Covalent Connection Research Articles

Binding Site for Blood Coagulation Factor Xa Involving Residues 311–325 in Factor Va

Factor Va inactivation by activated protein C is associated with cleavages at Arg306, Arg506, and Arg679 with Arg306 cleavage causing the major activity loss. To study functional roles of the Arg306 region, overlapping 15-mer peptides representing the sequence of factor Va residues 271-345 were synthesized and screened for anticoagulant activities. The peptide containing residues 311-325 (VP311) noncompetitively inhibited prothrombin activation by factor Xa, but only in the presence of factor Va. Fluorescence studies showed that VP311 bound to fluorescence-labeled 5-dimethylaminonaphthalene-1-sulfonyl-Glu-Gly-Arg factor Xa in solution with a Kd of 70 microM. Diisopropylphosphoryl factor Xa and factor Xa but not factor VII/VIIa or prothrombin bound to immobilized VP311 with relatively high affinity. These results support the hypothesis that residues 311-325, which are positioned between the A1 and A2 domains of factor Va and likely exposed to solvent, contribute to the binding of factor Xa by factor Va. Based on this hypothesis, it is suggested that cleavage by activated protein C at Arg306 in factor Va not only severs the covalent connection between the A1 and A2 domains but also disrupts the environment and structure of residues 311-325, thereby down-regulating the binding of factor Xa to factor Va.

Refolding of Bacteriorhodopsin from Expressed Polypeptide Fragments

Bacteriorhodopsin is a heptahelical membrane protein that can be refolded to the native state following denaturation. To analyze the in vitro folding process with independent structural domains, eight fragments comprising two (AB, FG), three (AC, EG), four (AD, DG) or five (AE, CG) of the transmembrane segments were produced by expression in Escherichia coli. The polypeptides were purified to homogeneity by solvent extraction of E. coli membranes, repeated phase separation, and anion-exchange chromatography employing the C-terminal tail of bacteriorhodopsin for adsorption. Upon reconstitution into phospholipid/detergent micelles pairs of complementary fragments (AB.CG, AC.DG, AD.EG, and AE.FG) assembled in the presence of retinal to regenerate the characteristic bacteriorhodopsin chromophore with high efficiency. Together with previous studies, these results demonstrate that the covalent connections in each of the six interhelical loops are dispensable for a correct association of the helices. The different loops, however, contribute to the stability of the folded structure, as shown by increased susceptibilities toward denaturation in SDS and at acidic pH, and decreased Schiff base pKa values for the AB.CG, AC. DG, AD.EG, and AE.FG complexes, compared with the intact protein. Notably, the heptahelical bundle structure was also generated by all possible combinations of pairs of overlapping fragments, containing one (AC.CG, AD.DG, AE.EG), two (AD.CG, AE.DG), or three (AE.CG) redundant helices. The spectral properties of the chromophores indicate that the retinal-binding pocket of the AC.CG, AD.CG, and AE. CG complexes is formed by helices A and B of the respective N-terminal fragment and the C-terminal CG fragment, whereas the AD. DG, AE.DG, and AE.EG complexes are likely to adopt a heptahelical bundle structure analogous to AD.EG. The combined data show that the specificity of the helix assembly of bacteriorhodopsin is influenced by connectivities provided by the C-D and E-F surface loops.

A “Mix and Match” Ionic−Covalent Strategy for Self-Assembly of Inorganic Multilayer Films

Multilayer thin films consisting of anionic α-zirconium phosphate (α-ZrP) sheets, tetrameric zirconium hydroxide cations [Zr4(OH)8(H2O)16]8+ (Zr48+), and alkanediylbis(phosphonic acid) (CnBPA) have been grown on silicon and gold surfaces by sequential adsorption reactions. The thin films were characterized by ellipsometry, X-ray diffraction, reflectance infrared spectroscopy, X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM). Alternately dipping cationic substrates into exfoliated α-ZrP-containing suspensions, aqueous zirconium oxychloride, and ethanolic C16BPA solutions generates a mixed ionic/covalent multilayer structure. The tetrameric Zr48+ cation adsorbs onto the α-ZrP surface, providing a covalent anchoring point for the growth of the C16BPA layer. Adsorbing a second layer of zirconium ions onto the C16BPA layer allows one to continue the layer growth sequence using either covalent (metal/phosphonate) or ionic (α-ZrP/polycation) interlayer connections. A multilayer film with a repeating α-ZrP/Zr48+/C16BPA/Zr48+ sequence is sufficiently well-ordered in the stacking direction to give a Bragg peak in the diffraction pattern. The intensities of infrared absorbances in the symmetric and asymmetric C−H stretching regions, which arise from C16BPA, are linear with the C16BPA layer number. This “mix and match” approach provides a versatile means of assembling multilayer heterostructures from both ionic and covalent building blocks, with essentially any desired sequence of layers.

Effect of the Monomer Ratio on the Strengthening of Polymer Phase Boundaries by Random Copolymers

The fracture toughness Gc of an interface between the immiscible homopolymers polystyrene (PS) and poly(2-vinylpyridine) (PVP) reinforced with random copolymers, dPSf−r-PVP1-f was measured as a function of the average monomer fraction f and the areal chain density Σ of the copolymer. Long symmetric random copolymers (f ≈ 0.48) are shown to be effective in strengthening the interfaces. The effectiveness of the random copolymer at low areal chain densities results from each chain establishing multiple covalent connections across the interface. Whether these connections result from each copolymer chain crossing the interface multiple times, entangling with the homopolymer on either side of the interface, or whether these connections result from a “pairing” of chains with different monomer fractions f (resulting from composition drift) is not yet certain. The interfacial Gc increases strongly with increasing Σ above Σ* ≈ 0.004 chains/nm2 where a transition from chain scission to crazing occurs. At a high areal density (Σ > Σsat, where Σsat is the saturation areal density of the copolymer, above which the random copolymer forms a distinct and continuous layer at the interface), the fracture toughness of the interface reinforced with f = 0.48 random copolymer becomes a constant. The effectiveness of the copolymer at high Σ may be due to the presence within the random copolymers of significant fractions of chains with f > 0.48 as well as f < 0.48. Such a spread in composition is caused by composition drift during the bulk copolymerization. In a thick layer of such a copolymer at the interface, the dPS-rich chains will preferentially segregate to the PS/random copolymer interface while the PVP-rich chains will preferentially segregate to the random copolymer/PVP interface, resulting in an overall interface that is graded in composition and highly entangled. For asymmetric random copolymers (f = 0.77, 0.60, 0.39, 0.25), the effectiveness decreases markedly as the copolymer becomes less entangled with the homopolymer (corresponding to the minor component in the copolymer) on one side of the interface. The maximum Gc for the interface saturated with the random copolymer decreases significantly as f deviates from 0.5.

Chemical Cleavage of 5′-Linked Protein from Tobacco Ringspot Virus Genomic RNAs and Characterization of the Protein–RNA Linkage

Each of the two genomic RNAs of tobacco ringspot nepovirus is known to have a 5′-linked protein, the VPg. We report a simplified analysis of the covalent VPg-RNA connection that allowed us to identify the 5′ nucleotide residue of each genomic RNA and its phosphodiester link to a specific serine residue of the VPg, without resorting toin vivolabeling with32P,in vitroradioiodination, or separation of the two genomic RNAs. Unfractionated genomic RNA was incubated with an oligodeoxyribonucleotide specific for the 5′ region of either RNA 1 or RNA 2 and ribonuclease H. Reaction products were 3′-end-labeled and were fractionated by gel electrophoresis. The most highly labeled product derived from each genomic RNA was identified as a VPg-oligoribonucleotide (VPg-5′-oligo) by its sensitivity to proteinase. In a presumed β-elimination reaction that apparently was more rapid than phosphodiester cleavage, incubation in alkaline sodium bicarbonate released a rapidly migrating product, 5′-oligo. Phosphatase-treated 5′-oligo accepted 5′-label in a polynucleotide kinase-catalyzed reaction, and uridylate was identified as the 5′ terminal residue for both RNA 1 and RNA 2. Results from Edman degradation of the VPg suggest that the VPg is linked at serine 5 to the 5′ uridylate of each genomic RNA.

Molecular Recognition and Phase Transfer of Underivatized Amino Acids by a Foldable Artificial Host

A novel molecular host 5 for amino acid zwitterions is described. The covalent connection of a chiral bicyclic guanidinium salt as an anchor function for the carboxylate and a triaza-crown ether as an ammonium binding moiety in combination with a strongly hydrophobic silyl ether serves to complex and transfer amino acids from water into dichloromethane with unprecedented efficiency. Quantitation of the extraction process by radiometry revealed a clean 1:1 stoichiometry and gives evidence for the zwitterion as the species undergoing phase transfer. Even small hydrophilic (Ser, Gly) but no charged amino acids were extracted better by factors up to 3000-fold than by a previously reported host of similar design (Galán, A.; Andreu, D.; Echavarren, A. M.; Prados, P.; de Mendoza, J. J. Am. Chem. Soc. 1992, 114, 1511−1512). Some enantioselection in the transfer of phenylalanine was observed (40% ee).

Photophysical properties of structurally deformed basket-handle prophyrins

The fluorescence properties of a series of structurally deformed basket-handle porphyrins in different solvents are reported. The deformation is induced by the covalent connection of the meso-phenyl groups of meso-5, 10, 15, 20-tetraphenylporphyrin (H2TPP)via bridging groups of varying nature and chain length. The structures calculated using molecular mechanics reveal the deformation in the porphyrin ring created by short bridging groups. The distortion in the macrocycle resulted in red shifts of the emission bands, low quantum yields, high intersystem crossing rates and marked perturbed lifetimes. Maximum effects are observed for the most distorted porphyrin. A possibility for an intramolecular electron transfer is invoked in one of the systems in which the bridging phenyl group acts as an acceptor owing to the presence of electron-withdrawing substituents.

Correlation effects in hydrogen-bonded polymer blends

In hydrogen-bonded polymer blends there are long range correlations that are a result of the combined covalent and hydrogen-bond connections. A mean field description of these mixtures is presented which indicates that there should be an infinite correlation length above some percolation threshold. There are various consequences of the compositional heterogeneities or clustering that results from these effects and these are discussed in this paper.

CONCEPTS: New dynamic algorithm for de novo drug suggestion

AbstractWe describe a new method for de novo design of molecules that bind to protein active sites. The method, CONCEPTS (Creation of Novel Compounds by Evaluation of Particles at Target Sites), places a group of atom‐like particles in the site. The particles are free to move within the site to improve binding to the protein. A key innovation of this technique is that covalent connections are made among the particles in a stochastic and dynamically reversible manner. These changes in the topology are either accepted or rejected depending on their ability to improve the total energy of the enzyme–inhibitor complex. The method is applied to two test systems: The FK506 binding protein (FKBP‐12) and HIV‐1 aspartyl protease. In both cases, we are able to predict, de novo, drugs that have striking similarities to known potent inhibitors and that can successfully be used to generate “hits” of the known inhibitors from a data base. © John Wiley &amp; Sons, Inc.

A non-lattice coordination theory of polymer solutions

Abstract We have examined the classic lattice theories of polymer solutions including the work of Chang (1939), Flory (1941), Huggins (1942), Miller (1942), and Guggenheim (1944a, b, 1952). By modifying Guggenheim's (1952) approach we have derived a generalized lattice like model without the necessity of invoking a lattice. The coordination number is logically defined in terms of the number of strongly interacting nearest neighbors, therefore it depends only upon specific solvent polymer interactions. This is accomplished by partitioning the polymer coordination number into an internal part, Zip, due to the covalent connections between monomers, and an external part, zep, due to the number of strong interactions between the polymer and the solvent. At very high polymer concentrations, where there is insufficient solvent to saturate the polymer, a modified Langmuir adsorption isotherm was used to model the solvent activities. Using experimentally determined enthalpies, we have successfully tested this model against experimental solvent activities for solutions of polyethylene glycol (PEG) (MW 150–6000) in water at temperatures from 20°C to 65°C with an average standard deviation from experiment of 0.012. The external coordination number for an effective monomer in a large polymer is 1 2 . Since an effective monomer is about one half the size of a PEG monomer, this corresponds to the interaction of one water molecule per PEG monomer. The neutron scattering study of Maconnachie et al. (1978) supports this number.

Bacteriorhodopsin can be refolded from two independently stable transmembrane helices and the complementary five-helix fragment.

This paper describes experimental tests of the hypothesis that bacteriorhodopsin (BR) can fold by the association of independently stable transmembrane helices. Peptides containing the first and second helical segments of BR were chemically synthesized. These two peptides and the complementary five-helix fragment of BR were reconstituted in three separate populations of native-lipid vesicles which were then mixed and fused to allow the fragments to interact. After addition of retinal, absorption spectroscopy of the reconstituted BR and X-ray diffraction of two-dimensional crystals of this material showed that the native structure of BR was regenerated. The first two helices of BR can therefore be considered as independent folding domains, and covalent connections in the loops connecting the helices to each other and to the rest of the molecule are not essential for the appropriate association of the helices.

Strong asymmetric induction without covalent bond connection

The reduction of ferrocenyl ketones using the aqueous suspension of β-cyclodextrin inclusion complex with sodium borohydride gave optically active alcohols.

Interdomain motion in liver alcohol dehydrogenase. Structural and energetic analysis of the hinge bending mode.

A study of the hinge bending mode in the enzyme liver alcohol dehydrogenase is made by use of empirical energy functions. The enzyme is a dimer, with each monomer composed of a coenzyme binding domain and a catalytic domain with a large cleft between the two. Superposition of the apoenzyme and holoenzyme crystal structures is used to determine a rigid rotation axis for closing of the cleft. It is shown that a rigid body transformation of the apoenzyme to the holoenzyme structure corresponds to a 10 degrees rotation of the catalytic domain about this axis. The rotation is not along the least-motion path for closing of the cleft but instead corresponds to the catalytic domain coming closer to the coenzyme binding domain by a sliding motion. Estimation of the energy associated with the interdomain motion of the apoenzyme over a range of 90 degrees (-40 to 50 degrees, where 0 degrees corresponds to the minimized crystal structure) demonstrates that local structural relaxation makes possible large-scale rotations with relatively small energy increments. A variety of structural rearrangements associated with the domain motion are characterized. They involve the hinge region residues that provide the covalent connections between the two domains and certain loop regions that are brought into contact by the rotation. Differences between the energy minimized and the holoenzyme structures point to the existence of alternative conformations for loops and to the importance of the ligands in the structural rearrangements.

Effect of papulacandin B and calcofluor white on the incorporation of mannoproteins in the wall of Candida albicans blastospores.

Incorporation of mannoproteins into the walls of Candida albicans blastospores (yeast phase) was followed by continuous labelling and pulse-chase experiments. The effect in the process of compounds that interfere with synthesis (papulacandin B) or assembly (calcofluor white) of structural polymers was also assessed. Mannoproteins which are kept in place by non-covalent bonds (mainly hydrogen bonds) were incorporated rapidly after their release into the periplasmic space, this process being blocked by calcofluor white. The stain had no effect on the incorporation of covalently linked mannoproteins. Papulacandin B inhibited formation of beta-glucans and incorporation of covalently linked mannoprotein molecules, whereas incorporation of hydrogen-bonded species took place normally. The results suggest that the formation of the non-covalent bonds between the mannoproteins occurs once they are secreted into the periplasmic space, whereas the formation of covalent connections between mannoproteins and wall glucan takes place at the level of the plasma membrane.

Proinsulin: a proposed three-dimensional structure

Empirical analysis of the primary structures of 10 mammalian C-peptides has indicated a conservation of conformation. The positioning of the C-peptide in the proinsulin molecule is essentially defined by the proposed secondary structure, the covalent connection to the A1 and B30 residues of insulin and the requirement that C-peptide lies against the exposed surface of the insulin hexamer, allowing a three-dimensional structure for proinsulin to be predicted. Conserved residues in the C-peptide are proximate to residues in insulin that are also conserved, suggesting that interactions between these residues are highly probable. Residues in insulin thought to be important for biological activity are principally those that interact with the C-peptide residues. The role of the C-peptide region in proinsulin in preventing expression of insulin activity and for nucleation of the folding of the prohormone are discussed.

Additional applications of the hexagonal conformation to calcitonin, myoglobin and other proteins of known sequence

The hexagonal concept of protein conformation has been extended in this paper to an additional number of proteins and peptides containing disulfide linkages. In most instances the formation of the required disulfide bonds can be readily achieved through the use of the hexagonal spiral structure but certain exceptions have been noted. Three zinc-containing proteins have been evaluated by this system and their metal-binding ligands have been found to be situated in the proper orientation to allow the desired complexing of the metal. The resulting conformations also fulfill other amino acid requirements for the active site in considerable detail. Myoglobin is another protein for which the known sequence allows the heme ligands to be co-ordinated with the hexagonal configuration of the chain. In addition to the “heme-binding” histidines of myoglobin, this model consigns a large fraction of the total histidines to this same segment of the structure. These new protein studies have also served to support the idea that in a large number of proteins the subunit structure may consist of two “half-subunits” arranged back to back in the form of two hexagonal disks having a covalent peptide connection near the midpoint of the complete sequence. It is pointed out that this “half and half” arrangement is also appearing as a feature of several known crystal structures.

The Structure of Plant Cell Walls

Degradative enzymes have been used to obtain defined fragments of the isolated cell walls of suspension-cultured sycamore cells. These fragments have been purified and structurally characterized. Fragments released from endopolygalacturonase-pretreated cell walls by a purified endoglucanase and the fragments extracted from these walls by urea and alkali provide evidence for a covalent connection between the xyloglucan and pectic polysaccharides. Fragments released by a protease from endopolygalacturonase-endoglucanase-pretreated cell walls provide evidence for a covalent connection between the pectic polysaccharides and the structural protein of the cell wall. Based on these interconnections and the strong binding which occurs between the xyloglucan and cellulose, a tentative structure of the cell wall is proposed.

Effect of cycloheximide on yeast cell wall synthesis

Incorporation of mannoproteins into the walls of Candida albicans blastospores (yeast phase) was followed by continuous lebelling and pulse-chase experiments. The effect in the process of compounds that interfere with synthesis (papulacandin B) or assembly (calcofluor white) of structural polymers was also assessed. Mannoproteins which are kept in place by non-covalent bonds (mainly hydrogen bonds) were incorporated rapidly after their release into the periplasmic space, this process being blocked by clacofluor white. The stain had no effect on the incorporation of covalently linked mannoproteins. Papulacandin B inhibited formation of β-glucans and incorporation of covalently linked mannoprotein molecules, whereas incorporation of hydrogen-bonded species took place normally. The results suggest that the formation of the non-covalent bonds between the mannoproteins occurs once they are secreted into the periplasmic space, whereas the formation of covalent connections between mannoproteins and wall glucan takes place at the level of the plasma membrane.

Peculiarities of the distribution of the hydrochloride of the diethylaminoethyl thioester of diphenylhydroxythioacetic acid in the body

The author studied the peculiarities of the distribution of thioester in the animal body in different ways of its administration in experiments in vivo and in vitro. It was demonstrated that no stechiometric relationship exists in the interaction of the thioester with the chemical structure of the brain tissue. The study of the distribution of the radioactive thioester in the animal body demonstrated that there is no stability in retention of this preparation by the biochemical structures of the brain tissue. Its interaction with the tissue substrates takes place not by the formation of the covalent connections where stechiometric relationships are imperative, but evidently as a result of adsorption.