Jim Warwicker

Calculation of the electric potential in the active site cleft due to alpha-helix dipoles.

Abstract A macroscopic dielectric model has been used to set up the electrostatic equation for the protein-solvent system. A numerical method of solution has been applied, enabling calculation of the electric potential outside a protein due to the charges within the protein. The glycolytic enzyme phosphoglycerate mutase, which is an α/β protein binding negatively charged substrates, has been studied. Modelling the helix dipoles with positive and negative charges shows that the α-helical structure could stabilize negatively charged substrates in the active site cleft of an enzyme with an energy of a few kT.

TAPAS-1, a novel microdomain within the unique N-terminal region of the PDE4A1 cAMP-specific phosphodiesterase that allows rapid, Ca2+-triggered membrane association with selectivity for interaction with phosphatidic acid

Here we identify an 11-residue helical module in the unique N-terminal region of the cyclic AMP-specific phosphodiesterase PDE4A1 that determines association with phospholipid bilayers and shows a profound selectivity for interaction with phosphatidic acid (PA). This module contains a core bilayer insertion unit that is formed by two tryptophan residues, Trp19 and Trp20, whose orientation is optimized for bilayer insertion by the Leu16:Val17 pairing. Ca2+, at submicromolar levels, interacts with Asp21 in this module and serves to gate bilayer insertion, which is completed within 10 ms. Selectivity for interaction with PA is suggested to be achieved primarily through the formation of a charge network of the form (Asp21−:Ca2+:PA2−:Lys24+) with overall neutrality at the bilayer surface. This novel phospholipid-binding domain, which we call TAPAS-1 (tryptophan anchoring phosphatidicacid selective-binding domain 1), is here identified as being responsible for membrane association of the PDE4A1 cAMP-specific phosphodiesterase. TAPAS-1 may not only serve as a paradigm for other PA-binding domains but also aid in detecting related phospholipid-binding domains and in generating simple chimeras for conferring membrane association and intracellular targeting on defined proteins.

Progress in the prediction of pKa values in proteins

The pKa‐cooperative aims to provide a forum for experimental and theoretical researchers interested in protein pKa values and protein electrostatics in general. The first round of the pKa‐cooperative, which challenged computational labs to carry out blind predictions against pKas experimentally determined in the laboratory of Bertrand Garcia‐Moreno, was completed and results discussed at the Telluride meeting (July 6–10, 2009). This article serves as an introduction to the reports submitted by the blind prediction participants that will be published in a special issue of PROTEINS: Structure, Function and Bioinformatics. Here, we briefly outline existing approaches for pKa calculations, emphasizing methods that were used by the participants in calculating the blind pKa values in the first round of the cooperative. We then point out some of the difficulties encountered by the participating groups in making their blind predictions, and finally try to provide some insights for future developments aimed at improving the accuracy of pKa calculations. Proteins 2011;

Buried Charged Surface in Proteins

BACKGROUND The traditional picture of charged amino acids in globular proteins is that they are almost exclusively on the outside exposed to the solvent. Buried charges, when they do occur, are assumed to play an essential role in catalysis and ligand binding, or in stabilizing structure as, for instance, helix caps. RESULTS By analyzing the amount and distribution of buried charged surface and charges in proteins over a broad range of protein sizes, we show that buried charge is much more common than is generally believed. We also show that the amount of buried charge rises with protein size in a manner which differs from other types of surfaces, especially aromatic and polar uncharged surfaces. In large proteins such as hemocyanin, 35% of all charges are greater than 75% buried. Furthermore, at all sizes few charged groups are fully exposed. As an experimental test, we show that replacement of the buried D178 of muconate lactonizing enzyme by N stabilizes the enzyme by 4.2 degrees C without any change in crystallographic structure. In addition, free energy calculations of stability support the experimental results. CONCLUSIONS Nature may use charge burial to reduce protein stability; not all buried charges are fully stabilized by a prearranged protein environment. Consistent with this view, thermophilic proteins often have less buried charge. Modifying the amount of buried charge at carefully chosen sites may thus provide a general route for changing the thermophilicity or psychrophilicity of proteins.

Side-chain conformational entropy at protein-protein interfaces.

Protein–protein interactions are the key to many biological processes. How proteins selectively and correctly associate with their required protein partner(s) is still unclear. Previous studies of this “protein‐docking problem” have found that shape complementarity is a major determinant of interaction, but the detailed balance of energy contributions to association remains unclear. This study estimates side‐chain conformational entropy (per unit solvent accessible area) for various protein surface regions, using a self‐consistent mean field calculation of rotamer probabilities. Interfacial surface regions were less flexible than the rest of the protein surface for calculations with monomers extracted from homodimer datasets in 21 of 25 cases, and in 8 of 9 for the large protomer from heterodimer datasets. In surface patch analysis, based on side‐chain conformational entropy, 68% of true interfaces were ranked top for the homodimer set and 66% for the large protomer/heterodimer set. The results indicate that addition of a side‐chain entropic term could significantly improve empirical calculations of protein–protein association.

A Quantitative Molecular Model for Modulation of Mammalian Translation by the eIF4E-binding Protein 1

Translation initiation is a key point of regulation in eukaryotic gene expression. 4E-binding proteins (4E-BPs) inhibit initiation by blocking the association of eIF4E with eIF4G, two integral components of the mRNA cap-binding complex. Phosphorylation of 4E-BP1 reduces its ability to bind to eIF4E and thereby to compete with eIF4G. A novel combination of biophysical and biochemical tools was used to measure the impact of phosphorylation and acidic side chain substitution at each potentially modulatory site in 4E-BP1. For each individual site, we have analyzed the effects of modification on eIF4E binding using affinity chromatography and surface plasmon resonance analysis, and on the regulatory function of the 4E-BP1 protein using a yeast in vivo model system and a mammalian in vitro translation assay. We find that modifications at the two sites immediately flanking the eIF4E-binding domain, Thr46 and Ser65, consistently have the most significant effects, and that phosphorylation of Ser65 causes the greatest reduction in binding affinity. These results establish a quantitative framework that should contribute to understanding of the molecular interactions underlying 4E-BP1-mediated translational regulation.

Improved pKa calculations through flexibility based sampling of a water-dominated interaction scheme.

Ionizable groups play critical roles in biological processes. Computation of pKas is complicated by model approximations and multiple conformations. Calculated and experimental pKas are compared for relatively inflexible active‐site side chains, to develop an empirical model for hydration entropy changes upon charge burial. The modification is found to be generally small, but large for cysteine, consistent with small molecule ionization data and with partial charge distributions in ionized and neutral forms. The hydration model predicts significant entropic contributions for ionizable residue burial, demonstrated for components in the pyruvate dehydrogenase complex. Conformational relaxation in a pH‐titration is estimated with a mean‐field assessment of maximal side chain solvent accessibility. All ionizable residues interact within a low protein dielectric finite difference (FD) scheme, and more flexible groups also access water‐mediated Debye‐Hückel (DH) interactions. The DH method tends to match overall pH‐dependent stability, while FD can be more accurate for active‐site groups. Tolerance for side chain rotamer packing is varied, defining access to DH interactions, and the best fit with experimental pKas obtained. The new (FD/DH) method provides a fast computational framework for making the distinction between buried and solvent‐accessible groups that has been qualitatively apparent from previous work, and pKa calculations are significantly improved for a mixed set of ionizable residues. Its effectiveness is also demonstrated with computation of the pH‐dependence of electrostatic energy, recovering favorable contributions to folded state stability and, in relation to structural genomics, with substantial improvement (reduction of false positives) in active‐site identification by electrostatic strain.

Modeling based on the structure of vicilins predicts a histidine cluster in the active site of oxalate oxidase.

It is known that germin, which is a marker of the onset of growth in germinating wheat, is an oxalate oxidase, and also that germins possess sequence similarity with legumin and vicilin seed storage proteins. These two pieces of information have been combined in order to generate a 3D model of germin based on the structure of vicilin and to examine the model with regard to a potential oxalate oxidase active site. A cluster of three histidine residues has been located within the conserved β-barrel structure. While there is a relatively low level of overall sequence similarity between the model and the vicilin structures, the conservation of amino acids important in maintaining the scaffold of the β-barrel lends confidence to the juxtaposition of the histidine residues. The cluster is similar structurally to those found in copper amine oxidase and other proteins, leading to the suggestion that it defines a metal-binding location within the oxalate oxidase active site. It is also proposed that the structural elements involved in intermolecular interactions in vicilins may play a role in oligomer formation in germin/oxalate oxidase.

Mechanisms for stabilisation and the maintenance of solubility in proteins from thermophiles

BackgroundThe database of protein structures contains representatives from organisms with a range of growth temperatures. Various properties have been studied in a search for the molecular basis of protein adaptation to higher growth temperature. Charged groups have emerged as key distinguishing factors for proteins from thermophiles and mesophiles.ResultsA dataset of 291 thermophile-derived protein structures is compared with mesophile proteins. Calculations of electrostatic interactions support the importance of charges, but indicate that increases in charge contribution to folded state stabilisation do not generally correlate with the numbers of charged groups. Relative propensities of charged groups vary, such as the substitution of glutamic for aspartic acid sidechains. Calculations suggest an energetic basis, with less dehydration for longer sidechains. Most other properties studied show weak or insignificant separation of proteins from moderate thermophiles or hyperthermophiles and mesophiles, including an estimate of the difference in sidechain rotameric entropy upon protein folding. An exception is increased burial of alanine and proline residues and decreased burial of phenylalanine, methionine, tyrosine and tryptophan in hyperthermophile proteins compared to those from mesophiles.ConclusionSince an increase in the number of charged groups for hyperthermophile proteins is separable from charged group contribution to folded state stability, we hypothesise that charged group propensity is important in the context of protein solubility and the prevention of aggregation. Accordingly we find some separation between mesophile and hyperthermophile proteins when looking at the largest surface patch that does not contain a charged sidechain. With regard to our observation that aromatic sidechains are less buried in hyperthermophile proteins, further analysis indicates that the placement of some of these groups may facilitate the reduction of folding fluctuations in proteins of the higher growth temperature organisms.

Electrostatic field of the large fragment of Escherichia coli DNA polymerase I

The electrostatic field of the large fragment of Escherichia coli DNA polymerase I (Klenow fragment) has been calculated by the finite difference procedure on a 2 A grid. The potential field is substantially negative at physiological pH (reflecting the net negative charge at this pH). The largest regions of positive potential are in the deep crevice of the C-terminal domain, which is the proposed binding site for the DNA substrate. Within the crevice, the electrostatic potential has a partly helical form. If the DNA is positioned to fulfil stereochemical requirements, then the positive potential generally follows the major groove and (to a lesser extent) the negative potential is in the minor groove. Such an arrangement could stabilize DNA configurations related by screw symmetry. The histidine residues of the Klenow fragment give the positive field of the groove a sensitivity to relatively small pH changes around neutrality. We suggest that the histidine residues could change their ionization states in response to DNA binding, and that this effect could contribute to the protein-DNA binding energy.