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Jim Warwicker

Publications and source records attributed to Jim Warwicker.

14 recordsLinked to original sources
PubMed2006

Subcellular pH and predicted pH-dependent features of proteins.

A characteristic of two-dimensional proteomics gels is a general bimodal distribution of isoelectric (pI) values. Discussion of this feature has focussed on the balance of acidic and basic ionisable residues, and potential relationships between pI distributions and organism classification or protein subcellular location. Electrostatics calculations on a set of protein structures with known subcellular location show that predicted folded state pI are similar to those calculated from sequence alone, but adjusted according to a general stabilising effect from interactions between ionisable groups. Bimodal distributions dominate both pI and the predicted pH of maximal stability. However, there are significant differences between these features. The average pH of maximal stability generally follows organelle pH. Average pI values are well removed from organelle pH in most subcellular environments, consistent with the view that proteins have evolved to carry (on average) net charge in a given subcellular location, and relevant to discussion of solubility in crowded environments. Correlation of the predicted pH of maximum stability with subcellular pH suggests an evolutionary pressure to adjust folded state interactions according to environment. Finally, our analysis of ionisable group contributions to stability suggests that Golgi proteins have the largest such term, although this dataset is small.

Cell Nucleus↗
PubMed2006

The CXXC motif at the N terminus of an alpha-helical peptide.

An active site containing a CXXC motif is always found in the thiol-disulphide oxidoreductase superfamily. A survey of crystal structures revealed that the CXXC motif had a very high local propensity (26.3 +/- 6.2) for the N termini of alpha-helices. A helical peptide with the sequence CAAC at the N terminus was synthesized to examine the helix-stabilizing capacity of the CXXC motif. Circular dichroism was used to confirm the helical nature of the peptide and study behavior under titration with various species. With DTT, a redox potential of E(o) = -230 mV was measured, indicating that the isolated peptide is reducing in nature and similar to native human thioredoxin. The pK(a) values of the individual Cys residues could not be separated in the titration of the reduced state, giving a single transition with an apparent pK(a) of 6.74 (+/-0.06). In the oxidized state, the N-terminal pK(a) is 5.96 (+/-0.05). Analysis of results with the modified helix-coil theory indicated that the disulfide bond stabilized the alpha-helical structure by 0.5 kcal/mol. Reducing the disulfide destabilizes the helix by 0.9 kcal/mol.

Amino Acid Motifs↗
PubMed2005

Simulation of non-specific protein-mRNA interactions.

Protein-nucleic acid interactions exhibit varying degrees of specificity. Relatively high affinity, sequence-specific interactions, can be studied with structure determination, but lower affinity, non-specific interactions are also of biological importance. We report simulations that predict the population of nucleic acid paths around protein surfaces, and give binding constant differences for changes in the protein scaffold. The method is applied to the non-specific component of interactions between eIF4Es and messenger RNAs that are bound tightly at the cap site. Adding a fragment of eIF4G to the system changes both the population of mRNA paths and the protein-mRNA binding affinity, suggesting a potential role for non-specific interactions in modulating translational properties. Generally, the free energy simulation technique could work in harness with characterized tethering points to extend analysis of nucleic acid conformation, and its modulation by protein scaffolds.

Computer Simulation↗
PubMed2005

Anomalies in the ionic properties of serum albumin.

The documented difference in the isoionic points of native and unfolded serum albumins is revisited with computational methods. Good agreement between calculated and measured DeltapIs is found, with the molecular origin appearing to reside in a diverse set of carboxyl group titrations. Although histidine ionization plays only a minor role in bringing about the DeltapI, the identification of three histidine residues with low computed pK(a)s (around pH 5) suggests an explanation for the mismatch between experimentally derived group pK(a)s and the pI of the native protein. Computed electrostatic properties (including pI) of native serum albumins are compared with a set of 178 nonenzyme proteins. We find that the degree of interdigitation of positive and negative charges is extreme for serum albumin, and discuss the relationship to protein solubility and pH-dependent structural transitions.

Animals↗
PubMed2005

Active site identification through geometry-based and sequence profile-based calculations: burial of catalytic clefts.

Electrostatics calculations with proteins that are uniformly charged over volume can aid enzyme/non-enzyme discrimination. For known enzymes, such methods locate active sites to within 5% on the enzyme surface, in 77% of a test set. We now report that removing the dielectric boundary improves active site location to 80%, with optimal discrimination between enzymes and non-enzymes of around 80% specificity and 80% sensitivity. This calculation quantifies burial of solvent-accessible regions. Many of the true enzymes incorrectly assigned as non-enzymes have active sites at subunit boundaries. These are missed in monomer-based calculations. Catalytic and non-catalytic antibodies are studied in this context of active/binding site burial. Whilst catalytic antibodies, on average, have marginally higher active site burial than non-catalytic antibodies, these values are generally smaller than for non-antibody enzymes, possibly contributing to their relatively low turnover. Prediction of active site location improves further when sequence profile-based weights replace the uniform charge distribution, so that a combination of burial and amino acid conservation is assessed. Accuracy rises to 93% of active sites to within 5%, in the test set, for the optimal profile weights scheme. The equivalent value in a separate validation set is 89% to within 5%. Enzyme/non-enzyme and enzyme functional site predictions are made for structural genomics proteins, suggesting that a substantial majority of these are non-enzymes.

Antibodies↗
PubMed2005

Identification of a very large Rab GTPase family in the parasitic protozoan Trichomonas vaginalis.

Rab proteins are pivotal components of the membrane trafficking machinery in all eukaryotes. Distinct Rab proteins locate to specific endomembrane compartments and genomic studies suggest that Rab gene diversity correlates with endomembrane system complexity; for example unicellular organisms generally possess 5-20 Rab family members and the size of the repertoire increases to 25-60 in multicellular systems. Here we report 65 open reading frames from the unicellular protozoan Trichomonas vaginalis that encode distinct Rab proteins (TvRabs), indicating a family with complexity that rivals Homo sapiens in number. The detection of gene transcripts for the majority of these genes and conservation of functional motifs strongly suggests that TvRabs retain functionality and likely roles in membrane trafficking. The T. vaginalis Rab family includes orthologues of the conserved subfamilies, Rab1, Rab5, Rab6, Rab7 and Rab11, but the majority of TvRabs are not represented by orthologues in other systems and includes six novel T. vaginalis specific Rab subfamilies (A-F). The extreme size of the T. vaginalis Rab family, the presence of novel subfamilies plus the divergent nature of many TvRab sequences suggest both the presence of a highly complex endomembrane system within Trichomonas and potentially novel Rab functionality. A family of more than 65 Rab genes in a unicellular genome is unexpected, but may be a requirement for progression though an amoeboid life-cycle phase as both Dictyostelium discoideum and Entamoeba histolytica share with T. vaginalis both an amoeboid life cycle stage and very large Rab gene families.

Amino Acid Sequence↗
PubMed2004

Prediction of pKa and redox properties in the thioredoxin superfamily.

Electrostatic interactions play important roles in diverse biological phenomena controlling the function of many proteins. Polar molecules can be studied with the FDPB method solving the Poisson-Boltzmann equation on a finite difference grid. A method for the prediction of pK(a)s and redox potentials in the thioredoxin superfamily is introduced. The results are compared with experimental pK(a) data where available, and predictions are made for members lacking such data. Studying CxxC motif variation in the context of different background structures permits analysis of contributions to cysteine DeltapK(a)s. The motif itself and the overall framework regulate pK(a) variation. The reported method includes generation of multiple side-chain rotamers for the CxxC motif and is an effective predictive tool for functional pK(a) variation across the superfamily. Redox potential follows the trend in cysteine pK(a) variation, but some residual discrepancy indicates that a pH-independent factor plays a role in determining redox potentials for at least some members of the superfamily. A possible molecular basis for this feature is discussed.

Amino Acid Motifs↗
PubMed2004

Enzyme/non-enzyme discrimination and prediction of enzyme active site location using charge-based methods.

Calculations of charge interactions complement analysis of a characterised active site, rationalising pH-dependence of activity and transition state stabilisation. Prediction of active site location through large DeltapK(a)s or electrostatic strain is relevant for structural genomics. We report a study of ionisable groups in a set of 20 enzymes, finding that false positives obscure predictive potential. In a larger set of 156 enzymes, peaks in solvent-space electrostatic properties are calculated. Both electric field and potential match well to active site location. The best correlation is found with electrostatic potential calculated from uniform charge density over enzyme volume, rather than from assignment of a standard atom-specific charge set. Studying a shell around each molecule, for 77% of enzymes the potential peak is within that 5% of the shell closest to the active site centre, and 86% within 10%. Active site identification by largest cleft, also with projection onto a shell, gives 58% of enzymes for which the centre of the largest cleft lies within 5% of the active site, and 70% within 10%. Dielectric boundary conditions emphasise clefts in the uniform charge density method, which is suited to recognition of binding pockets embedded within larger clefts. The variation of peak potential with distance from active site, and comparison between enzyme and non-enzyme sets, gives an optimal threshold distinguishing enzyme from non-enzyme. We find that 87% of the enzyme set exceeds the threshold as compared to 29% of the non-enzyme set. Enzyme/non-enzyme homologues, "structural genomics" annotated proteins and catalytic/non-catalytic RNAs are studied in this context.

Binding Sites↗
PubMed2004

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

Ionizable groups play critical roles in biological processes. Computation of pK(a)s is complicated by model approximations and multiple conformations. Calculated and experimental pK(a)s 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 pK(a)s 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 pK(a) 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.

Amino Acids↗
PubMed2002

Delineation of RAID1, the RACK1 interaction domain located within the unique N-terminal region of the cAMP-specific phosphodiesterase, PDE4D5.

BACKGROUND: The cyclic AMP specific phosphodiesterase, PDE4D5 interacts with the beta-propeller protein RACK1 to form a signaling scaffold complex in cells. Two-hybrid analysis of truncation and mutant constructs of the unique N-terminal region of the cAMP-specific phosphodiesterase, PDE4D5 were used to define a domain conferring interaction with the signaling scaffold protein, RACK1. RESULTS: Truncation and mutagenesis approaches showed that the RACK1-interacting domain on PDE4D5 comprised a cluster of residues provided by Asn-22/Pro-23/Trp-24/Asn-26 together with a series of hydrophobic amino acids, namely Leu-29, Val-30, Leu-33, Leu-37 and Leu-38 in a 'Leu-Xaa-Xaa-Xaa-Leu' repeat. This was done by 2-hybrid analyses and then confirmed in biochemical pull down analyses using GST-RACK1 and mutant PDE4D5 forms expressed in COS cells. Mutation of Arg-34, to alanine, in PDE4D5 attenuated its interaction with RACK1 both in 2-hybrid screens and in pull down analyses. A 38-mer peptide, whose sequence reflected residues 12 through 49 of PDE4D5, bound to RACK1 with similar affinity to native PDE4D5 itself (Ka circa 6 nM). CONCLUSIONS: The RACK1 Interaction Domain on PDE4D5, that we here call RAID1, is proposed to form an amphipathic helical structure that we suggest may interact with the C-terminal beta-propeller blades of RACK1 in a manner akin to the interaction of the helical G-gamma signal transducing protein with the beta-propeller protein, G-beta.

Adaptor Proteins, Signal Transducing↗
PubMed2002

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, Trp(19) and Trp(20), whose orientation is optimized for bilayer insertion by the Leu(16):Val(17) pairing. Ca(2+), at submicromolar levels, interacts with Asp(21) 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 (Asp(21-):Ca(2+):PA(2-):Lys(24+)) with overall neutrality at the bilayer surface. This novel phospholipid-binding domain, which we call TAPAS-1 (tryptophan anchoring phosphatidic acid 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.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2002

Effect of phosphorylation on alpha-helix stability as a function of position.

We have investigated the effect of placing phosphoserine at the N-cap, N1, N2, N3, and interior position in alanine-based alpha-helical peptides. Helix contents of each peptide were measured by CD spectroscopy and titrations performed to determine pK(a) values. Data were analyzed with modified Lifson-Roig theory to determine helix-coil parameters (n, n(1), n(2), n(3), and w) and free energy changes for phosphoserine at each helical position. Results are given for a -1 and -2 phosphoserine charge state. Results show that phosphoserine stabilizes at the N-terminal positions by as much as 2.3 kcal.mol(-1), while destabilizes in the helix interior by 1.2 kcal.mol(-1), relative to serine. The rank order of free energies relative to serine at each position is N2 > N3 > N1 > N-cap > interior. Moreover, -2 phosphoserine is the most preferred residue known at each of these N-terminal positions. Experimental pK(a) values for the -1 to -2 phosphoserine transition are in the order N2 < N-cap < N1 < N3 < interior. This order agrees well with electrostatics calculations carried out with phosphoserine at the N-terminal positions and interior positions. Combining these with calculations at the C3, C2, C1, and C-cap positions gives results for phosphoserine along the length of the helix. We see a transition from phosphoserine stabilization at the N-terminus to destabilization at the C-terminus and can explain this in terms of the balance of protein solvation, favorable interactions, and dehydration. These results give insight into the phosphorylatable control of biological systems through positive or negative changes in stability.

Amino Acid Sequence↗
PubMed2002

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.

Binding Sites↗