DNA bending by bHLH charge variants

We wish to understand the role of electrostatics in DNA stiffness and bending. The DNA charge collapse model suggests that mutual electrostatic repulsions between neighboring phosphates significantly contribute to DNA stiffness. According to this model, placement of fixed charges near the negatively charged DNA surface should induce bending through asymmetric reduction or enhancement of these inter-phosphate repulsive forces. We have reported previously that charged variants of the elongated basic-leucine zipper (bZIP) domain of Gcn4p bend DNA in a manner consistent with this charge collapse model. To extend this result to a more globular protein, we present an investigation of the dimeric basic-helix–loop–helix (bHLH) domain of Pho4p. The 62 amino acid bHLH domain has been modified to position charged amino acid residues near one face of the DNA double helix. As observed for bZIP charge variants, DNA bending toward appended cations (away from the protein:DNA interface) is observed. However, unlike bZIP proteins, DNA is not bent away from bHLH anionic charges. This finding can be explained by the structure of the more globular bHLH domain which, in contrast to bZIP proteins, makes extensive DNA contacts along the binding face.     

A method to identify distinctive charge configurations in protein sequences, with application to human herpesvirus polypeptides

Charge interactions are of great importance for protein function and structure, and for a variety of cellular and biochemical processes. We present a systematic approach to the detection of distinctive clusters, runs and periodic patterns of charged residues in a protein sequence. Criteria and formulae are set forth to assess statistical significance of these charge configurations. For the 80-odd proteins potentially encoded by the Epstein-Barr virus, only the major nuclear antigens of the latent state and the transactivator of the lytic cycle contain separated charge clusters of opposite sign as well as periodic charge patterns. From our studies of the polypeptides of the human herpesviruses and of a broad collection of human and other viral protein sequences, distinctive charge configurations appear to be associated with viral capsid and core proteins (positive clusters or runs, mostly at the carboxyl terminus), with many viral glycoproteins and membrane-associated proteins (negative charge clusters), and with transactivators and transforming proteins (multiple charge structures). The statistics developed in this paper apply more generally to other than charge properties of a protein and should aid in the evaluation of a large variety of sequence features.     

Surface, subunit interfaces and interior of oligomeric proteins

The solvent-accessible surface area (As) of 23 oligomeric proteins is calculated using atomic co-ordinates from high-resolution and well-refined crystal structures. As is correlated with the protein molecular weight, and a power law predicts its value to within 5% on average. The accessible surface of the average oligomer is similar to that of monomeric proteins in its hydropathy and amino acid composition. The distribution of the 20 amino acid types between the protein surface and its interior is also the same as in monomers. Interfaces, i.e. surfaces involved in subunit contacts, differ from the rest of the subunit surface. They are enriched in hydrophobic side-chains, yet they contain a number of charged groups, especially from Arg residues, which are the most abundant residues at interfaces except for Leu. Buried Arg residues are involved in H-bonds between subunits. We counted H-bonds at interfaces and found that several have none, others have one H-bond per 200 A2 of interface area on average (1 A = 0.1 nm). A majority of interface H-bonds involve charged donor or acceptor groups, which should make their contribution to the free energy of dissociation significant, even when they are few. The smaller interfaces cover about 700 A2 of the subunit surface. The larger ones cover 3000 to 10,000 A2, up to 40% of the subunit surface area in catalase. The lower value corresponds to an estimate of the accessible surface area loss required for stabilizing subunit association through the hydrophobic effect alone. Oligomers with small interfaces have globular subunits with accessible surface areas similar to those of monomeric proteins. We suggest that these oligomers assemble from preformed monomers with little change in conformation. In oligomers with large interfaces, isolated subunits should be unstable given their excessively large accessible surface, and assembly is expected to require major structural changes.     

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.     

Effect of spatial coupling of amino acid residues on the possibility of mutational substitution in globular proteins

Amino acid residues can be divided into similar groups by frequencies of interreplacements in the evolutionary pathway and by trends to spatial contacts at the tertiary structures of globular proteins. Each residue was compared to the cluster of spatial surrounding--the totality of residues spacially drawn together. 5210 clusters in 32 unhomologous proteins with established tertiary structure and 6447 clusters formed only by variables amino acid residues were analysed. Spatial contacts among residues were studied depending on the secondary structure and the amount of residues in a cluster. It was assumed that functionally admissible mutations may be defined, first of all, by the degree of neighboring of amino acid residues in the spatial surrounding.     

Voltage-dependent orientation of membrane proteins

In order to study the influence of electrostatic forces on the disposition of proteins in membranes, we have examined the interaction of a receptor protein and of a membrane-active peptide with black lipid membranes. In the first study we show that the hepatic asialoglycoprotein receptor can insert spontaneously into lipid bilayers from the aqueous medium. Under the influence of a trans-positive membrane potential, the receptor, a negatively charged protein, appears to change its disposition with respect to the membrane. In the second study we consider melittin, an amphipathic peptide containing a generally hydrophobic stretch of 19 amino acids followed by a cluster of four positively charged residues at the carboxy terminus. The hydrophobic region contains two positively charged residues. In response to trans-negative electrical potential, melittin appears to assume a transbilayer position. These findings indicate that electrostatic forces can influence the disposition, and perhaps the orientation, of membrane proteins. Given the inside-negative potential of most or all cells, we would expect transmembrane proteins to have clusters of positively charged residues adjacent to the cytoplasmic ends of their hydrophobic transmembrane segments, and clusters of negatively charged residues just to the extracytoplasmic side. This expectation has been borne out by examination of the few transmembrane proteins for which there is sufficient information on both sequence and orientation. Surface and dipole potentials may similarly affect the orientation of membrane proteins.     

Formation of metal nanoclusters on specific surface sites of protein molecules

During vacuum condensation of metals on frozen proteins, nanoclusters are preferentially formed at specific surface sites (decoration). Understanding the nature of metal/protein interaction is of interest for structure analysis and is also important in the fields of biocompatibility and sensor development. Studies on the interaction between metal and distinct areas on the protein which enhance or impede the probability for cluster formation require information on the structural details of the protein's surface underlying the metal clusters. On three enzyme complexes, lumazine synthase from Bacillus subtilis, proteasome from Thermoplasma acidophilum and GTP cyclohydrolase I from Escherichia coli, the decoration sites as determined by electron microscopy (EM) were correlated with their atomic surface structures as obtained by X-ray crystallography. In all three cases, decoration of the same protein results in different cluster distributions for gold and silver. Gold decorates surface areas consisting of polar but uncharged residues and with rough relief whereas silver clusters are preferentially formed on top of protein pores outlined by charged and hydrophilic residues and filled with frozen buffer under the experimental conditions. A common quality of both metals is that they strictly avoid condensation on hydrophobic sites lacking polar and charged residues. The results open ways to analyse the binding mechanism of nanoclusters to small specific sites on the surface of hydrated biomacromolecules by non-microscopic, physical-chemical methods. Understanding the mechanism may lead to advanced decoration techniques resulting in fewer background clusters. This would improve the analysis of single molecules with regard to their symmetries and their orientation in the adsorbed state and in precrystalline assemblies as well as facilitate the detection of point defects in crystals caused by misorientation or by impurities.     

A contribution to the theory of biological staining based on the principles for structural organization of biological macromolecules

Biological staining is to a large degree explainable based on the principles governing folding and aggregation of macromolecules in aqueous solution. Most macromolecules are polyions, which, except for heteropolysaccharides, have a large proportion of nonpolar or only slightly polar residues. Because they are amphiphilic, they react in water by a complex set of hydrophobic interactions involving charged residues, nonpolar residues and water molecules. The hydrophobic interactions lead to complex folding systems or micelle-like structures. Dyes are amphiphilic molecules with a tendency to form micelles, but with limitations due to geometric constraints and charge repulsion. Macromolecules and dyes react with each other in aqueous solution following the same principles as for the structural organization of macromolecules, as in protein folding for example. Dye binding requires near contact between nonpolar groups in both the dye and macromolecule, and this is accomplished by choosing a pH at which the dye and macromolecule have opposite net charges. Charge attraction is insufficient for binding in most cases, but it is directive because it determines which macromolecules a given dye ion is able to contact. These considerations apply to the staining of globular (cytoplasmic) proteins and to nucleic acid staining. The staining mechanism is by hydrophobic interactions. Above approximately pH 3.5, DNA may also bind dyes by hydrophobic intercalation between the bases of the double helix; at lower pH the double helix opens and dye binding is as for RNA and globular proteins. Heteroglycans (mucins) have virtually no nonpolar groups, so nonpolar interactions are restricted to the dye molecules. Metachromatic staining of heteroglycans is due to hydrophobic bonding or micelle formation between the monovalent planar dye molecules aided by charge neutralization by the negatively charged heteroglycans. Alternatively, as the charge attraction increases with the number of closely placed charges, acidic heteroglycans may be stained by a polycation such as alcian blue or colloidal iron. For elastic fiber and collagen staining, actual hydrophobic interactions are less important and hydrogen bonding and simple nonpolar interactions play a major role. These macromolecules may therefore be stained using a nonaqueous alcoholic solution.     

A contribution to the theory of biological staining based on the principles for structural organization of biological macromolecules

Biological staining is to a large degree explainable based on the principles governing folding and aggregation of macromolecules in aqueous solution. Most macromolecules are polyions, which, except for heteropolysaccharides, have a large proportion of nonpolar or only slightly polar residues. Because they are amphiphilic, they react in water by a complex set of hydrophobic interactions involving charged residues, nonpolar residues and water molecules. The hydrophobic interactions lead to complex folding systems or micelle-like structures. Dyes are amphiphilic molecules with a tendency to form micelles, but with limitations due to geometric constraints and charge repulsion. Macromolecules and dyes react with each other in aqueous solution following the same principles as for the structural organization of macromolecules, as in protein folding for example. Dye binding requires near contact between nonpolar groups in both the dye and macromolecule, and this is accomplished by choosing a pH at which the dye and macromolecule have opposite net charges. Charge attraction is insufficient for binding in most cases, but it is directive because it determines which macromolecules a given dye ion is able to contact. These considerations apply to the staining of globular (cytoplasmic) proteins and to nucleic acid staining. The staining mechanism is by hydrophobic interactions. Above approximately pH 3.5, DNA may also bind dyes by hydrophobic intercalation between the bases of the double helix; at lower pH the double helix opens and dye binding is as for RNA and globular proteins. Heteroglycans (mucins) have virtually no nonpolar groups, so nonpolar interactions are restricted to the dye molecules. Metachromatic staining of heteroglycans is due to hydrophobic bonding or micelle formation between the monovalent planar dye molecules aided by charge neutralization by the negatively charged heteroglycans. Alternatively, as the charge attraction increases with the number of closely placed charges, acidic heteroglycans may be stained by a polycation such as alcian blue or colloidal iron. For elastic fiber and collagen staining, actual hydrophobic interactions are less important and hydrogen bonding and simple nonpolar interactions play a major role. These macromolecules may therefore be stained using a nonaqueous alcoholic solution.     

Aromatic cluster mutations produce focal modulations of β-sheet structure

Site-directed mutagenesis is a powerful tool for altering the structure and function of proteins in a focused manner. Here, we examined how a model β-sheet protein could be tuned by mutation of numerous surface-exposed residues to aromatic amino acids. We designed these aromatic side chain “clusters” at highly solvent-exposed positions in the flat, single-layer β-sheet of Borrelia outer surface protein A (OspA). This unusual β-sheet scaffold allows us to interrogate the effects of these mutations in the context of well-defined structure but in the absence of the strong scaffolding effects of globular protein architecture. We anticipated that the introduction of a cluster of aromatic amino acid residues on the β-sheet surface would result in large conformational changes and/or stabilization and thereby provide new means of controlling the properties of β-sheets. Surprisingly, X-ray crystal structures revealed that the introduction of aromatic clusters produced only subtle conformational changes in the OspA β-sheet. Additionally, despite burying a large degree of hydrophobic surface area, the aromatic cluster mutants were slightly less stable than the wild-type scaffold. These results thereby demonstrate that the introduction of aromatic cluster mutations can serve as a means for subtly modulating β-sheet conformation in protein design.     

Solution NMR structure of the iron-sulfur cluster assembly protein U (IscU) with zinc bound at the active site

IscU is a highly conserved protein that serves as the scaffold for IscS-mediated assembly of iron-sulfur ([Fe-S]) clusters. We report the NMR solution structure of monomeric Haemophilus influenzae IscU with zinc bound at the [Fe-S] cluster assembly site. The compact core of the globular structure has an alpha-beta sandwich architecture with a three-stranded antiparallel beta-sheet and four alpha-helices. A nascent helix is located N-terminal to the core structure. The zinc is ligated by three cysteine residues and one histidine residue that are located in and near conformationally dynamic loops at one end of the IscU structure. Removal of the zinc metal by chelation results in widespread loss of structure in the apo form. The zinc-bound IscU may be a good model for iron-loaded IscU and may demonstrate structural features found in the [Fe-S] cluster bound form. Structural and functional similarities, genomic context in operons containing other homologous genes, and distributions of conserved surface residues support the hypothesis that IscU protein domains are homologous (i.e. derived from a common ancestor) with the SufE/YgdK family of [Fe-S] cluster assembly proteins.     

The potential of differential mobility analysis coupled to MS for the study of very large singly and multiply charged proteins and protein complexes in the gas phase

As previously demonstrated by the technique of gas-phase electrophoretic mobility molecular analyzer (GEMMA) introduced by Kaufman and colleagues, differential mobility analysis (DMA) of charge-reduced electrospray ions in the gas phase is a useful complement to MS for studying large proteins and their weakly bound complexes. Several limitations of GEMMA, the solutions for which have the potential to greatly improve its performance, are discussed here, including DMA resolution and transmission. A quantitative theory of charge reduction kinetics for dried multiply charged globular proteins at atmospheric pressures is also presented, showing that the charge reduction time must be carefully chosen to maximize a singly charged ion signal, while avoiding survival of contaminating multiply charged species. Because charge reduction limits the range of masses analyzable by MS, we also consider the potential of a parallel-plate DMA coupled in series to an MS for DMA-MS studies without charge reduction.     

Protein stability and electrostatic interactions between solvent exposed charged side chains

To investigate the contribution to protein stability of electrostatic interactions between charged surface residues, we have studied the effect of substituting three negatively charged solvent exposed residues with their side-chain amide analogs in bovine calbindin D9k--a small (Mr 8,500) globular protein of the calmodulin superfamily. The free energy of urea-induced unfolding for the wild-type and seven mutant proteins has been measured. The mutant proteins have increased stability towards unfolding relative to the wild-type. The experimental results correlate reasonably well with theoretically calculated relative free energies of unfolding and show that electrostatic interactions between charges on the surface of a protein can have significant effects on protein stability.     

Exploring proteome‐wide occurrence of clusters of charged residues in eukaryotes

Clusters of charged residues are one of the key features of protein primary structure since they have been associated to important functions of proteins. Here, we present a proteome wide scan for the occurrence of Charge Clusters in Protein sequences using a new search tool (FCCP) based on a score‐based methodology. The FCCP was run to search charge clusters in seven eukaryotic proteomes: Arabidopsis thaliana, Caenorhabditis elegans, Danio rerio, Drosophila melanogaster, Homo sapiens, Mus musculus, and Saccharomyces cerevisiae. We found that negative charge clusters (NCCs) are three to four times more frequent than positive charge clusters (PCCs). The Drosophila proteome is on average the most charged, whereas the human proteome is the least charged. Only 3 to 8% of the studied protein sequences have negative charge clusters, while 1.6 to 3% having PCCs and only 0.07 to 0.6% have both types of clusters. NCCs are localized predominantly in the N‐terminal and C‐terminal domains, while PCCs tend to be localized within the functional domains of the protein sequences. Furthermore, the gene ontology classification revealed that the protein sequences with negative and PCCs are mainly binding proteins. Proteins 2015; 83:1252–1261. © 2015 Wiley Periodicals, Inc.     

Planar charged clusters--structural invariants of the gamma-crystallin family of the crystalline lens: functional role and evolutionary conservatism]

Plane charge clusters from the calf eye lens protein gamma-crystallin are considered. The clusters consist of four to six side chain charged groups with interatomic distances in ionic pairs from 4 to 7 A. The charge clusters appear to decrease the hydrophilic potential of the molecular surface which maintains the transparent refracting lens medium of vertebrates with a very high protein concentration. It is shown that the charge pattern for different gene products of one species is conservative as well as for whole set of 25 sequences of vertebrates, including carp, frog, mouse, rat, calf and human. Taking into account "neutral mutations", Asp-Glu and Arg-Lys the homology of those charge positions is equal to 95-100%. Functionally important charge clusters are absent in the ancient structural motifs of gamma-crystallin.     

Solution structure of ribosomal protein S28E from Methanobacterium thermoautotrophicum

The ribosomal protein S28E from the archaeon Methanobacterium thermoautotrophicum is a component of the 30S ribosomal subunit. Sequence homologs of S28E are found only in archaea and eukaryotes. Here we report the three-dimensional solution structure of S28E by NMR spectroscopy. S28E contains a globular region and a long C-terminal tail protruding from the core. The globular region consists of four antiparallel beta-strands that are arranged in a Greek-key topology. Unique features of S28E include an extended loop L2-3 that folds back onto the protein and a 12-residue charged C-terminal tail with no regular secondary structure and greater flexibility relative to the rest of the protein. The structural and surface resemblance to OB-fold family of proteins and the presence of highly conserved basic residues suggest that S28E may bind to RNA. A broad positively charged surface extending over one side of the beta-barrel and into the flexible C terminus may present a putative binding site for RNA.     

Electrostatic contributions to the stability of hyperthermophilic proteins.

Electrostatic contributions to the folding free energy of several hyperthermophilic proteins and their mesophilic homologs are calculated. In all the cases studied, electrostatic interactions are more favorable in the hyperthermophilic proteins. The electrostatic free energy is found not to be correlated with the number of ionizable amino acid residues, ion pairs or ion pair networks in a protein, but rather depends on the location of these groups within the protein structure. Moreover, due to the large free energy cost associated with burying charged groups, buried ion pairs are found to be destabilizing unless they undergo favorable interactions with additional polar groups, including other ion pairs. The latter case involves the formation of stabilizing ion pair networks as is observed in a number of proteins. Ion pairs located on the protein surface also provide stabilizing interactions in a number of cases. Taken together, our results suggest that many hyperthermophilic proteins enhance electrostatic interactions through the optimum placement of charged amino acid residues within the protein structure, although different design strategies are used in different cases. Other physical mechanisms are also likely to contribute, however optimizing electrostatic interactions offers a simple means of enhancing stability without disrupting the core residues characteristic of different protein families.     

Prediction of natively unfolded regions in protein chain

We have shown that the ability of a protein to be in globular or in natively unfolded state (under native conditions) may be determined (besides low overall hydrophobicity and a large net charge) by such a property as the average environment density, the average number of residues enclosed at the given distance. A statistical scale of the average number of residues enclosed at the given distance for 20 types of amino acid residues in globular state has been created on the basis of 6626 protein structures. Using this scale for separation of 80 globular and 90 natively unfolded proteins we fail only in 11% of proteins (compared with 17% of errors which are observed if to use hydrophobicity scale). The present scale may be used both for prediction of form (folded or unfolded) of the native state of protein and for prediction of natively unfolded regions in protein chains. The results of comparison of our method of predicting natively unfolded regions with the other known methods show that our method has the highest fraction of correctly predicted natively unfolded regions (that is 87% and 77% if to make averaging over residues and over proteins correspondingly).     

Understanding on the residue contact network using the log‐normal cluster model and the multilevel wheel diagram

Residue clusters play essential role in stabilizing protein structures in the form of complex networks. We show that the cluster sizes in a native protein follow the log‐normal distribution for a dataset consisting of 424 proteins. To our knowledge, this is the first time of such fitting for the native structures. Based on log‐normal model, the asymptotically increasing mean cluster sizes produce a critical protein chain length of about 200 amino acids, beyond which length most globular proteins have nearly the same mean cluster sizes. This suggests that the larger proteins use a different packing mechanism than the smaller proteins. We confirmed the scale‐free property of the residue contact network for most of the protein structures in the dataset, although the violations were observed for the tightly packed proteins. Residue cluster network wheel (RCNW) is proposed to visualize the relationship between the multiple properties of the residue network such as the cluster size, the residue types and contacts, and the flexibility of the residue. We noticed that the residues with large cluster size have smaller Cα displacement measured using the normal mode analysis. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 904–916, 2010.