Design and synthesis of AB(3)-type (A = 1,3,5-benzenetricarbonyl unit; B = Glu diOME or Glu(7) octa OMe) peptide dendrimers: crystal structure of the first generation

The first generation molecule of glutamic acid-based dendrons on a 1, 3,5-benzenetricarbonyl core leads to a cylindrical assembly as demonstrated by single crystal x-ray diffraction. The benzene pi-pi stack (A) is stabilized by vertical NH...O===C hydrogen bonding with each subunit participating in three intermolecular hydrogen bonds related by three-fold rotation symmetry.     

Broadly distributed nucleophilic reactivity of proteins coordinated with specific ligand binding activity

Covalent nucleophile-electrophile interactions have been established to be important for recognition of substrates by several enzymes. Here, we employed an electrophilic amidino phosphonate ester (EP1) to study the nucleophilic reactivity of the following proteins: albumin, soluble epidermal growth factor receptor (sEGFR), soluble CD4 (sCD4), calmodulin, casein, alpha-lactalbumin, ovalbumin, soybean trypsin inhibitor and HIV-1 gp120. Except for soybean trypsin inhibitor and alpha-lactalbumin, these proteins formed adducts with EP1 that were not dissociated by denaturing treatments. Despite their negligible proteolytic activity, gp120, sEGFR and albumin reacted irreversibly with EP1 at rates comparable to the serine protease trypsin. The neutral counterpart of EP1 reacted marginally with the proteins, indicating the requirement for a positive charge close to the electrophilic group. Prior heating resulted in altered rates of formation of the EP1-protein adducts accompanied by discrete changes in the fluorescence emission spectra of the proteins, suggesting that the three-dimensional protein structure governs the nucleophilic reactivity. sCD4 and vasoactive intestinal peptide (VIP) containing phosphonate groups (EP3 and EP4, respectively) reacted with their cognate high-affinity binding proteins gp120 and calmodulin, respectively, at rates exceeding the corresponding reactions with EP1. Reduced formation of EP3-gp120 adducts and EP4-calmodulin adducts in the presence of sCD4 and VIP devoid of the phosphonate groups was evident, suggesting that the nucleophilic reactivity is expressed in coordination with non-covalent recognition of peptide determinants. These observations suggest the potential of EPs for specific and covalent targeting of proteins, and raise the possibility of nucleophile-electrophile pairing as a novel mechanism stabilizing protein-protein complexes.     

A robust hybrid peptide crystal formed with weak hydrogen bonds

In the course of our work relating to the design of a bihelical structure (I) from diphenic anhydride by tethering with cystine di-OMe, stable, hard, and rigid crystals, mp 215-218 degrees C were isolated in low yields ( approximately 2%). The crystal structure established that it was a bis amide (II) arising from diphenic acid and cystine di-OMe [(II), C(22)H(22)N(2)O(6)S(2) (a = 9.897 (1) A, b = 12.210 (1) A, c = 18.192 (1) A, sp. gr. P2(1)2(1)2(1))]. An authentic sample of (II) was subsequently prepared in 47% yields by condensation of diphenic acid dichloride with cystine di-OMe. A most surprising feature of II was, despite its high density, rigidity, and hardness, it did not exhibit any normal hydrogen bonds. The nearest approximation to a "usual" hydrogen bond was the single NH...OC linkage that occurred between molecules along a twofold screw axis. In this linkage, N...O = 3.265 A and H...O = 2.43 A, values that are at least 10% longer than those usually observed in peptides. The rigidity of the crystals appears to depend upon many weak hydrogen bonds of the type CH...O, CH...pi, CH...S, and NH...S working in concert. Even these attractions have separations that are at the high end of the range of previously observed values, although some of the weak hydrogen bonds have been rarely reported and have poorly defined ranges. The attractive effect of each of these weak bonds may be enhanced by the occurrence of a number of them in a parallel fashion like rungs in a ladder.     

Nitration of the pollen allergen bet v 1.0101 enhances the presentation of bet v 1-derived peptides by HLA-DR on human dendritic cells

Nitration of pollen derived allergens can occur by NO(2) and ozone in polluted air and it has already been shown that nitrated major birch (Betula verrucosa) pollen allergen Bet v 1.0101 (Bet v 1) exhibits an increased potency to trigger an immune response. However, the mechanisms by which nitration might contribute to the induction of allergy are still unknown. In this study, we assessed the effect of chemically induced nitration of Bet v 1 on the generation of HLA-DR associated peptides. Human dendritic cells were loaded with unmodified Bet v 1 or nitrated Bet v 1, and the naturally processed HLA-DR associated peptides were subsequently identified by liquid chromatography-mass spectrometry. Nitration of Bet v 1 resulted in enhanced presentation of allergen-derived HLA-DR-associated peptides. Both the copy number of Bet v 1 derived peptides as well as the number of nested clusters was increased. Our study shows that nitration of Bet v 1 alters antigen processing and presentation via HLA-DR, by enhancing both the quality and the quantity of the Bet v 1-specific peptide repertoire. These findings indicate that air pollution can contribute to allergic diseases and might also shed light on the analogous events concerning the nitration of self-proteins.     

An asymmetric conformation of 1,3,5-benzene tricarbonyl [Aib4OMe]3.

Molecules of 1,3,5-benzene tricarbonyl [Aib(4)OMe](3) do not possess any internal symmetry, neither exact nor approximate, in the crystalline state. The Aib(4)OMe moieties each form a 3(10)-helix with an appropriate pair of hydrogen bonds but the sense of rotation is right-handed for two of the helices and left-handed for the third one. The helices are not evenly positioned around the benzene ring, and their helix axes are inclined toward one side of the plane of the benzene ring, giving the molecule the shape of a shallow bowl with an irregular periphery. The molecules are largely surrounded by water and dimethyl sulfoxide (DMSO) solvent molecules that form hydrogen bonds with the CO and NH moieties that protrude from the surfaces of the peptide molecule. The space group is Cc with a = 23.618(4) A, b = 19.708(6) A, c = 17.939(7) A and beta = 100.09(3) degrees.     

Genetic linkage map in sour cherry using RFLP markers

 Restriction fragment length polymorphism (RFLP) linkage maps of two tetraploid sour cherry (Prunus cerasus L., 2n=4x=32) cultivars, Rheinische Schattenmorelle (RS) and Erdi Botermo (EB), were constructed from 86 progeny from the cross RS×EB. The RS linkage map consists of 126 single-dose restriction fragment (SDRF, Wu et al. 1992) markers assigned to 19 linkage groups covering 461.6 cM. The EB linkage map has 95 SDRF markers assigned to 16 linkage groups covering 279.2 cM. Fifty three markers mapped in both parents were used as bridges between both maps and 13 sets of homologous linkage groups were identified. Homoeologous relationships among the sour cherry linkage groups could not be determined because only 15 probes identified duplicate loci. Fifty nine of the markers on the linkage maps were detected with probes used in other Prunus genetic linkage maps. Four of the sour cherry linkage groups may be homologous with four of the eight genetic linkage groups identified in peach and almond. Twenty one fragments expected to segregate in a 1 : 1 ratio segregated in a 2 : 1 ratio. Three of these fragments were used in the final map construction because they all mapped to the same linkage group. Six fragments exhibited segregation consistent with the expectations of intergenomic pairing and/or recombination. Received: 1 April 1998 / Accepted: 9 June 1998     

Peptide design: influence of a guest Aib-Pro segment on the stereochemistry of an oligo-val sequence--solution conformations and crystal structure of Boc-(Val)2-Aib-Pro-(Val)3-OMe

The peptide Boc-Val-Val-Aib-Pro-Val-Val-Val-OMe has been synthesized to investigate the effect of introduction of a strong beta-turn promoting guest segment into an oligopeptide with a tendency to form extended structures. 1H-nmr studies in solution using analysis of NH group solvent accessibility and nuclear Overhauser effects suggest an appreciable solvent dependence of conformations. In chloroform a 3(10)-helical structure is favored, while in dimethylsulfoxide an Aib-Pro beta-turn with extended arms on either side is suggested. In the crystal, the backbone forms a somewhat distorted 3(10)-helix despite the presence of a Pro residue in the middle. Among the four possible intrahelical hydrogen bonds three are of the 4----1 type and one 5----1. Head-to-tail NH...O = C hydrogen bonds link the helical molecules into continuous columns. The space group is P2(1)2(1)2(1) a = 11.320(2), b = 19.889(3), and c = 21.247(3) A.     

Medullary cystic kidney disease type 1: mutational analysis in 37 genes based on haplotype sharing

Medullary cystic kidney disease type 1 (MCKD1) is an autosomal dominant, tubulo-interstitial nephropathy that causes renal salt wasting and end-stage renal failure in the fourth to seventh decade of life. MCKD1 was localized to chromosome 1q21. We demonstrated haplotype sharing and confirmed the telomeric border by a recombination of D1S2624 in a Belgian kindred. Since the causative gene has been elusive, high resolution haplotype analysis was performed in 16 kindreds. Clinical data and blood samples of 257 individuals (including 75 affected individuals) from 26 different kindreds were collected. Within the defined critical region mutational analysis of 37 genes (374 exons) in 23 MCKD1 patients was performed. In addition, for nine kindreds RT-PCR analysis for the sequenced genes was done to screen for mutations activating cryptic splice sites. We found consistency with the haplotype sharing hypothesis in an additional nine kindreds, detecting three different haplotype subsets shared within a region of 1.19 Mb. Mutational analysis of all 37 positional candidate genes revealed sequence variations in 3 different genes, AK000210, CCT3, and SCAMP3, that were segregating in each affected kindred and were not found in 96 healthy individuals, indicating, that a single responsible gene causing MCKD1 remains elusive. This may point to involvement of different genes within the MCKD1 critical region.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Purification of lumazine proteins from Photobacterium leiognathi and Photobacterium phosphoreum: bioluminescence properties

Bright strains of the marine bioluminescent bacterium Photobacterium leiognathi produce a "lumazine protein" in amounts comparable to that previously found in Photobacterium phosphoreum. New protocols are developed for the purification to homogeneity of the proteins from both species in yields up to 60%. In dimmer strains the amounts of lumazine protein in extracts are less, and also there is an accompanying shift of the bioluminescence spectral maximum to longer wavelength, 492 nm. Both types of lumazine proteins have identical fluorescence spectra, with maxima at 475 nm, so it is suggested that, whereas lumazine protein is the major emitter in bright strains, there is a second emitter also present with a fluorescence maximum at longer wavelength. The two species of lumazine protein have the same 276 nm/visible absorbance ratio, 2.2, but differ in visible maxima: P. phosphoreum, 417 nm; P. leiognathi, 420 nm. For the latter the bound lumazine has epsilon 420 = 10 100 M-1 cm-1, practically the same as in free solution. The two lumazine proteins also differ quantitatively in their effect on the in vitro bioluminescence reaction, i.e., at blue shifting the bioluminescence spectrum or altering the kinetics. The P. phosphoreum lumazine protein is more effective with its homologous luciferase or with P. leiognathi luciferase than is the lumazine protein from P. leiognathi. These differences may have an electrostatic origin.     

Conformation of the cyclic tetrapeptide dihydrochlamydocin. Iabu-L-Phe D-Pro-LX, and experimental values for 3 leads to 1 intramolecular hydrogen bonds by X-ray diffraction.

Chlamydocin, Iabu-L -Phe-D -Pro-L X, is a naturally occurring cyclic tetrapeptide that exhibits high cytostatic activity. The conformation of the peptide ring, as well as the stereo configuration in the vicinity of the epoxide ring, have been established by a single-crystal X-ray study of dihydrochlamydocin: C₂₈H₄₀N₄O₆·H₂O. It crystallizes in the monoclinic space group P2₁ with a = 12.616(6) Å, b = 12.355(6) Å, c = 9.442(5) Å, and β = 99.5(1)°. The structure was solved by the symbolic addition procedure for phase determination followed by the tangent formula method of phase refinement. This structure represents the first cyclic tetrapeptide in which all four peptide units have been found in the trans conformation; however, each peptide unit is significantly nonplanar with ω angles deviating by 14–24° from the ideal value of 180°. This molecule contains two intramolecular 3 → 1 hydrogen bonds and experimentally determined parameters for these seven-membered turns are presented.