Apolar peptide models for conformational heterogeneity, hydration, and packing of polypeptide helices: crystal structure of hepta- and octapeptides containing alpha-aminoisobutyric acid

The crystal structures of two helical peptides Boc-Val-Ala-Leu-Aib-Val-ala-Leu-OMe (VALU-7) and Boc-Val-Ala-Leu-Aib-Val-Ala-Leu-Aib-OMe (VALU-8) have been determined to a resolution of 1.0 and 0.9 A, respectively. Both the seven and eight residue peptides crystallize with two conformers per asymmetric unit. The VALU-8 conformers are completely helical and differ only at the C-terminus by a sign reversal of the phi, psi angles of the last residue. One of the VALU-7 conformers occurs as a normal alpha-helix, whereas in the other, the N(7)--O(3) alpha-type hydrogen bond is ruptured by the entry of a water molecule (W) into the helix, which in turn makes hydrogen bonds N(7)...W = 2.97 A and W...O(3) = 2.77 A. The other side of the water molecule is surrounded by a hydrophobic pocket. These two conformers give a static representation of a step in a possible helix unwinding or folding process. In the VALU-8 crystal the helices aggregate in a parallel mode, whereas the aggregation is anti-parallel in the VALU-7 crystal. The crystal parameters are VALU-7, P2(1), a = 10.203 (3) A, b = 19.744 (6) A, c = 22.561 (6) A, beta = 96.76 degrees, Z = 4, C38H69N7O10.0.5H2O, R = 6.65% for 3674 reflections observed greater than 3 sigma (F); and VALU-8, P2(1), a = 10.593 (4) A, b = 27.57 (6) A, c = 17.745 (5) A, beta = 95.76 (3) degrees, Z = 4, C42H76N8O11.0.25 CH3OH, R = 6.63% for 4701 reflections observed greater than 3 sigma (F).     

Fourth polymorph of [Phe4 Val6] antamanide (pentahydrate)

The cyclic decapeptide antamanide and the synthetic, biologically active analog [Phe4 Val6]antamanide (cyclic[ValProProPhePhe]2) crystallize in various crystal forms as a function of the solvent. The present crystalline polymorph obtained from acetone/water (also from ethanol/water and DMSO/water) crystallizes in space group P2(1)2(1)2(1) with a = 20.194 (30) A, b = 21.118 (31) A, c = 16,132 (25) A and four molecules of peptide in the unit cell. There are five cocrystallized water molecules per peptide molecule, of which four water molecules are intrinsic to the peptide molecule. Although the molecular packing is entirely different in each of the polymorphs, the conformation of the peptide molecule, including the intrinsic water molecules, is very similar in all the polymorphs.     

Transport of Cryptosporidium parvum Oocysts through Vegetated Buffer Strips and Estimated Filtration Efficiency

Vegetated buffer strips were evaluated for their ability to remove waterborne Cryptosporidium parvum from surface and shallow subsurface flow during simulated rainfall rates of 15 or 40 mm/h for 4 h. Log₁₀ reductions for spiked C. parvum oocysts ranged from 1.0 to 3.1 per m of vegetated buffer, with buffers set at 5 to 20% slope, 85 to 99% fescue cover, soil textures of either silty clay (19:47:34 sand-silt-clay), loam (45:37:18), or sandy loam (70:25:5), and bulk densities of between 0.6 to 1.7 g/cm³. Vegetated buffers constructed with sandy loam or higher soil bulk densities were less effective at removing waterborne C. parvum (1- to 2-log₁₀ reduction/m) compared to buffers constructed with silty clay or loam or at lower bulk densities (2- to 3-log₁₀ reduction/m). The effect of slope on filtration efficiency was conditional on soil texture and soil bulk density. Based on these results, a vegetated buffer strip comprised of similar soils at a slope of ≤20% and a length of ≥3 m should function to remove ≥99.9% of C. parvum oocysts from agricultural runoff generated during events involving mild to moderate precipitation.     

Controls exerted by the Aib residue: helix formation and helix reversal

The helix forming properties of the achiral alpha-amino isobutyric residue (Aib) have been demonstrated by numerous crystal structure analyses of designed and naturally occurring peptides containing one or more Aib residues in the sequence. Experimental and computational results concerning the type of helix obtained, whether the 3(10)-helix with 4 --> 1 type hydrogen bonds or the alpha-helix with 5 --> 1 hydrogen bonds or mixtures of the two, have been published. This paper deals with residues that, if inserted into a sequence, could perturb the helix-forming propensity afforded by the presence of Aib residues. Examples of structures will be presented in which Pro, Hyp, Gly-Gly, d-Ala-Gly, and Lac have been centrally placed in the sequence. In addition to the formation of helices, detailed experimentally obtained conformation information is presented for the role of the Aib residue in reversing the sense of the helix (the Schellman motif) with the consequent formation of the 6 --> 1 type hydrogen bond or a solvated 6 --> 1 hydrogen bond. Data are presented for 13 molecules with helix reversals at the C-terminus or near the center of the sequence.     

Specific HIV gp120-cleaving antibodies induced by covalently reactive analog of gp120

We report the results of efforts to strengthen and direct the natural nucleophilic activity of antibodies (Abs) for the purpose of specific cleavage of the human immunodeficiency virus-1 coat protein gp120. Phosphonate diester groups previously reported to form a covalent bond with the active site nucleophile of serine proteases (Paul, S., Tramontano, A., Gololobov, G., Zhou, Y. X., Taguchi, H., Karle, S., Nishiyama, Y., Planque, S., and George, S. (2001) J. Biol. Chem. 276, 28314-28320) were placed on Lys side chains of gp120. Seven monoclonal Abs raised by immunization with the covalently reactive analog of gp120 displayed irreversible binding to this compound (binding resistant to dissociation with the denaturant SDS). Catalytic cleavage of biotinylated gp120 by three monoclonal antibodies was observed. No cleavage of albumin and the extracellular domain of the epidermal growth factor receptor was detected. Cleavage of model peptide substrates occurred on the C-terminal side of basic amino acids, and Km for this reaction was approximately 200-fold greater than that for gp120 cleavage, indicating Ab specialization for the gp120 substrate. A hapten phosphonate diester devoid of gp120 inhibited the catalytic activity with exceptional potency, confirming that the reaction proceeds via a serine protease mechanism. Irreversible binding of the hapten phosphonate diester by polyclonal IgG from mice immunized with gp120 covalently reactive analog was increased compared with similar preparations from animals immunized with control gp120, indicating induction of Ab nucleophilicity. These findings suggest the feasibility of raising antigen-specific proteolytic antibodies on demand by covalent immunization.     

Smn, the spinal muscular atrophy-determining gene product, modulates axon growth and localization of beta-actin mRNA in growth cones of motoneurons

Spinal muscular atrophy (SMA), a common autosomal recessive form of motoneuron disease in infants and young adults, is caused by mutations in the survival motoneuron 1 (SMN1) gene. The corresponding gene product is part of a multiprotein complex involved in the assembly of spliceosomal small nuclear ribonucleoprotein complexes. It is still not understood why reduced levels of the ubiquitously expressed SMN protein specifically cause motoneuron degeneration. Here, we show that motoneurons isolated from an SMA mouse model exhibit normal survival, but reduced axon growth. Overexpression of Smn or its binding partner, heterogeneous nuclear ribonucleoprotein (hnRNP) R, promotes neurite growth in differentiating PC12 cells. Reduced axon growth in Smn-deficient motoneurons correlates with reduced beta-actin protein and mRNA staining in distal axons and growth cones. We also show that hnRNP R associates with the 3' UTR of beta-actin mRNA. Together, these data suggest that a complex of Smn with its binding partner hnRNP R interacts with beta-actin mRNA and translocates to axons and growth cones of motoneurons.     

Covalent reactivity of phosphonate monophenyl esters with serine proteinases: an overlooked feature of presumed transition state analogs

Phosphonate monoesters have been assumed to serve as noncovalent transition state analogs for enzymes capable of catalyzing transacylation reactions. Here, we present evidence for the covalent reaction of certain serine proteinases and peptidase antibody fragments with monophenyl amino(4-amidinophenyl)methanephosphonate derivatives. Stable adducts of the N-biotinylated monophenyl ester with trypsin and antibody fragments were evident under conditions that disrupt noncovalent interactions. The reaction was inhibited by the active-site-directed reagent diisopropyl fluorophosphate. Mass spectrometry of the fragments from monoester-labeled trypsin indicated phosphonylation of the active site. Irreversible inhibition of trypsin- and thrombin-catalyzed hydrolysis of model substrates was observed. Kinetic analysis of inactivation of trypsin by the N-benzyloxycarbonylated monoester suggested that the first-order rate constant for formation of covalent monoester adducts is comparable to that of the diester adducts (0.47 vs 2.0 min(-1)). These observations suggest that the covalent reactivity of phosphonate monoesters contributes to their interactions with serine proteinases, including certain proteolytic antibodies.     

Stereoelectronic features of the cinchona alkaloids determine their differential antimalarial activity.

For most potent antimalarial activity, the cinchona alkaloids appear to require certain electronic features, particularly a sufficiently acidic hydroxyl proton and an electric field direction pointing from the aliphatic nitrogen atom towards the quinoline ring. These observations are the result of an analysis of molecular electronic properties of eight cinchona alkaloids and an in vivo metabolite calculated using ab initio 3-21G quantum chemical methods in relation to their in vitro IC50 values against chloroquine-sensitive and chloroquine-resistant Plasmodium falciparum parasites. The purpose is to provide a profile of the electronic characteristics necessary for potent antimalarial activity for use in the design of new antimalarial agents and to gain insight into the mechanistic path for antimalarial activity. Distinguishing features of the weakly active epiquinine and epiquinidine include a higher dipole moment, a different direction of the electric field, a greater intrinsic nucleophilicity, lower acidity of the hydroxyl proton, a lesser electron affinity of the lowest unoccupied molecular orbitals, and a higher proton affinity than the active cinchona alkaloids. A moderately potent quinine metabolite possesses some, but not all, of the same electronic features as the most potent cinchona alkaloids. Both the positioning of the hydroxyl and aliphatic amine groups and their electronic features appear to play a crucial role for antimalarial potency of the cinchona alkaloids, most likely by controlling the ability of these groups to form effective intermolecular hydrogen bonds.     

Discovery of benzothiazole guanidines as novel inhibitors of thrombin and trypsin IV

In a project to find novel neutral P1 fragments for the synthesis of thrombin inhibitors with improved pharmacokinetic properties, fragments containing a benzothiazole guanidine scaffold were identified as weak thrombin inhibitors. WaterLOGSY (Water-Ligand Observed via Gradient SpectroscopY) NMR was used to detect fragments binding to thrombin and these fragments were followed up by Biacore A100 affinity measurements and enzyme assays. A crystal structure of the most potent compound with thrombin was obtained and revealed an unexpected binding mode as well as the key interactions of the fragment with the protein. Based on these results, the structure-based design and synthesis of a small series of optimized novel substituted benzothiazole guanidines with comparatively low pK(a) values was accomplished. Testing of these compounds against human trypsin I and human trypsin IV revealed unexpected inhibitory activity and selectivity of some of the compounds, making them attractive starting points for selective trypsin inhibitors.     

Model for immunologic testing of biomaterials]

Corrosion products and electric fields are capable of changing proteins to antigens, thus permitting the immunological system to identify the biomaterial as foreign. The reaction between corrosion products and a macro-molecule also leads to an antigen (carrier antigen), such as conformational changes of a macro-molecule, e.g. a protein, caused by the electric field at the implant surface (modified macro-molecule antigen). While the sensitivity to corrosion and the effectiveness of galvanic elements is measurable by electrochemical methods, suitable methods of determining the field strength in the vicinity of biomaterial surfaces are still unavailable. The influence of the double layer of uncoated and coated titanium surfaces on the conformation of proteins and their conversion to antigens are investigated with polyclonal antibodies capable of identifying the unchanged protein despite adsorption to the surface. 14C-marked Bovine Serum Albumin serves as a model protein. Determination of the total number of protein molecules adsorbed is effected via the detection of the emitted electrons. The quotient of the concentration of natural proteins to the concentration of adsorbed molecules gives the biocompatibility index, which is independent of the surface area, and gives an indication of the expected biocompatibility of the material. The results of the biological tests of titanium and two coating materials on titanium were confirmed in an animal experiment. It is possible that in the future immunological tests may replace experiments in animals.