realistic catalytic site including a re-constructed pseudo-cofactor with an attached formyl group, in an environment with optimal local van der Waals interactions (locally minimized) that imitates closely the catalytic reactants, has been used for pKa calculations. Strong electrostatic coupling among catalytic residues His108, Asp144, and substrate GAR was observed, which is extremely sensitive to the initial protonation and imidazole ring flip state of His108 and small structural changes. We demonstrate that a proton can be exchanged between GAR and His108 depending on their relative geometry and their distance to Asp144, and when the proton is attached on His108 catalysis could be possible. Using the formylated locally minimized construct of GART, a high pKa for His108 was calculated indicating a protonated histidine, and a low pKa for GAR(NHa) was calculated indicating that GAR is in neutral form. Our results are in qualitative agreement with the current mechanistic picture of the catalytic process of GART, deduced from the experimental data, but do not reproduce the absolute magnitude of the pKa's extracted from fits of kcat-pH profiles, possibly because the static time averaged crystallogrphic structure does not describe adequately the dynamic nature of the catalytic site during binding and catalysis. In addition, a strong effect on the pKa of GAR(NH2) is produced by the theoretical mutations of His108Ala and Asp144Ala, which is not in agreement with the observed insensitivity of the pKa of GAR(NH2) modeled from the experimental data, using similar mutations. Finally, we show that important three-way electrostatic interactions between highly conserved His137, with His108 and Asp144 are responsible for stabilizing the electrostatic microenvironment of the catalytic site. In conclusion, our data suggest that further detailed computational and experimental work is necessary.
Kinetic Mechanism of End to End Annealing of Actin Filaments
Ernesto Andrianantoandro, Laurent Blanchoin, David Sept, J. Andrew McCammon and Thomas D. Pollard
We investigated the effect of actin filament length and capping protein on the rate of end to end annealing of actin filaments. Long filaments were fragmented by shearing and allowed to recover. Stabilizing filaments with phalloidin in most experiments eliminated any contribution of subunit dissociation and association to redistribution of lengths but did not affect the results. Two different assays, fluorescence microscopy to measure filament lengths and polymerization to measure concentration of barbed filament ends, gave the same time course of annealing. The rate of annealing declines with time as the average filament length increases. Longer filaments also anneal slower than short filaments. The second order annealing rate constant is inversely proportional to mean polymer length with a value of 1.1 mM-1s-1/length in subunits. Capping protein slows but does not prevent annealing. Annealing is a highly favorable reaction with a strong influence on the length of polymers produced by spontaneous polymerization and should be considered in thinking about polymer dynamics in cells.