Intermolecular interactions and phase structures of plasticized wheat proteins materials

The intermolecular interactions and phase structures of thermally processed wheat proteins with glycerol and water as plasticizers were studied by dynamic mechanical analysis and solid-state high-resolution NMR spectroscopy. The results of phase structures at scales of molecular level to tens of nanometers were correlated with the mechanical properties of the materials. The strong hydrogen bonding intermolecular interactions between the components in wheat proteins and the plasticizers resulted in a significant change in molecular motions of wheat protein materials. The plasticized systems, however, still presented a wide distribution of chain mobility at a scale from the molecular level to 20-30 nm, and the plasticizing effect was different for each wheat protein system. High protein content systems tended to be plasticized relatively easily especially when lipid content is high, but the existence of residual starch would require more plasticizers to reach a similar level of chain mobility. On a scale of 20-30 nm, plasticized vital wheat gluten (WG) and the deamidated wheat proteins (WP-I) were heterogeneous with each component exhibiting its individual mobility, whereas the plasticized insoluble protein system (WP-II) with poor mechanical properties was homogeneous. Both WG and WP-I systems showed excellent mechanical polymeric properties in tensile strength and elasticity despite the heterogeneity. The strong intermolecular hydrogen bonding interactions and soluble protein components in the materials could provide an adhesion among different components and act as a continuous matrix in the systems. Therefore, these materials displayed excellent mechanical properties via coordination effects among different components.     

Ordered water structure around a B-DNA dodecamer: A quantitative study

The crystal structure of the double-helical B-DNA dodecamer of sequence C-G-C-G-A-A-T-T-C-G-C-G has been solved and refined independently in three forms: (1) the parent sequence at room temperature; (2) the same sequence at 16 K; and (3) the 9-bromo variant C-G-C-G-A-A-T-T^BrC-G-C-G at 7 °C in 60% (v/v) 2-methyl-2.4-pentanediol. The latter two structures show extensive hydration along the phosphate backbone, a feature that was invisible in the native structure because of high temperature factors (indicating thermal or static disorder) of the backbone atoms. Sixty-five solvent peaks are associated with the phosphate backbone, or an average of three per phosphate group. Nineteen other molecules form a first shell of hydration to base edge N and O atoms within the major groove, and 36 more are found in upper hydration layers. The latter tend to occur in strings or clusters spanning the major groove from one phosphate group to another. A single spermine molecule also spans the major groove. In the minor groove, the zig-zag spine of hydration that we believe to be principally responsible for stabilizing the B form of DNA is found in all three structures. Upper level hydration in the minor groove is relatively sparse, and consists mainly of strings of water molecules extending across the groove, with few contacts to the spine below. Sugar O-1′ atoms are closely associated with water molecules, but these are chiefly molecules in the spine, so the association may reflect the geometry of the minor groove rather than any intrinsic attraction of O-1′ atoms for hydration. The phosphate O-3′ and O-5′ atoms within the backbone chain are least hydrated of all, although no physical or steric impediment seems to exist that would deny access to these oxygen atoms by water molecules.     

Water and ions in a high resolution structure of B-DNA.

A detailed picture of hydration and counterion location in the B-DNA duplex d(GCGAATTCG) is presented. Detailed data have been obtained by single crystal x-ray diffraction at atomic resolution (0.89 A) in the presence of Mg(2+). The latter is the highest resolution ever obtained for a B-DNA oligonucleotide. Minor groove hydration is compared with that found in the Na(+) and Ca(2+) crystal forms of the related dodecamer d(CGCGAATTCGCG). High resolution data (1.45 A) of the Ca(2+) form obtained in our laboratory are used for that purpose. The central GAATTC has a very stable hydration spine identical in all cases, independent of duplex length and crystallization conditions (counterions, space group). However, the organization of the water molecules (tertiary and quaternary layers) associated with the central spine vary in each case.     

Intermolecular interactions in type I collagens.

X-PLOR modelling of collagen dimers containing Gly-Glu-Arg in each chain has been carried out. The interaction between molecules when two Gly-Glu-Arg are present on each chain is found to be substantially less than two times that obtained with one per chain, implying that relative tilting of two collagen molecules does not offset the disadvantages of misalignment of the interacting moieties. This implies that if multiple (Glu(-)-Arg+)3 interactions are important in fibril formation, their lateral separations must be large enough to insure that they act independently.     

Intermolecular interactions in solutions of serum albumin

The mechanisms of intermolecular protein complex formation were studied by the example of monomers, oligomers and aggregates of bovine serum albumin (BSA) depending on the protein concentration, pH and urea concentration. Using dynamic light scattering (DLS), analytical ultracentrifugation (AUC) and PAG electrophoresis we have shown the existence of dynamic equilibrium between monomers and aggregates in BSA solution. Decreasing pH of the solution (4.0–1.0) resulted in increasing sizes of the aggregates. In the solutions with low urea concentrations (below 2 M) the sizes of aggregates decreased, while higher urea concentrations (2–8 M) induced formation of larger aggregates due to the unfolding of the protein.     

Transient HMGB protein interactions with B-DNA duplexes and complexes

HMGB proteins are abundant, non-histone proteins in eukaryotic chromatin. HMGB proteins contain one or two conserved “HMG boxes” and can be sequence-specific or nonspecific in their DNA binding. HMGB proteins cause strong DNA bending and bind preferentially to deformed DNAs. We wish to understand how HMGB proteins increase the apparent flexibility of non-distorted B-form DNA. We test the hypothesis that HMGB proteins bind transiently, creating an ensemble of distorted DNAs with rapidly interconverting conformations. We show that binding of B-form DNA by HMGB proteins is both weak and transient under conditions where DNA cyclization is strongly enhanced. We also detect novel complexes in which HMGB proteins simultaneously bind more than one DNA duplex.     

Mediation of the A/B-DNA helix transition by G-tracts in the crystal structure of duplex CATGGGCCCATG

The crystal structure of the DNA dodecamer duplex CATGGGCCCATG lies on a structural continuum along the transition between A- and B-DNA. The dodecamer possesses the normal vector plot and inclination values typical of B-DNA, but has the crystal packing, helical twist, groove width, sugar pucker, slide and x-displacement values typical of A-DNA. The structure shows highly ordered water structures, such as a double spine of water molecules against each side of the major groove, stabilizing the GC base pairs in an A-like conformation. The different hydration of GC and AT base pairs provides a physical basis for solvent-dependent facilitation of the A↔B helix transition by GC base pairs. Crystal structures of CATGGGCCCATG and other A/B-DNA intermediates support a ‘slide first, roll later’ mechanism for the B→A helix transition. In the distribution of helical parameters in protein–DNA crystal structures, GpG base steps show A-like properties, reflecting their innate predisposition for the A conformation.     

Refined crystal structure of deoxyhemoglobin S. II. Molecular interactions in the crystal

The refined crystal structure of deoxyhemoglobin S (Padlan, E. A., and Love, W. E. (1985) J. Biol. Chem. 260, 8272-8279) was used to analyze in detail the molecular interactions between hemoglobin tetramers in the crystal. The analysis confirms the close similarity and also the nonequivalence of the molecular interactions involving the two independent tetramers in the asymmetric unit of the crystal. The residue at the site of the hemoglobin S mutation, beta 6, is intimately involved in the lateral contacts between adjacent molecules. The molecular contacts in the crystals of deoxyhemoglobin S, deoxyhemoglobin A, and deoxyhemoglobin F were compared; some contacts involve the same regions of the molecule although the details of the interactions are very different. The effect of introducing an R state tetramer into the deoxyhemoglobin S strands was investigated using the known structure of carbon monoxyhemoglobin A. It was found that substituting a molecule of carbon monoxyhemoglobin A for one of the deoxyhemoglobin S tetramers results in extensive molecular interpenetration.     

Intermolecular histone H4 interactions in core nucleosomes

Chicken histone H4, labeled at methionine-84 with 1-N-pyrenyliodoacetamide, has been incorporated into a nucleosome-like particle with core length DNA and unmodified histones H2A, H2B, and H3. These synthetic nucleosomes exhibit properties very similar to those displayed by native particles and those labeled with other fluors. The emission spectrum of the pyrene-labeled nucleosome was characteristic of excited dimer (excimer) fluorescence, indicating that the single pyrene groups on the two H4 molecules are in close proximity in the reconstituted particle. Histone H4 was also labeled randomly at lysines with a group that contains two pyrene moieties separated by 12 A at most. Incorporation of this histone into nucleosome-like particles provides an excimer standard which does not depend on intermolecular interactions. The properties of the pyrene-containing nucleosome were examined as a function of ionic strength. It was found that the H4-H4 pyrene excimer fluorescence exhibited a cooperative disruption centered at 0.1 M NaCl which preceded increases in accessibility and environment polarity revealed by other fluors attached at the same site.     

Intermolecular interactions in a two-layered viral capsid that requires a complex symmetry mismatch

The surface of the bluetongue virus core forms a T=13 quasiequivalent icosahedral protein shell with 260 trimers of a single gene product: VP7 protein. Underneath is a smooth layer, made up of VP3 protein, which appears to guide and nucleate the assembly of VP7 trimers. The contacts between the two shells are extensive but nonspecific, and construction of the T=13 icosahedral shell requires polymorphism in the association of the VP7 subunits, each of which has two domains that contribute to trimer formation. We used structural and relative sequence information to guide an investigation of how such a complex structure is achieved during virus assembly and what residues are required to form a stable capsid. Fifteen single or multiple site-specific substitution mutations were introduced into the helical domain of VP7, which is closely associated with the VP3 layer, and the effects on capsid assembly were analyzed. Our data show that both the position and the nature of single residues are critical for the attachment of VP7 to VP3 and that formation of a stable VP7 lattice is not the automatic consequence of trimer formation.     

Intermolecular interactions between retroviral Gag proteins in the nucleus

The retroviral Gag polyprotein directs virus particle assembly, resulting in the release of virions from the plasma membranes of infected cells. The earliest steps in assembly, those immediately following Gag synthesis, are very poorly understood. For Rous sarcoma virus (RSV), Gag proteins are synthesized in the cytoplasm and then undergo transient nuclear trafficking before returning to the cytoplasm for transport to the plasma membrane. Thus, RSV provides a useful model to study the initial steps in assembly because the early and later stages are spatially separated by the nuclear envelope. We previously described mutants of RSV Gag that are defective in nuclear export, thereby isolating these “trapped” Gag proteins at an early assembly step. Using the nuclear export mutants, we asked whether Gag protein-protein interactions occur within the nucleus. Complementation experiments revealed that the wild-type Gag protein could partially rescue export-defective Gag mutants into virus-like particles (VLPs). Additionally, the export mutants had a trans-dominant negative effect on wild-type Gag, interfering with its release into VLPs. Confocal imaging of wild-type and mutant Gag proteins bearing different fluorescent tags suggested that complementation between Gag proteins occurred in the nucleus. Additional evidence for nuclear Gag-Gag interactions was obtained using fluorescence resonance energy transfer, and we found that the formation of intranuclear Gag complexes was dependent on the NC domain. Bimolecular fluorescence complementation allowed the direct visualization of intranuclear Gag-Gag dimers. Together, these experimental results strongly suggest that RSV Gag proteins are capable of interacting within the nucleus.     

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.     

X-ray studies of water structure in 2 Zn insulin crystal

The water structure of rhombohedral 2 Zn insulin crystal which contains about 280 water molecules and 0.55-0.60 mol citrate molecules per dimer has been studied by X-ray crystallographic refinement with 1.1 A resolution data. Atomic parameters of 83 fully occupied and 258 partially occupied water molecules and 0.3 mol of citrate were obtained. Full matrix least-squares method with isotropic temperature factor was used for the refinement of partially occupied water molecules. The water molecules in this crystal exist in one of the three states: fully occupied water, partially occupied water and water continuum, and a schematic model of water structure in protein crystal was proposed. The flexibility of water molecules is described.     

Non-additive interactions of nucleobases in model dinucleotide steps occurring in B-DNA crystals

Non-additivity of base-base interactions in all ten possible model dinucleotide steps were analyzed on MP2/aug-cc-pvDZ quantum chemistry level. Conformations of four nucleobases exactly matched to ones occurring in B-DNA crystals. In most of thw 162 analyzed tetramers both three- and four-body contributions are negligible except for d(GpG) steps. However, in these dinucleotides both contributions are always of opposite signs and in all cases the sum of all non-additive part of intermolecular interactions do not exceed 2.6 kcal mol^-1. This stands for less than 5% of the overall binding energy of dinucleotide steps. Also replacements of guanine with 8-oxoguanine in d(GpG) systems introduces non-additivity of the same magnitude as for canonical dinucleotides. It is observed linear relationships between values of total binding energy obtained in the tetramer basis set and estimated energy exclusively in dimers basis sets with assumption of pairwise additivities. For all analyzed dinucleotides steps there are also linear correlations between amount of non-additive contributions and magnitude of pairs interactions. Based on differences in electrostatic contribution to the total binding energy of four nucleobases and polarity of dinucleotide steps three distinct classes of dinucleotide steps were identified.