Polyunsaturated fatty acids inhibit Kv1.4 by interacting with positively charged extracellular pore residues.

Polyunsaturated fatty acids (PUFAs) modulate voltage-gated K(+) channel inactivation by an unknown site and mechanism. The effects of ω-6 and ω-3 PUFAs were investigated on the heterologously expressed Kv1.4 channel. PUFAs inhibited wild-type Kv1.4 during repetitive pulsing as a result of slowing of recovery from inactivation. In a mutant Kv1.4 channel lacking N-type inactivation, PUFAs reversibly enhanced C-type inactivation (Kd, 15-43 µM). C-type inactivation was affected by extracellular H(+) and K(+) as well as PUFAs and there was an interaction among the three: the effect of PUFAs was reversed during acidosis and abolished on raising K(+) Replacement of two positively charged residues in the extracellular pore (H508 and K532) abolished the effects of the PUFAs (and extracellular H(+) and K(+)) on C-type inactivation but had no effect on the lipoelectric modulation of voltage sensor activation, suggesting two separable interaction sites/mechanisms of action of PUFAs. Charge calculations suggest that the acidic head group of the PUFAs raises the pKa of H508 and this reduces the K(+) occupancy of the selectivity filter, stabilizing the C-type inactivated state.

Specific ion and buffer effects on protein-protein interactions of a monoclonal antibody.

Better predictive ability of salt and buffer effects on protein-protein interactions requires separating out contributions due to ionic screening, protein charge neutralization by ion binding, and salting-in(out) behavior. We have carried out a systematic study by measuring protein-protein interactions for a monoclonal antibody over an ionic strength range of 25 to 525 mM at 4 pH values (5, 6.5, 8, and 9) in solutions containing sodium chloride, calcium chloride, sodium sulfate, or sodium thiocyante. The salt ions are chosen so as to represent a range of affinities for protein charged and noncharged groups. The results are compared to effects of various buffers including acetate, citrate, phosphate, histidine, succinate, or tris. In low ionic strength solutions, anion binding affinity is reflected by the ability to reduce protein-protein repulsion, which follows the order thiocyanate > sulfate > chloride. The sulfate specific effect is screened at the same ionic strength required to screen the pH dependence of protein-protein interactions indicating sulfate binding only neutralizes protein charged groups. Thiocyanate specific effects occur over a larger ionic strength range reflecting adsorption to charged and noncharged regions of the protein. The latter leads to salting-in behavior and, at low pH, a nonmonotonic interaction profile with respect to sodium thiocyanate concentration. The effects of thiocyanate can not be rationalized in terms of only neutralizing double layer forces indicating the presence of an additional short-ranged protein-protein attraction at moderate ionic strength. Conversely, buffer specific effects can be explained through a charge neutralization mechanism, where buffers with greater valency are more effective at reducing double layer forces at low pH. Citrate binding at pH 6.5 leads to protein charge inversion and the formation of attractive electrostatic interactions. Throughout the report, we highlight similarities in the measured protein-protein interaction profiles with previous studies of globular proteins and of antibodies providing evidence that the behavior will be common to other protein systems.

The role of electrostatics in protein-protein interactions of a monoclonal antibody.

Understanding how protein-protein interactions depend on the choice of buffer, salt, ionic strength, and pH is needed to have better control over protein solution behavior. Here, we have characterized the pH and ionic strength dependence of protein-protein interactions in terms of an interaction parameter kD obtained from dynamic light scattering and the osmotic second virial coefficient B22 measured by static light scattering. A simplified protein-protein interaction model based on a Baxter adhesive potential and an electric double layer force is used to separate out the contributions of longer-ranged electrostatic interactions from short-ranged attractive forces. The ionic strength dependence of protein-protein interactions for solutions at pH 6.5 and below can be accurately captured using a Deryaguin-Landau-Verwey-Overbeek (DLVO) potential to describe the double layer forces. In solutions at pH 9, attractive electrostatics occur over the ionic strength range of 5-275 mM. At intermediate pH values (7.25 to 8.5), there is a crossover effect characterized by a nonmonotonic ionic strength dependence of protein-protein interactions, which can be rationalized by the competing effects of long-ranged repulsive double layer forces at low ionic strength and a shorter ranged electrostatic attraction, which dominates above a critical ionic strength. The change of interactions from repulsive to attractive indicates a concomitant change in the angular dependence of protein-protein interaction from isotropic to anisotropic. In the second part of the paper, we show how the Baxter adhesive potential can be used to predict values of kD from fitting to B22 measurements, thus providing a molecular basis for the linear correlation between the two protein-protein interaction parameters.

Freezing and folding behavior in simple off-lattice heteropolymers.

We have performed parallel tempering Monte Carlo simulations using a simple continuum heteropolymer model for proteins. All 10 heteropolymer sequences which we have studied have shown first-order transitions at low temperature to ordered states dominated by single chain conformations. These results are in contrast with the theoretical predictions of the random energy model for heteropolymers, from which we would expect continuous transitions to glassy behavior at low temperatures.

Biochemical and genetic evidence for a family of heterotrimeric G-proteins in Trichomonas vaginalis.

We have cloned a single copy gene from the human parasite Trichomonas vaginalis that encodes a putative protein of 402 amino acids with approximately 35% sequence identity to known alpha subunits of heterotrimeric G-proteins. It contains the characteristic GTP binding domains G-1 to G-5 with the key residues conserved. The new sequence has an unusual N-terminal extension of approximately 70 residues that cannot be aligned to reference G-proteins and which is characterised by proline-rich repeats. To investigate the expression and cellular localisation of the protein we produced specific antisera against a recombinant fusion protein. The antisera recognised a protein of an apparent molecular mass of 51 kDa in protein extracts from T. vaginalis and immunofluorescent microscopy established that the protein is localised to discrete endomembranes. Using a protocol designed to purify mammalian heterotrimeric G-proteins incorporating a GTPgammaS binding assay, we isolated two proteins from Trichomonas that are recognised by an heterologous GA/1 antisera raised to a peptide of the conserved G-1 domain of G-protein alpha subunits. These two proteins have an apparent molecular mass of 61 and 48 kDa, respectively, larger and smaller than the translation product of the cloned gene. Consistent with these results, the GA/1 antisera did not cross-react with the fusion protein produced from the gene we have cloned. These data suggest T. vaginalis possesses more than one heterotrimeric G-protein alpha subunit. Based on the sequence features of the cloned gene and the biochemical properties of the purified proteins, we suggest that these alpha subunits are likely to be part of classic heterotrimeric G-protein complexes.

A second eIF4E protein in Schizosaccharomyces pombe has distinct eIF4G-binding properties.

The eukaryotic cap-binding proteins belonging to the eIF4E family are generally involved in mediating the recruitment of ribosomes to capped mRNA. We described previously a cap-binding protein (now called eIF4E1) in Schizosaccharomyces pombe that appears to have all of the usual structural and functional attributes of an eIF4E. We have now characterised a new type of cap-binding protein (eIF4E2) from this organism, which at the amino acid sequence level, is 52% identical and 59% similar to eIF4E1. eIF4E2 is not essential in S.pombe but has some novel properties that may be related to a special function in the cell. The ratio of eIF4E2:eIF4E1 in the cell shifts in favour of eIF4E2 at higher temperatures. Despite having all of the dorsal face amino acids that have so far been associated with eIF4G binding to eIF4E1, eIF4E2 binds the eIF4E-binding domain of S.pombe eIF4G >10(2)-times weaker than eIF4E1 in vitro. The eIF4E2 cap-binding affinity is in the typical micromolar range. The results suggest that eIF4E2 is not active on the main pathway of translation initiation in fission yeast but might play a role in the adaptation strategy of this organism under specific growth conditions. Moreover, they provide insight into the molecular characteristics required for tight binding to eIF4G.

Modelling of auxin-binding protein 1 suggests that its C-terminus and auxin could compete for a binding site that incorporates a metal ion and tryptophan residue 44.

Sequence comparison indicates that auxin-binding protein 1 (ABP1) belongs to a family of proteins with the core beta-barrel structure of the vicilins. Previous modelling within this family correctly predicted metal-ion binding and oligomeric properties of oxalate oxidase. ABP1 also contains a putative metal-ion-binding cluster of amino acids, adjacent to a tryptophan side chain, leading to a proposed auxin-binding site that incorporates metal-ion interaction with the auxin carboxylate. Modelling implicates W44 (Zea mays ABP1) in auxin binding, rather than W136 or W151. Reduced sequence similarity for the C-terminal region prevents model building. It is proposed that one of these C-terminal tryptophans, along with a neighbouring negatively charged side chain, occupies the binding pocket in the absence of auxin, thereby linking auxin binding to conformational change and C-terminal involvement in signalling.

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, Thr(46) and Ser(65), consistently have the most significant effects, and that phosphorylation of Ser(65) 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.

Modeling a prion protein dimer: predictions for fibril formation.

Models of structural transition in prion protein (PrP) focus on the domain visualised by solution NMR. Accumulating evidence suggests that the adjacent and highly conserved nonpolar segment, as well as PrP-membrane interactions, should also be considered. Calculations predict that membrane-induced structural destabilisation is mediated by stabilisation of the unfolded form. Comparative analysis of PrP structures leads to a model for PrP dimerisation that incorporates the nonpolar segment. A prediction that PrP will interact with the PrP-like protein (Dpl) to form a heterodimer, but that Dpl will not form a homodimer, can be tested. Modelling is discussed in the context of ataxias associated with the expression of Dpl or truncated PrP in transgenic animals lacking wild-type PrP. A PrP(C) dimer model forms the basis for considering the geometry of PrP(Sc) fibril formation.

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.

Modelling charge interactions in the prion protein: predictions for pathogenesis.

Calculations are presented for the pH-dependence of stability and membrane charge complementarity of prion protein fragments. The theoretical results are compared with reported characterisations of prion protein folding in vitro. Discussion of models for conformational change and pathogenesis in vivo leads to the prediction of amino acids that could mediate sensitivity to the endosomal pH and to a design strategy for recombinant prion proteins with an increased susceptibility to prion proteinSc-like properties in vitro. In this model, the protective effect of certain basic polymorphisms can be interpreted in terms of oligomerisation on a negatively-charged surface.

Simplified methods for pKa and acid pH-dependent stability estimation in proteins: removing dielectric and counterion boundaries.

Much computational research aimed at understanding ionizable group interactions in proteins has focused on numerical solutions of the Poisson-Boltzmann (PB) equation, incorporating protein exclusion zones for solvent and counterions in a continuum model. Poor agreement with measured pKas and pH-dependent stabilities for a (protein, solvent) relative dielectric boundary of (4,80) has lead to the adoption of an intermediate (20,80) boundary. It is now shown that a simple Debye-Huckel (DH) calculation, removing both the low dielectric and counterion exclusion regions associated with protein, is equally effective in general pKa calculations. However, a broad-based discrepancy to measured pH-dependent stabilities is maintained in the absence of ionizable group interactions in the unfolded state. A simple model is introduced for these interactions, with a significantly improved match to experiment that suggests a potential utility in predicting and analyzing the acid pH-dependence of protein stability. The methods are applied to the relative pH-dependent stabilities of the pore-forming domains of colicins A and N. The results relate generally to the well-known preponderance of surface ionizable groups with solvent-mediated interactions. Although numerical PB solutions do not currently have a significant advantage for overall pKa estimations, development based on consideration of microscopic solvation energetics in tandem with the continuum model could combine the large deltapKas of a subset of ionizable groups with the overall robustness of the DH model.

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 beta-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 beta-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.

Modeling charge interactions and redox properties in DsbA.

Accurate prediction of charge interactions in macromolecules presents a significant challenge for computational biology. A model for the low Cys30 pKa and oxidizing power of DsbA (Gane, P. J., Freedman, R. B., and Warwicker, J. (1995) J. Mol. Biol. 249, 376-387) has been investigated experimentally (Hennecke, J., Spleiss, C., and Glockshuber, R. (1997) J. Biol. Chem. 272, 189-195), with substitutions for Glu37 and Glu38 and with residues 38-40 removed. Measured changes in Cys30 pKa and redox potential were relatively small and reported to be in contrast to model predictions. It is now shown, particularly with calculations of wild-type:mutant differences for a range of salt concentrations, that the data are consistent with the model and support the key finding that a number of different factors contribute to the oxidizing power of DsbA, so that any particular one need not necessarily be large. A feature of the model is a low protein dielectric, and higher values (which are becoming popular in predictions of pH dependence) are inconsistent with both the difference data and the wild-type Cys30 pKa.

A hypothesis describing a potential link between molecular structure and TSE strains.

In considering a protein-only model for prion pathogenesis in TSEs, one key challenge is to explain the existence of strains. These have traditionally been characterised by neuropathology and incubation times and more recently through biochemical analysis of prion protein (PrP), which shows differences in protease-resistant fragment size and glycoform ratios. It is now suggested that PrP possesses two faces which on the basis of conservation and non-polar nature could each (physiologically) interact either with membrane or with neighbouring protein. This model leads to the construction of two clearly different membrane-attached PrP orientations, with consequences for protease resistance and glycoform incorporation that qualitatively match to experiment.

Improving pKa calculations with consideration of hydration entropy.

Continuum dielectric modelling of electrostatics interactions in macromolecules provides a valuable tool in the study of structure-function relationships, but falls short of providing consistently accurate calculated pKas. It is suggested that the model can be significantly improved with the inclusion of a term that estimates the entropy associated with first hydration shell solvent ordering, with reference to computed results for cysteines in DsbA and thioredoxin, and aspartic and glutamic acids in a number of proteins. The modification is based on the geometry of charge burial and an hydration number, which is adjustable (by fit to experiment), and is uniform within each class of ionizable group studied. The potential for further development is clear within this framework, since experiment and simulation can furnish non-adjustable, ionizable group-specific, hydration numbers.

A molecular model for interfacial activation in phospholipase A2.

Electrostatic calculations predict that amino-terminal conformation and ionisation contribute significantly to transition state stability in phospholipase A2, so that control of these factors by binding to aggregated substrate provides a plausible mechanism for interfacial activation. In particular, it is suggested that a part of the pH dependence of interfacial activity may arise from transient deprotonation of an ordered amino-terminus. Interface charge and the detailed structure of the interfacial complex are also predicted to influence catalytic activity. The model is compared with available biochemical data.

Species barriers in a model for specific prion protein dimerisation.

It has been proposed that the most highly conserved sequence segment within the prion protein (PrP) may be involved in dimer formation within both the normal (PrPC) and misfolded (PrPSc) forms. This hypothesis is now examined in the context of amino acids known to be involved in species barriers or in disease modifying polymorphisms, and the structure of a mouse PrP fragment. These locations can be plausibly explained on the basis of the specific dimer model, so that a potential role for a conserved dimerisation element in prion disease progression cannot be excluded.

A model for prion protein dimerisation based on alpha-helical packing.

Residues 109-122 of the human prion protein (PrP) are highly conserved across species, and are predicted to be alpha-helical in PrPc, the cellular form. A computational search of the potential for alpha-helical dimerisation has been made for residues 109-122. The conformation which consistently scores highest in terms of burying non-polar surface area is a tight association involving alanine, glycine and valine residues. A model of heterodimerisation for PrPc and PrPSc (the misfolded form) is presented in which species barrier mutations would arise from interaction specificities that would follow, at least in part, the same framework as formation of a putative homodimer.

Calculation of Cys 30 delta pKa's and oxidising power for DsbA mutants.

DsbA possesses a redox active disulphide, with the equilibrium strongly shifted towards the reduced form as compared to its structural homologue, thioredoxin. It is widely believed that the two amino acids that separate the active site cysteines play a crucial role in determining oxidising power within the thioredoxin family. Data concerning redox and pKa properties for DsbA mutants in this region are available. Electrostatics calculations show reasonable agreement with the experimental data, and support the suggestion that amino acids outside of the CXXC active site sequence are as important in determining oxidising power within the thioredoxin family as are those within it.