Secondary structure predictions and medium range interactions

Several authors have proposed that predictions of protein secondary structure derived from statistical information about the known structures can be improved when information about neighboring residues participating in short and medium range interactions is included. A substantial improvement shown here indicates that current methods of including this information are not more successful than methods that do not. Evaluations of the Chou and Fasman method (Adv. Enzymol. 47 (1978) 45-148), that does not include information about interactions (except in averaging), have shown it to be about 49% correct for three states (helix, beta-sheet and undefined). In comparison, the method of Garnier et al. (J. Mol. Biol. 120 (1978) 97-120), that explicitly includes information about neighboring residues, has an accuracy of 57% residues correct for three states. However, we have obtained an 8% improvement for predictions of secondary structure based on the algorithm by Chou and Fasman. The improvements are obtained by eliminating many rules and by choosing the best decision constants for structure assignments. The simplified method described here is 57% correct for three states using preference values calculated in 1978.     

Species-specific long range interactions between receptor/ligand pairs.

Total internal reflection microscopy (TIRM) monitors Brownian fluctuations in elevation as small as 1 nm by measuring the scattering of a single sphere illuminated by an evanescent wave when the sphere is levitated by colloidal forces such as electrostatic double-layer repulsion. From the Boltzmann distribution of elevations sampled by the sphere over time, the potential energy profile can be determined with a resolution of approximately 0.1 of the thermal energy kT. Thus, the interaction between a receptor-coated (goat, horse, or rabbit immunoglobulin G (IgG)) latex sphere and a protein A (SpA)-coated glass microscope slide was studied. A typical TIRM potential energy profile measured between a bare sphere and a bare glass plate, where the sphere fluctuates around the secondary potential energy minimum formed between double-layer repulsion and gravitational attraction, agrees well with DLVO theory. The interactions measured between IgG-coated spheres and SpA-coated slides, on the other hand, displayed a weaker repulsion compared with that observed between bare surfaces under the same conditions. Analysis of the results obtained between the coated surfaces suggests an additional attractive force. The decay length of this attraction correlates with the known dissociation constants for the binding of IgG with SpA in free solution.     

Formins regulate actin filament flexibility through long range allosteric interactions

The members of the formin family nucleate actin polymerization and play essential roles in the regulation of the actin cytoskeleton during a wide range of cellular and developmental processes. In the present work, we describe the effects of mDia1-FH2 on the conformation of actin filaments by using a temperature-dependent fluorescence resonance energy transfer method. Our results revealed that actin filaments were more flexible in the presence than in the absence of formin. The effect strongly depends on the mDia1-FH2 concentration in a way that indicates that more than one mechanism is responsible for the formin effect. In accordance with the more flexible filament structure, the thermal stability of actin decreased and the rate of phosphate dissociation from actin filaments increased in the presence of formin. The interpretation of the results supports a model in which formin binding to barbed ends makes filaments more flexible through long range allosteric interactions, whereas binding of formin to the sides of the filaments stabilizes the protomer-protomer interactions. These results suggest that formins can regulate the conformation of actin filaments and may thus also modulate the affinity of actin-binding proteins to filaments nucleated/capped by formins.     

Influence of medium and long range interactions in protein folding.

Protein structures are stabilized by both local and long range interactions. In this work, we analyze the residue-residue contacts and the role of medium- and long-range interactions in globular proteins belonging to different structural classes. The results show that while medium range interactions predominate in all-alpha class proteins, long-range interactions predominate in all-beta class. Based on this, we analyze the performance of several structure prediction methods in different structural classes of globular proteins and found that all the methods predict the secondary structures of all-alpha proteins more accurately than other classes. Also, we observed that the residues occurring in the range of 21-30 residues apart contributes more towards long-range contacts and about 85% of residues are involved in long-range contacts. Further, the preference of residue pairs to the folding and stability of globular proteins is discussed.     

Amido binding to ReO core: Synthesis, structure and intermolecular interactions

The light green coloured complexes of general formula [Re^VO(L)Cl(OH₂)]Cl have been synthesised in good yields by reacting [Re^VOCl₃(AsPh₃)₂] with HL in dichloromethane in dinitrogen atmosphere. Here, L⁻ is the deprotonated form of N,N-bis(2-pyridylmethyl)amine (HL¹); N-(2-pyridylmethyl)-N′,N′-dimethylethylenediamine (HL²) and N-(2-pyridylmethyl)-N′,N′-diethylethylenediamine (HL³). Single crystal X-ray structure determination of [Re^VO(L¹)Cl(OH₂)]Cl confirms the amido binding of ReO³⁺ species. In the solid state of [Re^VO(L¹)Cl(OH₂)]Cl, the coordinated and counter chloride ions are engaged in Re-Cl…H-C(ring), Cl…H-C(ring) and Re-(OH₂)…Cl hydrogen bonding and forming of a supramolecular network in the solid state. The subunit of the supramolecular network consists of one eight-membered and two nine-membered hydrogen bonded rings. The average diameters of eight-membered and nine-membered rings are ∼3.70 and ∼5.26 Å, respectively.     

Phosphate stabilization of intermolecular interactions

Receptor heteromerization is an important phenomenon that results from the interaction of epitopes on two receptors. Previous studies have suggested the possibility of Dopamine D2-NMDA receptors' interaction. We believe that the interaction is through an acidic epitope of the NMDA NR1 subunit (KVNSEEEEEDA) and a basic epitope of the D2 third intracellular loop (VLRRRRKRVN), which was shown to also interact with the Adenosine A2A receptor. In previous work, we highlighted the role of certain amino acid residues, mainly two or more adjacent arginine on one peptide and two or more adjacent glutamate, or aspartate, or a phosphorylated residue on the other in the formation of noncovalent complexes (NCX) between epitopes. In the present work, we use the phosphorylated (KVNSpEEEEEDA), nonphosphorylated (KVNSEEEEEDA) and modified (KVNpSAAAAAAA) forms of the NMDA epitope that possibly interact with the D2 epitope to investigate the gas-phase stability of the NCXs as a function of the nominal energy given to the NCX ion as it enters the collision cell. In addition to theoretical calculations, the experimental data was used to calculate the stability of each electrostatic complex versus that of the dimer of KVNSpEEEEEDA. Our results demonstrate the importance of the phosphate group in stabilizing molecular interactions and that appreciably higher collision energies are required to completely dissociate any of the three different NCX ions that are formed through electrostatic interaction in comparison to the energy required to dissociate the KVNpSEEEEEDA dimer ion, which is mainly kept together by hydrogen bonding. This study emphasizes ionic bonds stability and their importance to protein structure as their potent electrostatic attractions can in the gas-phase surpass the strength of covalent bonds.     

Molecular packing and intermolecular interactions in N-acylethanolamines: crystal structure of N-myristoylethanolamine.

N-Acylethanolamines elicited much interest in recent years owing to their occurrence in biological membranes under conditions of stress as well as under normal conditions. The molecular conformation, packing properties and intermolecular interactions of N-myristoylethanolamine (NMEA) have been determined by single crystal X-ray diffraction analysis. The lipid crystallized in the space group P21/a with unit cell dimensions: a=9.001, b=4.8761, c=39. 080. There are four symmetry-related molecules in the monoclinic unit cell. The molecules are organized in a tail-to-tail fashion, similar to the arrangement in a bilayer membrane. The hydrophobic acyl chain of the NMEA molecule is tilted with respect to the bilayer normal by an angle of 37 degrees. Each hydroxy group forms two hydrogen bonds, one as a donor and the other as an acceptor, with the hydroxy groups of molecules in the opposing leaflet. These O-H...O hydrogen bonds form an extended, zig-zag type network along the b-axis. In addition, the N-H and C=O groups of adjacent molecules are involved in N-H...O hydrogen bonds, which also connect adjacent molecules along the b-axis.     

Saccharomyces species assignment by long range ribotyping

Type strains of 10 genotypically distinctSaccharomyces species are differentiated by ribosomal DNA restriction fragment analysis (ribotyping). The full length of the chromosomal ribosomal repeat was amplified in two parts, the 18SrDNA including both ITS regions (2600 bp) and the 25SrDNA (3300 bp). Restriction fragments generated by 9 enzymes from these two products yield characteristic patterns, by which unknownSaccharomyces isolates are assigned to the type strains. For convenient separation and detection only fragments longer than 200 bp were monitored. In contrast to molecular differentiation methods of highest resolution as RAPD-PCR or fingerprinting, the results from ribotyping are absolutely reproducible and thereby suitable for databases. The phylogeny computed from the discrete character matrix for presence/absence of fragments by the PHYLIP program package is in complete accordance to the phylogeny derived from ribosomal RNA sequence analysis. By this the field of application of the long range ribotyping can be regarded basically as equal to DNA sequence analysis of the same locus. Because distant relationships are recognized, missidentified genera were detected upon the species assignment. This cannot be done by methods of higher resolution like RAPD-PCR or fingerprinting.     

Macrophage-specific up-regulation of apolipoprotein E gene expression by STAT1 is achieved via long range genomic interactions

In atherogenesis, macrophage-derived apolipoprotein E (apoE) has an athero-protective role by a mechanism that is not fully understood. We investigated the regulatory mechanisms involved in the modulation of apoE expression in macrophages. The experiments showed that the promoters of all genes of the apoE/apoCI/apoCIV/apoCII gene cluster are enhanced by multienhancer 2 (ME.2), a regulatory region that is located 15.9 kb downstream of the apoE gene. ME.2 interacts with the apoE promoter in a macrophage-specific manner. Transient transfections in RAW 264.7 macrophages showed that the activity of ME.2 was strongly decreased by deletion of either 87 bp from the 5' end or 131 bp from the 3' end. We determined that the minimal fragment of this promoter that can be activated by ME.2 is the proximal -100/+73 region. The analysis of the deletion mutants of ME.2 revealed the importance of the 5' end of ME.2 in apoE promoter transactivation. Chromatin conformational capture assays demonstrated that both ME.2 and ME.1 physically interacted with the apoE promoter in macrophages. Our data showed that phorbol 12-myristate 13-acetate-induced differentiation of macrophages is accompanied by a robust induction of apoE and STAT1 expression. In macrophages (but not in hepatocytes), STAT1 up-regulated apoE gene expression via ME.2. The STAT1 binding site was located in the 174-182 region of ME.2. In conclusion, the specificity of the interactions between the two multienhancers (ME.1 and ME.2) and the apoE promoter indicates that these distal regulatory elements play an important role in the modulation of apoE gene expression in a cell-specific manner.     

Collective properties of hydration: long range and specificity of hydrophobic interactions.

We report results of molecular dynamics (MD) simulations of composite model solutes in explicit molecular water solvent, eliciting novel aspects of the recently demonstrated, strong many-body character of hydration. Our solutes consist of identical apolar (hydrophobic) elements in fixed configurations. Results show that the many-body character of PMF is sufficiently strong to cause 1) a remarkable extension of the range of hydrophobic interactions between pairs of solute elements, up to distances large enough to rule out pairwise interactions of any type, and 2) a SIF that drives one of the hydrophobic solute elements toward the solvent rather than away from it. These findings complement recent data concerning SIFs on a protein at single-residue resolution and on model systems. They illustrate new important consequences of the collective character of hydration and of PMF and reveal new aspects of hydrophobic interactions and, in general, of SIFs. Their relevance to protein recognition, conformation, function, and folding and to the observed slight yet significant nonadditivity of functional effects of distant point mutations in proteins is discussed. These results point out the functional role of the configurational and dynamical states (and related statistical weights) corresponding to the complex configurational energy landscape of the two interacting systems: biomolecule + water.     

Conformation and intermolecular interactions of carbohydrate chains

For consideration of their conformations and interactions, carbohydrate chains can conveniently be divided into 3 classes on the basis of their covalent structure; namely periodic (a), interrupted periodic (b), and aperiodic (c) types. In aqueous solution carbohydrate chains often exist as highly disordered random coils. Under appropriate conditions, however, polysaccharides of types (a) and (b) can adopt a variety of ordered conformations. Physical methods, and in particular optical rotation, circular dichroism, and nuclear magnetic resonance, provide sensitive probes for the study of the mechanism and specificity of these disorder-order transitions in aqueous solution. Intermolecular interactions between such polysaccharide chains arise from co-operative associations of long structurally regular regions which adopt the ordered conformations. For acidic polysaccharides these cooperative associations may involve alignment of extended ribbons with cations sandwhiched between them. In other systems the interactions involve double belices which may then aggregate further, and geometric “matching” of different polysaccharide chains can also occur. These ordered, associated regions are generally terminated by deviations from structural regularity or by “kinks” which prevent complete aggregation of the molecules. The complex carbohydrate chains which occur at the periphery of animal cells have very different, aperiodic structures and although their conformations are as yet poorly understood, preliminary indications are considered.     

Molecular structure and intra- and intermolecular magnetic interactions in chloro-bridged copper(II) dimers

Magnetic interactions in binuclear copper(II) complexes, [Cu₂(apyhist)₂Cl₂](ClO₄)₂ (1) and [Cu₂(2-pyhist)₂Cl₂](ClO₄)₂ (2) with tridentate diimine ligands and chloro-bridged groups (where apyhist=(4-imidazolyl)ethylene-2-amino-1-ethylpyridine and 2-pyhist=(4-imidazolyl)ethylene-2-aminomethylpyridine) were studied with the aim of better elucidating magneto-structural correlations in such species, both in solution and in solid state. X-ray analyses revealed that chloro-bridged ligands keep the copper(II) ion coordinated to adjacent unit, at Cu-Cl distances of 2.271 and 2.737 Å, and a Cu-Cl-Cu angle of 87.46° in compound 1. Each Cu^II atom is also coordinated to three N atoms from the imine ligand, in a distorted tetragonal pyramidal environment. Magnetic measurements carried out in temperatures from 0.8 to 290 K and in magnetic field up to 170 kOe indicated that besides the intramolecular magnetic coupling between the copper centers [J/k=−(1.93±0.05) K] further interactions between adjacent dimers [J^′z/k=−(1.3±0.1) K] should be taken into account. Similar results were observed for compound 2, for which [J/k=−(4.27±0.05) K] and [J^′z/k=−(3.7±0.1) K]. In solution, the interconversion of the dimer 1 and the related monomer species [Cu(apyhist)(H₂O)₂] (ClO₄)₂ (3) monitored by EPR spectra, was verified to be very dependent on the solvent.     

HSP27 multimerization mediated by phosphorylation-sensitive intermolecular interactions at the amino terminus

Distinct biochemical activities have been reported for small and large molecular complexes of heat shock protein 27 (HSP27), respectively. Using glycerol gradient ultracentrifugation and chemical cross-linking, we show here that Chinese hamster HSP27 is expressed in cells as homotypic multimers ranging from dimers up to 700-kDa oligomers. Treatments with arsenite, which induces phosphorylation on Ser15 and Ser90, provoked a major change in the size distribution of the complexes that shifted from oligomers to dimers. Ser90 phosphorylation was sufficient and necessary for causing this change in structure. Dimer formation was severely inhibited by replacing Ser90 with Ala90 but not by replacing Ser15 with Ala15. Using the yeast two-hybrid system, two domains were identified that were responsible for HSP27 intermolecular interactions. One domain was insensitive to phosphorylation and corresponded to the C-terminal alpha-crystallin domain. The other domain was sensitive to serine 90 phosphorylation and was located in the N-terminal region of the protein. Fusion of this N-terminal domain to firefly luciferase conferred luciferase with the capacity to form multimers that dissociated into monomers upon phosphorylation. A deletion within this domain of residues Arg5-Tyr23, which contains a WDPF motif found in most proteins of the small heat shock protein family, yielded a protein that forms only phosphorylation-insensitive dimers. We propose that HSP27 forms stable dimers through the alpha-crystallin domain. These dimers further multimerize through intermolecular interactions mediated by the phosphorylation-sensitive N-terminal domain.     

Direct measurement of the intermolecular forces between counterion-condensed DNA double helices. Evidence for long range attractive hydration forces.

Rather than acting by modifying van der Waals or electrostatic double layer interactions or by directly bridging neighboring molecules, polyvalent ligands bound to DNA double helices appear to act by reconfiguring the water between macromolecular surfaces to create attractive long range hydration forces. We have reached this conclusion by directly measuring the repulsive forces between parallel B-form DNA double helices pushed together from the separations at which they have self organized into hexagonal arrays of parallel rods. For all of the wide variety of "condensing agents" from divalent Mn to polymeric protamines, the resulting intermolecular force varies exponentially with a decay rate of 1.4-1.5 A, exactly one-half that seen previously for hydration repulsion. Such behavior qualitatively contradicts the predictions of all electrostatic double layer and van der Waals force potentials previously suggested. It fits remarkably well with the idea, developed and tested here, that multivalent counterion adsorption reorganizes the water at discrete sites complementary to unadsorbed sites on the apposing surface. The measured strength and range of these attractive forces together with their apparent specificity suggest the presence of a previously unexpected force in molecular organization.     

Regulation of RhoGEF activity by intramolecular and intermolecular SH3 domain interactions

RhoGEFs are central controllers of small G-proteins in cells and are regulated by several mechanisms. There are at least 22 human RhoGEFs that contain SH3 domains, raising the possibility that, like several other enzymes, SH3 domains control the enzymatic activity of guanine nucleotide exchange factor (GEF) domains through intra- and/or intermolecular interactions. The structure of the N-terminal SH3 domain of Kalirin was solved using NMR spectroscopy, and it folds much like other SH3 domains. However, NMR chemical shift mapping experiments showed that this Kalirin SH3 domain is unique, containing novel cooperative binding site(s) for intramolecular PXXP ligands. Intramolecular Kalirin SH3 domain/ligand interactions, as well as binding of the Kalirin SH3 domain to the adaptor protein Crk, inhibit the GEF activity of Kalirin. This study establishes a novel molecular mechanism whereby intramolecular and intermolecular Kalirin SH3 domain/ligand interactions modulate GEF activity, a regulatory mechanism that is likely used by other RhoGEF family members.