Channel-forming, self-assembling, bishelical amphiphilic peptides: design, synthesis and crystal structure of Py(Aibn)2, n=2,3,4.

The design, synthesis, characterization and self-assembling properties of a new class of amphiphilic peptides, constructed from a bifunctional polar core attached to totally hydrophobic arms, are presented. The first series of this class, represented by the general structure Py(Aibn)2 (Py=2,6-pyridine dicarbonyl unit; Aib=alpha, alpha'-dimethyl glycine; n=1-4), is prepared in a single step by the condensation of commercially available 2,6-pyridine dicarbonyl dichloride with the methyl ester of homo oligoAib peptide (Aibn-OMe) in the presence of triethyl amine. 1H NMR VT and ROESY studies indicated the presence of a common structural feature of 2-fold symmetry and an NH...N hydrogen bond for all the members. Whereas the Aib3 segment in Py(Aib3)2 showed only the onset of a 3(10)-helical structure, the presence of a well-formed 3(10)-helix in both Aib4 arms of Py(Aib4)2 was evident in the 1H NMR of the bispeptide. X-ray crystallographic studies have shown that in the solid state, whereas Py(Aib2)2 molecules organize into a sheet-like structure and Py(Aib3)2 molecules form a double-stranded string assembly, the tetra Aib bispeptide, Py(Aib4)2, is organized to form a tetrameric assembly which in turn extends into a continuous channel-like structure. The channel is totally hydrophobic in the interior and can selectively encapsulate lipophilic ester (CH3COOR, R=C2H5, C5H11) molecules, as shown by the crystal structures of the encapsulating channel. The crystal structure parameters are: 1b, Py(Aib2)2, C25H37N5O8, sp. gr. P2(1)2(1)2(1), a=9.170(1) A, b=16.215(2) A, c=20.091(3) A, R=4.80; 1c, Py(Aib3)2, C33H51N7O10H2O, sp. gr. P1, a=11.040(1) A, b=12.367(1) A, c=16.959(1) A, alpha =102.41 degrees, beta =97.29 degrees, gamma =110.83 degrees, R1=6.94; 1 da, Py(Aib4)2.et ac, C41H65N9O12.1.5H2O.C4H8O2, sp. gr. P1, a=16.064(4) A, b=16.156 A, c=21.655(5) A, alpha =90.14(1)degrees, beta=101.38(2) degrees, gamma=97.07(1)degrees, Z=4, R1=9.03; 1db, Py(Aib4)2.amylac, C41H65N9O12.H2O.C7H14O2, P2(1)/c, a=16.890(1) A, b=17.523(1)A, c=20.411(1) A, beta=98.18 degrees, Z=4, R=11.1 (with disorder).     

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.     

Conformational characteristics of peptides and unanticipated results from crystal structure analyses

Preferred conformation and types of molecular folding are some of the topics that can be addressed by structure analysis using x-ray diffraction of single crystals. The conformations of small linear peptide molecules with 2-6 residues are affected by polarity of solvent, presence of water molecules, hydrogen bonding with neighboring molecules, and other packing forces. Larger peptides, both cyclic and linear, have many intramolecular hydrogen bonds, the effect of which outweighs any intermolecular attractions. Numerous polymorphs of decapeptides grown from a variety of solvents, with different cocrystallized solvents, show a constant conformation for each peptide. Large conformational changes occur, however, upon complexation with metal ions. A new form of free valinomycin grown from DMSO exhibits near three-fold symmetry with only three intramolecular hydrogen bonds. The peptide is in the form of a shallow bowl with a hydrophobic exterior. Near the bottom of the interior of the bowl are three carbonyl oxygens, spaced and directed so that they are in position to form three ligands to a K+, e.g., complexation can be completed by the three lobes containing the beta-bends closing over and encapsulating the K+ ion. In another example, free antamanide and the biologically inactive perhydro analogue, in which four phenyl groups become cyclic hexyl groups, have essentially the same folding of backbone and side chains. The conformation changes drastically upon complexation with Li+ or Na+. However, the metal ion complex of natural antamanide has a hydrophobic globlar form whereas the metal ion complex of the inactive perhydro analogue has a polar band around the middle. The structure results indicate that the antamanide molecule is in a complexed form during its biological activity. Single crystal x-ray diffraction structure analyses have identified the manner in which water molecules are essential to creating minipolar areas on apolar helices. Completely apolar peptides, such as membrane-active peptides, can acquire amphiphilic character by insertion of a water molecule into the helical backbone of Boc-Aib-Ala-Leu-Aib-Ala-Leu-Aib-Ala-Leu-Aib-OMe, for example. The C-terminal half assumes an alpha-helix conformation, whereas the N-terminal half is distorted by an insertion of a water molecule W(1) between N(Ala5) and O(Ala2), forming hydrogen bonds N(5)H...W(1) and W(1)...O(2). The distortion of the helix exposes C = O(Aib1) and C = O(Aib4) to the outside environment with the consequence of attracting additional water molecules. The leucyl side chains are on the other side of the molecule. Thus a helix with an apolar sequence can mimic an amphiphilic helix.     

Crystal structure analysis of sea lamprey hemoglobin at 2 Å resolution

The crystal structure of the predominant hemoglobin component of blood from the sea lamprey, Petromyzon marinus, has been determined by X-ray diffraction analysis. Crystals for this analysis were grown from cyanide methemoglobin V as crystal type D₂. These crystals are in space group P2₁2₁2₁ and have unit cell dimensions of $\text{a = 44.57}\text{A}\text{?}\text{, b = 96.62}\text{A}\text{?}\text{and}\text{c = 31.34}\text{A}\text{?}$. Isomorphous heavyatom derivatives were prepared by soaking crystals in solutions of Hg(CN)₂, K₂Hg(CNS)₄ and KAu(CN)₂. Diffracted intensities to as far as 2 Å spacings were measured on a diffractometer. Phases were found by means of the isomorphous replacements and anomalous scattering, with supplementary information provided by the tangent formula. An atomic model was fitted to the final electron density map in a Richards optical comparator. The lamprey hemoglobin molecule is generally similar in structure to other globins, but differs in many details. Each molecule is in contact with ten neighboring molecules in the crystal lattice. The nature of the binding of the heavy atoms to lamprey hemoglobin has been interpreted.     

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.     

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.     

Towards irreversible HIV inactivation: stable gp120 binding by nucleophilic antibodies

Conventional antibodies react with antigens reversibly. We report the formation of unusually stable complexes of HIV gp120 and nucleophilic antibodies raised by immunization with an electrophilic HIV gp120 analog (E-gp120). The stability of the complexes was evident from their very slow dissociation in a nondenaturing solvent (approximate t(1/2) 18.5 days) and their resistance to dissociation by a denaturant commonly employed to disrupt noncovalent protein-protein binding (sodium dodecyl sulfate). Kinetic studies indicated time-dependent and virtually complete progression of the antibody-gp120 complexes from the initial noncovalent state to a poorly dissociable state. The antibodies to E-gp120 displayed improved covalent reactivity with an electrophilic phosphonate probe compared to control antibodies, suggesting their enhanced nucleophilicity. One of the stably binding antibodies neutralized the infectivity of CCR5-dependent primary HIV strains belonging to clades B and C. These findings suggest the feasibility of raising antibodies capable of long-lasting inactivation of antigens by electrophilic immunization.     

Naturally occurring proteolytic antibodies: selective immunoglobulin M-catalyzed hydrolysis of HIV gp120

We report the selective catalytic cleavage of the HIV coat protein gp120, a B cell superantigen, by IgM antibodies (Abs) from uninfected humans and mice that had not been previously exposed to gp120. The rate of IgM-catalyzed gp120 cleavage was greater than of other polypeptide substrates, including the bacterial superantigen protein A. The kinetic parameters of gp120 cleavage varied over a broad range depending on the source of the IgMs, and turnover numbers as great as 2.1/min were observed, suggesting that different Abs possess distinct gp120 recognition properties. IgG Abs failed to cleave gp120 detectably. The Fab fragment of a monoclonal IgM cleaved gp120, suggesting that the catalytic activity belongs to the antibody combining site. The electrophoretic profile of gp120 incubated with a monoclonal human IgM suggested hydrolysis at several sites. One of the cleavage sites was identified as the Lys(432)-Ala(433) peptide bond, located within the region thought to be the Ab-recognizable superantigenic determinant. A covalently reactive peptide analog (CRA) corresponding to gp120 residues 421-431 with a C-terminal amidino phosphonate diester mimetic of the Lys(432)-Ala(433) bond was employed to probe IgM nucleophilic reactivity. The peptidyl CRA inhibited the IgM-catalyzed cleavage of gp120 and formed covalent IgM adducts at levels exceeding a control hapten CRA devoid of the peptide sequence. These observations suggest that IgMs can selectively cleave gp120 by a nucleophilic mechanism and raise the possibility of their role as defense enzymes.     

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