Protein Charge Distribution Research Articles

Application of the Poisson-Nernst-Planck Theory with Space-Dependent Diffusion Coefficients to KcsA

The Poisson-Nernst-Planck electrodiffusion theory serves to compute charge fluxes and is here applied to the ion current through a protein channel. KcsA was selected as an example because of the abundance of experimental and theoretical data. The potassium channels MthK and KvAP were used as templates to define two open channel models for KcsA. Channel boundary surfaces and protein charge distributions were defined according to atomic radii and partial atomic charges. To establish the sensitivity of the results to these parameters, two different sets were used. Assigning the potassium diffusion coefficients equal to the value for free-diffusion in water (1.96 × 10 −9 m 2/s), the computed currents overestimated the experimental data. Ion distributions inside the channel suggest that the overestimate is not due to an excess of charge shielding. A good agreement with the experimental data was achieved by reducing the potassium diffusion coefficient inside the channel to 1.96 × 10 −10 m 2/s, a value of substantial motility but nonetheless in accord with the intuitive notion that the channel has a high affinity for the ions and therefore slows them down. These results are independent of the open channel model and the parameterization adopted for atomic radii and partial atomic charges. The method offers a reliable estimate of the channel current with low computational effort.

Use of polysaccharides to control protein adsorption to the air–water interface

In order to understand foaming behaviour of mixed protein/anionic polysaccharide solutions, we investigated the effect of β-lactoglobulin/pectin interaction in the bulk on β-lactoglobulin adsorption to the air–water interface. Adsorption kinetics were evaluated by following surface pressure development in time of several pure protein solutions and of mixed protein/polysaccharide solutions using an automated drop tensiometer (ADT). It was found that complexation of proteins with polysaccharides can slow down the kinetics of surface pressure development by at least a factor 100, and greatly diminish foam formation. In contrast, a five times acceleration in the increase of surface pressure was observed in other cases. We propose a mechanism for protein adsorption from mixed protein/polysaccharide solutions. Effects of ionic strength, pH and mixing ratio on this mechanism were studied for mixtures of β-lactoglobulin and low methoxyl pectin, whereas other proteins and anionic polysaccharides were used to explore the role of protein and polysaccharide charge density and distribution. Whereas the possibilities to change system parameters like ionic strength or pH are limited in food related systems, selecting a suitable combination of protein and polysaccharide offers a broad opportunity to control protein adsorption kinetics and with that foam formation.

A quantum-mechanical model for the determination of internal electric fields at protein active sites from the Stark effect on persistent spectral holes

The Stark effect of spectral holes burnt in protoporphyrin IX (H 2-PPIX) substituted myoglobin is employed to determine quantitatively the internal electric fields generated by protein charge distribution at the H 2-PPIX site. The procedure for extracting the internal field in molecular resolution from the measured hole profiles involves the numerical diagonalization of the H 2-PPIX Hamiltonian. The resulting values for the internal fields depend on the number of electronic states used in the diagonalization. In this paper, we extend earlier work by carrying out the diagonalization by successively adding more electronic states based on their energy in order to determine the convergence behavior of the internal-field magnitudes. We show that a different sequence of adding states improves the convergence behavior of the internal fields.

Improved 3D continuum calculations of ion flux through membrane channels.

A continuum model, based on the Poisson-Nernst-Planck (PNP) theory, is applied to simulate steady-state ion flux through protein channels. The PNP equations are modified to explicitly account (1) for the desolvation of mobile ions in the membrane pore and (2) for effects related to ion sizes. The proposed algorithm for a three-dimensional self-consistent solution of PNP equations, in which final results are refined by a focusing technique, is shown to be suitable for arbitrary channel geometry and arbitrary protein charge distribution. The role of the pore shape and protein charge distribution in formation of basic electrodiffusion properties, such as channel conductivity and selectivity, as well as concentration distributions of mobile ions in the pore region, are illustrated by simulations on model channels. The influence of the ionic strength in the bulk solution and of the externally applied electric field on channel properties are also discussed.

Profiling Charge Complementarity and Selectivity for Binding at the Protein Surface

A novel analysis and representation of the protein surface in terms of electrostatic binding complementarity and selectivity is presented. The charge optimization methodology is applied in a probe-based approach that simulates the binding process to the target protein. The molecular surface is color coded according to calculated optimal charge or according to charge selectivity, i.e., the binding cost of deviating from the optimal charge. The optimal charge profile depends on both the protein shape and charge distribution whereas the charge selectivity profile depends only on protein shape. High selectivity is concentrated in well-shaped concave pockets, whereas solvent-exposed convex regions are not charge selective. This suggests the synergy of charge and shape selectivity hot spots toward molecular selection and recognition, as well as the asymmetry of charge selectivity at the binding interface of biomolecular systems. The charge complementarity and selectivity profiles map relevant electrostatic properties in a readily interpretable way and encode information that is quite different from that visualized in the standard electrostatic potential map of unbound proteins.

Metal-dependent Folding and Stability of Nuclear Hormone Receptor DNA-binding Domains

The nuclear/hormone receptors are an extensive family of ligand-activated transcription factors that recognise DNA targets through a highly conserved, structurally autonomous DNA-binding domain. The compact structure of the DNA-binding domain is supported by two zinc ions, each of which is co-ordinated by the tetrahedral arrangement of thiol groups from four cysteine residues. Metal binding is expected to be linked with deprotonation of the co-ordinating thiol groups and folding of the polypeptide. Using a variety of biophysical approaches, we characterise these linked equilibria for the isolated DNA-binding domains (DBD) of the receptors for estrogen and glucocorticoid. Mass spectrometry and equilibrium denaturation indicate that, near neutral pH, approximately four of the eight co-ordinating thiol groups release protons with zinc uptake, in agreement with the expected p K a change for the –SH group in the presence of the metal. Mass spectrometry reveals that the protein charge distribution changes with the uptake of zinc and that metal binding is co-operative. The co-operativity is consistent with observations from equilibrium denaturation, which indicate that the folding event is a two-state process. A crucial residue that stabilises the equilibrium structure of the DBD fold itself is a cysteine residue situated in the hydrophobic core of all known nuclear hormone receptors (but not involved in metal binding): it appears to be conserved absolutely for its unique combination of size and hydrophobicity. Stabilisation of the DBDs could be achieved by truncating the flexible, basic termini, suggesting that like-charge clusters may have deleterious effects on protein folds. While the metal-free apo protein and the chemically denatured state have little defined secondary structure, these states were expanded only partially in comparison with the native structure, according to data from small-angle X-ray scattering. The comparatively compact shapes of the denatured and apo forms may explain, in part, the marginal stability of the native fold.

Transitions in Chain Entanglement and Compactness Associated with in Vacuo Unfolding of Lysozyme Ions

Chain entanglement and compactness are two a priori independent properties that convey large-scale shape features of polymer conformations. In this work, we use these properties to monitor the initial step for the in vacuo unfolding of charged lysozyme ions. Using molecular dynamics simulations and a statistical model for the protein charge distribution, we show the existence of a narrow range of total charge within which compact (quasinative) and extended (partly unfolded) conformers can coexist. Denatured conformers are always found above the critical charge for unfolding, whereas quasinative structures are associated with low-charge states. We find that the global molecular shape of the accessible conformers is conserved over a range of temperatures, despite a shift in the critical charge as temperature increases. Within our model, in vacuo unfolding occurs for lysozyme ions with charges +7 and +8, when a thermal bath of T = 500 K is considered. This result is compatible with gas-phase experiments on lysozyme and suggests that unfolding results from the combined effect of heating and the Coulomb repulsion between charged residues.

1P171タンパク質の電荷分布から見た生物ゲノム比較

1P171タンパク質の電荷分布から見た生物ゲノム比較

Comparative Analysis of Protein Charge Distribution in Genome Scale

Biological cells are living in various environments: Halobacteria are in saturated salt solution and thermophilic bacteria are living at high temperature, while the environment of eukaryotic cells is kept constant by homeostasis. Therefore, each biological species is under the evolutionary pressure from its environment. It is well known that the amino acid sequence of each protein determines the 3D-structure, which is related to its biological function. For example, the amino acid sequence of membrane proteins have the specific hydrophobic distribution compared to soluble proteins and can be predicted [1]. The fraction of ORFs coding membrane proteins is 25% of total ORFs on whole genomes of 15 unicellular organisms [2]. Environments of living cells may not affect membrane proteins because this kind of protein is not subjected to cytoplasm. The environmental effect of all of ORFs on whole genomes has not been studied so far. In this work, we analyzed the electric net charge of total genomes for comparing the charge distribution among various biological organisms. Because the surface of proteins in aqueous solvent is mainly comprised of polar residues, the charge distribution of proteins has to be a good parameter to monitor the effect of environment on the physicochemical properties of proteins.

Effects of Surface Charge Distribution of Proteins in Their Complexation with Polyelectrolytes in an Aqueous Salt-Free System

Complexation between protein and polyelectrolyte was studied by a combination of three main experimental techniques: turbidimetric titration, quasi-elastic light scattering (QELS), and static light scattering (SLS). Proteins of two sorts, lysozyme (Lyz) and ribonuclease (RNase), were chosen by considering the following characteristics: (i) Both proteins have the same number (19) of basic groups; (ii) their distribution is almost homogeneous on Lyz but not on RNase; (iii) there is little difference in the molar mass between both proteins. Potassium poly(vinyl alcohol) sulfate (KPVS) with different molecular weights (MPE) and various degrees of esterification (De) was used as the polyelectrolyte. We employed a salt-free aqueous medium and adjusted it to pH 2, the level of which forces to completion the protonation of all of the basic groups. As the titration of proteins with KPVS proceeded, the absorbance (A) as an indication of turbidity increased linearly and then rapidly at a certain titrant volume, r...

Linear scaling molecular orbital calculations of biological systems using the semiempirical divide and conquer method

A “linear-scaling revolution” is occurring in quantum chemistry. This development is allowing for the first time the routine examination of large molecular assembles (e.g., proteins and DNA in water) using electronic structure methods. One of these approaches is the divide and conquer method and, in this article, we review the implementation of this approach for semiempirical Hamiltonians. This is then followed by brief reviews of three application areas. First, we will discuss the charge distribution of biological molecules in solution as described by quantum mechanics. In particular, the role polarization and charge transfer plays in affecting the charge distribution of proteins will be discussed. Next, we will examine the energetic consequences of charge transfer and polarization on biomolecular solvation. The final section will describe the computation of solvation free energies using a combined divide and conquer/Poisson–Boltzmann approach. The application of linear scaling quantum mechanical methods to biology is only just beginning, but the future is very bright, and it is our opinion that quantum mechanics will have a profound influence on our understanding of biological systems in the coming years. © 2000 John Wiley & Sons, Inc. J Comput Chem 21: 1494–1504, 2000

Influence of Structural Details in Modeling Electrostatically Driven Protein Adsorption

Mechanistic modeling of protein adsorption has evolved to include increasingly detailed descriptions of protein structure in an effort to capture experimentally observed behavior. This has been especially true of electrostatically driven adsorption, for which colloidal models have been used frequently. These efforts have focused on adsorption of proteins to oppositely charged surfaces and often capture the experimental trends even with gross simplification of protein structure. As a more stringent test of model sensitivity to structural details, we have modeled the patch-controlled adsorption of basic proteins on anion-exchange surfaces, where a small number of negative charges on the protein surface lead to a net attraction between the net positively charged protein and the positively charged surface. We account in detail for the protein shape and charge distribution and examine the role of the assumed surface description. A model assuming a uniformly charged surface is unable to predict electrostaticall...

Effect of a Protein Electric Field on the CO Stretch Frequency. Finite Difference Poisson−Boltzmann Calculations on Carbonmonoxycytochromesc

We report electrostatic potential and electric field calculations at the CO ligand of the heme in both horse heart and yeast cytochrome c, obtained from a finite difference solution to the Poisson−Boltzmann equation. This method takes into account the protein shape and charge distribution, as well as the solvent and generalized ionic strength effects. The calculations support recent experimental and theoretical evidence suggesting that polar interactions can significantly affect the vibrational frequency of the ligand. Our work shows that the ionizable amino acid residues and polar contributions of the protein matrix can induce a Stark effect on the CO stretch frequency of the carbonmonoxycytochromes. The observed CO stretches, at 1965.9 and 1960.1 cm-1 for horse and yeast cytochromes c, respectively, show that the experimental shift is of the order of 6 cm-1. This is in good agreement with a calculated value of 8 cm-1 (±1 cm-1) for a vibrational Stark shift due to the different charge distributions in bo...

Computation of the electrophoretic mobility of proteins

A scheme is presented for computing the electrophoretic mobility of proteins in free solution, accounting for the details of the protein shape and charge distribution. The method of Teubner is implemented using a boundary integral formulation within which the velocity distribution, the equilibrium electrical potential around the molecule, and the potential distribution due to the applied field are solved for numerically using the boundary element method. Good agreement of the numerical result is obtained for spheres with the corresponding semi-analytical specialization of Henry's analysis. For protein systems, the method is applied to lysozyme and ribonuclease A. In both cases, the predicted mobility tensors are fairly isotropic, with the resulting scalar mobilities being significantly smaller than for spheres of equal volume and net charge. Comparisons with previously published experimental results for ribonuclease show agreement to be excellent in the presence of a net charge, but poorer at the point of zero charge. The approach may be useful for evaluating approximate methods for estimating protein electrophoretic mobilities and for using electrophoretic measurements to obtain insight into charge distributions on proteins.

Convergent evolution of amino acid usage in archaebacterial and eubacterial lineages adapted to high salt

Chemical composition and physical properties of the total protein of Haloferax mediterranei, a halophilic archaebacterium requiring high salt concentration for growth, of Halomonas elongata, a halotolerant eubacterium able to grow at any concentration of salt, and of Escherichia coli B, a eubacterium related to H. elongata, unable to grow at high salt concentration, were compared using robust standard biochemical methods. The distribution of amino acid abundancies in the bulk protein from H. elongata was found to be intermediate between that from H. mediterranei and that from E. coli. The two high-salt-adapted organisms displayed an enrichment in aspartic acid and glutamic acid together with an impoverishment in lysine as compared to E. coli. This signature in amino acid usage is reflected in the charge distribution of proteins, as revealed by anion exchange chromatography of crude cell extracts. Since H. elongata diverged from H. mediterranei more than three billion years ago, the resemblance of their amino acid usages can be interpreted as a convergent imprint of their common habitats onto the chemical constitution of their proteins.

Protein transfer from an aqueous phase into reversed micelles. The effect of protein size and charge distribution.

Proteins are spontaneously transferred from an aqueous solution into reversed micelles, provided the aqueous phase has the proper composition. Besides the composition of the aqueous phase, the composition of the organic phase and the properties of the proteins also play a role. We studied uptake profiles of 19 proteins as a function of pH of the aqueous solution. The organic phase consisted of trioctylmethylammonium chloride and nonylphenol pentaethoxylate (Rewopal HV5) as surfactant, octanol as cosurfactant and isooctane as continuous phase. In all cases, except for rubredoxin, proteins were transferred at pH values above their isoelectric point. The pH where maximal solubilization takes place can be described by the relationship: pHoptimum = isoelectric point +0.11 x 10(-3) Mr -0.97. So, the larger the protein, the more charge is needed to provide the energy required for the adaptation of the micellar size to the protein size. For protein transfer into sodium di-(2-ethylhexyl)sulphosuccinate (AOT) reversed micelles a similar relationship was found. The percentage of protein transferred could be related to the symmetry of charge distribution over the protein. This symmetry was expressed as the % of random electric moments on a protein that is larger than the effective electric moment of the protein (% S) [Barlow, D. J. and Thornton, J. M. (1986) Biopolymers 25, 1717]. The larger the value of % S, the more homogeneously the charges are distributed and the lower the percentage transfer.

Laser flash photolysis studies of the kinetics of reduction of spinach and Clostridium ferredoxins by a viologen analogue: electrostatically controlled nonproductive complex formation and differential reactivity among the iron-sulfur clusters.

We have studied the transient kinetics of electron transfer from a positively charged viologen analogue (propylene diquat), reduced by pulsed laser excitation of the deazariboflavin/EDTA system, to the net negatively charged ferredoxins from spinach and Clostridium pasteurianum. Spinach ferredoxin showed monophasic kinetics over the ionic strength range studied, consistent with the presence of only a single iron-sulfur center. Clostridium ferredoxin at low ionic strength showed biphasic kinetics, which indicates a differential reactivity of the two iron-sulfur centers of this molecule toward the electron donor. The kobsd values for the initial fast phase observed with Clostridium ferredoxin were ionic strength dependent, whereas the slow-phase kinetics were ionic strength independent. This correlates with the highly asymmetric charge distribution on the surface of the bacterial protein relative to the two iron-sulfur clusters. The kinetics corresponding to spinach ferredoxin reduction were also ionic strength dependent, and the results obtained with these kinetics and with the fast phase of the bacterial ferredoxin reduction were consistent with a mechanism involving electrostatically stabilized complex formation. For spinach ferredoxin, the second-order rate constant extrapolated to infinite ionic strength was 2-fold smaller, and the extrapolated limiting first-order rate constant was 10-fold smaller, than for Clostridium ferredoxin, indicating a smaller intrinsic reactivity of the spinach protein toward the electron donor. Differences in the rate constant values and the ionic strength dependencies with both ferredoxins are consistent with differences in cluster structure and environment and protein size and charge distribution. For both proteins, the total amount of ferredoxin reduced increased with the ionic strength.(ABSTRACT TRUNCATED AT 250 WORDS)

Electric charge distribution in proteins and ionic strength dependence of reaction rate.

The ionic strength dependence of the reaction rate between protein and dichloride anion radical has been investigated by flash photolysis of aqueous chloride-containing lysozyme, ribonuclease A, or insulin. The rate constant for the reaction of lysozyme or ribonuclease A with dichloride anion radicals decreases with increasing ionic strength, while it increases for insulin. The dependence was found to obey an equation derived from the theory of Debye and Hückel or the equation of Wherland and Gray for lysozyme within experimental errors. For ribonuclease A, however, it deviates largely from these equations. In the case of insulin a moderate deviation was observed. The different behavior in the ionic strength dependence is discussed in terms of the electric charge distribution in the protein molecules.

Effect of traditional methods of processing on the electrophoretic patterns of faba bean water soluble proteins

A number of national dishes are prepared from the dry seeds of faba bean by soaking, germination (nabet), cooking (bisara and medammis) or frying a dough (falafel). Soaking (12 h) decreased the intensity of bands in the standard PAGE and increased the charge heterogeneity of neutral proteins in PAGIF. Germination (24 or 60 h) changed the patterns for esterases, peroxidases and amylases, but not for phosphorylases. The charge distribution of proteins was markedly changed in the basic region. Roasting did not affect the distribution in standard PAGE. All bands in the basic region of PAGIF disappeared. High MW-proteins wre degraded to low MW-proteins as seen in SDS-PAGE. The cooking broth and the cooked beans of nabet soup and medammis had more or less similar patterns in the standard PAGE. The broth of nabet soup had larger number of low MW-proteins than the bean extract in SDS-PAGE. Bisara proteins showed almost the same patterns as the corresponding proteins of the cooking broth of nabet soup. Falafel proteins showed patterns very similar to that obtained from roasted beans in the SDS-PAGE. PAGIF gave a different but characteristic charge distribution for falafel.

Cuticular proteins: The neglected component

AbstractThis paper emphasizes the importance of the protein component of cuticles. Correlation of electrophoretic charge distribution of individual cuticular proteins and physical properties of the cuticles from which they were extracted, as well as interpopulation and interspecies conservation of electrophoretic patterns, are used to argue that individual proteins play precise roles in the cuticle. Glycosylation of cuticular proteins is described, but no function for these modifications is yet known. Analogy is drawn to analyses of chorion proteins and the case is made that analysis of amino acid sequence data is likely to provide insights into how cuticular proteins and chitin interact to construct the diverse types of cuticles.