Hydrogen bonding in peptide helices - Analysis of two independent helices in the crystal structure of a peptide Boc-Val-Ala-Leu-Aib-Val-Ala-Phe-OMe

The crystal structure determination of the heptapeptide Boc-Val-Ala-Leu-Aib-Val-Ala-Phe-OMe reveals two peptide helices in the asymmetric unit, Crystal parameters are: space group P2(1), a = 10.356(2) Angstrom, b = 19.488(5) Angstrom, c = 23.756(6) Angstrom, beta = 102.25(2)degrees), V = 4685.4 Angstrom(3), Z = 4 and R = 5.7% for 7615 reflections [I>3 sigma(I)]. Both molecules adopt largely alpha-helical conformations with variations at the C-terminus, Helix type Is determined by analysing both 4-->1 and 5-->1 hydrogen-bond interactions and comparison with the results of analysis of protein structures. The presence of two 4-->1 hydrogen-bond interactions, besides four 5-->1 interact ions in both the conformations provides an opportunity to characterize bifurcated hydrogen bonds at high resolution, Comparison of the two helical conformations with related peptide structures suggests that distortions at the C-terminus are more facile than at the N-terminus.

Peptide models for b-turns.A circular dichroism study

The circular dichroism spectra of four 0-turn model peptides, Z-Aib-Pro-Aib-Pro- OMe (l), Piv-Pro-Aib-NHMe (2), Piv-Pro-D-Ala-NHMe (3) and Piv-Pro-Val-NHMe (4) have been examined under a wide range of solvent conditions, using methanol, hexafluoroisopropanol and cyclohexane. Type I and Type I1 0-turns have been observed for peptides 1 and 2 respectively, in the solid state, while the Pro-D-Ala sequence adopts a Type I1 Sturn in a related peptide crystal structure. A class C spectrum is observed for 1 in various solvents, suggesting a variant of a Type I(II1) structure. The Type I1 f3-turn is characterized by a CD spectrum having two positive CD bands at - 230 nm and - 202 nm, a feature observed in Piv-Pro- D-Ala-NHMe in cyclohexane and methanol and for Piv-Pro-Aib-NHMe in methanol. Peptide 2 exhibits solvent dependent CD spectra, which may be rationalized by considering Type 11, I11 and V reverse turn structures. Piv-Pro- Val-NHMe adopts nonaturn structures in polar solvents, but exhibits a class B spectrum in cyclohexane suggesting a population of Type I &turns.

Solid state and solution conformation of Boc-L-Met-Aib-L-Phe-OMe. Beta-turn conformation of a sequence related to an active chemotactic peptide analog

The conformation of the peptide Boc-L-Met-Aib-L-Phe-OMe has been studied in the solid state and solution by X-ray diffraction and 1H n.m.r., respectively. The peptide differs only in the N-terminal protecting group from the biologically active chemotactic peptide analog formyl-L-Met-Aib-L-Phe-OMe. The molecules adopt a type-II beta-turn in the solid state with Met and Aib as the corner residues (phi Met = -51.8 degrees, psi Met = 139.5 degrees, phi Aib = 58.1 degrees, psi Aib = 37.0 degrees). A single, weak 4----1 intramolecular hydrogen bond is observed between the Boc CO and Phe NH groups (N---O 3.25 A, N-H---O 128.4 degrees). 1H n.m.r. studies, using solvent and temperature dependencies of NH chemical shifts and paramagnetic radical induced line broadening of NH resonances, suggest that the Phe NH is solvent shielded in CDCl3 and (CD3)2SO. Nuclear Overhauser effects observed between Met C alpha H and Aib NH protons provide evidence of the occurrence of Met-Aib type-II beta-turns in these solvents.

Identification of a novel nucleolin related protein (NRP) gene expressed during rat spermatogenesis

Nucleolin is a major nucleolar phosphoprotein involved in various steps of ribosome biogenesis in eukaryotic cells. As nucleolin plays a significant role in ribosomal RNA transcription we were interested in examining in detail the expression of nucleolin across different stages of spermatogenesis and correlate with the transcription status of ribosomal DNA in germ cells.

The nonplanar peptide unit III. Quantum chemical calculations for related compounds and experimental X-ray diffraction data

The possible nonplanar distortions of the amide group in formamide, acetamide, N-methylacetamide, and N-ethylacetamide have been examined using CNDO/2 and INDO methods. The predictions from these methods are compared with the results obtained from X-ray and neutron diffraction studies on crystals of small open peptides, cyclic peptides, and amides. It is shown that the INDO results are in good agreement with observations, and that the dihedral angles N and defining the nonplanarity of the amide unit are correlated approximately by the relation N = -2, while C is small and uncorrelated with . The present study indicates that the nonplanar distortions at the nitrogen atom of the peptide unit may have to be taken into consideration, in addition to the variation in the dihedral angles (,), in working out polypeptide and protein structures.

Gene expression profiling during Forskolin induced differentiation of BeWo cells by differential display RT-PCR

The differentiation of cytotrophoblasts into syncytiotrophoblasts in the placenta has been employed as a model to investigate stage specific expression as well as regulation of genes during this process. While the cytotrophoblasts are highly invasive and proliferative with relatively less capacity to synthesize pregnancy related proteins, the multinucleated syncytiotrophoblasts are non-proliferative and non-invasive. However, syncytiotrophoblasts are the site of synthesis of a variety of protein, peptide and steroid hormones as well as several growth factors. Both the freshly isolated cytotrophoblasts from human placenta as well as the BeWo cell, a choriocarcinoma cell line model which retain several characteristic of cytotrophoblasts has been employed by us to study regulation of differentiation. In the present study, we have employed the differential display RT-PCR analysis (DD-RT-PCR) to evaluate gene expression changes during Forskolin induced in vitro differentiation of BeWo cells. We have identified several genes which are differentially expressed during differentiation and the differential expression of 10 transcripts was confirmed by Northern blot analysis. Based on the identity of the transcripts an attempt has been made to relate the known function of the gene products, to changes observed during differentiation. Of the several transcripts, one of the transcripts, namely Secretory Leukocyte Protease Inhibitor (SLPI) which is known to have multiple functions was found to increase 15-fold in the syntiotrophoblast.

Co-liposomes comprising a lipidated multivalent RGD-peptide and a cationic gemini cholesterol induce selective gene transfection in alpha v beta 3 and alpha v beta 5 integrin receptor-rich cancer cells

The alpha v beta 3 and alpha v beta 5 integrins, transmembrane glycoprotein receptors, are over-expressed in numerous tumors and in endothelial cells that constitute tumor blood vessels. As this protein selectively binds to the Arg-Gly-Asp (RGD) sequence containing peptides, it is an attractive way to target tumors. Herein we have developed novel formulations for integrin mediated selective gene delivery. These formulations are composed of a novel palmitoylated tetrameric RGD containing scaffold (named RAFT-RGD), cationic gemini cholesterol (GL5) and a natural helper lipid 1,2-dioleoyl-L-alpha-glycero-3-phosphatidylethanolamine (DOPE). We have optimized a co-liposomal formulation to introduce the multivalent RGD-containing macromolecule in GL5: DOPE (GL5D) mixture to produce GL5D-RGD. We have unambiguously shown the selectivity of these formulations towards cancer cells that over express alpha v beta 3 and alpha v beta 5 integrins. Two reporter plasmids, pEGFP-C3 and PGL-3, were employed for the transfection experiments and it was shown that GL5D-RGD Liposomes increased exclusively the transfection in alpha v beta 3 and alpha v beta 5 overexpressing HeLa cells.

Peptide-utilizing carbon starvation gene yjiY is required for flagella-mediated infection caused by Salmonella

Peptide metabolism forms an important part of the metabolic network of Salmonella and to acquire these peptides the pathogen possesses a number of peptide transporters. Whilst various peptide transporters known in Salmonella are well studied, very little is known about the carbon starvation (cst) genes cstA and yjiY, which are also predicted to be involved in peptide metabolism. We investigated the role of these genes in the metabolism and pathogenesis of Salmonella, and demonstrated for the first time, to the best of our knowledge, that cst genes actually participate in transport of specific peptides in Salmonella. Furthermore, we established that the carbon starvation gene yjiY affects the expression of flagella, leading to poor adhesion of the bacterium to host cells. In contrast to the previously reported role of cstA in virulence of Salmonella in Caenorhabditis elegans, we showed that yjiY is required for successful colonization of Salmonella in the mouse gut. Thus, cst genes not only contribute to the metabolism of Salmonella, but also influence its virulence.

Peptide-utilizing carbon starvation gene yjiY is required for flagella-mediated infection caused by Salmonella

Peptide metabolism forms an important part of the metabolic network of Salmonella and to acquire these peptides the pathogen possesses a number of peptide transporters. Whilst various peptide transporters known in Salmonella are well studied, very little is known about the carbon starvation (cst) genes cstA and yjiY, which are also predicted to be involved in peptide metabolism. We investigated the role of these genes in the metabolism and pathogenesis of Salmonella, and demonstrated for the first time, to the best of our knowledge, that cst genes actually participate in transport of specific peptides in Salmonella. Furthermore, we established that the carbon starvation gene yjiY affects the expression of flagella, leading to poor adhesion of the bacterium to host cells. In contrast to the previously reported role of cstA in virulence of Salmonella in Caenorhabditis elegans, we showed that yjiY is required for successful colonization of Salmonella in the mouse gut. Thus, cst genes not only contribute to the metabolism of Salmonella, but also influence its virulence.

The crystal structure of the amino-terminal pentapeptide of suzukacillin. Occurrence of a four-fold peptide helix

The monohydrate of the protected amino-terminal pentapeptide of suzukacillin, t-butoxycarbonyl--aminoisobutyryl-L-prolyl-L-valyl--aminoisobutyryl-L-valine methyl ester, C29H51N5O8, crystallizes in the orthorhombic space group P212121 with a= 10.192, b= 10.440, c= 32.959 Å, and Z= 4. The structure has been solved by direct methods and refined to an R value of 0.101 for 1 827 observed reflections. The molecule exists as a four-fold helix with a pitch of 5.58 Å. The helix is stabilised by N–H O hydrogen bonds, two of the 51 type (corresponding to the -helix) and the third of the 41 type (310 helix). The carbonyl oxygen of the amino-protecting group accepts two hydrogen bonds, one each from the amide NH groups of the third (41) and fourth (51) residues. The remaining 51 hydrogen bond is between the two terminal residues. The lone water molecule in the structure is hydrogen bonded to carbonyl oxygens of the prolyl residue in one molecule and the non-terminal valyl residue in a symmetry-related molecule.

Alamethicin and related membrane channel forming polypeptides

Alamethicin and several related microbial polypeptides, which contain a high proportion of agr-aminoisobutyric acid (Aib) residues, possess the ability to modify the permeability properties of phospholipid bilayer membranes. Alamethicin induces excitability phenomena in model membranes and has served as an excellent model for the study of voltage sensitive transmembrane channels. This review summarizes various aspects of the structural chemistry and membrane modifying properties of alamethicin and related Alb containing peptides. The presence of Aib residues in these sequences, constrains the polypeptides to 310 or agr-helical conformations. Functional membrane channels are formed by aggregation of cylindrical peptide helices, which span the lipid bilayer, forming a scaffolding for an aqueous column across the membrane. After consideration of the available data on the conductance characteristics of alamethicin channels, a working, hypothesis for a channel model is outlined. Channel aggregates in the lipid phase may be stabilized by intermolecular hydrogen bonding, involving a central glutamine residue and also by interactions between the macro-dipoles of proximate peptide helices. Fluctuations between different conductance states are rationalized by transitions between states of different aggregation and hence altered dimensions of the aqueous core or by changes in net dipole moment of the aggregate. Ion fluxes through the channel may also be affected by the electric field within the aqueous core.

Distress call-induced gene expression in the brain of the Indian short-nosed fruit bat, Cynopterus sphinx

Individuals in distress emit audible vocalizations to either warn or inform conspecifics. The Indian short-nosed fruit bat, Cynopterus sphinx, emits distress calls soon after becoming entangled in mist nets, which appear to attract conspecifics. Phase I of these distress calls is longer and louder, and includes a secondary peak, compared to phase II. Activity-dependent expression of egr-1 was examined in free-ranging C. sphinx following the emissions and responses to a distress call. We found that the level of expression of egr-1 was higher in bats that emitted a distress call, in adults that responded, and in pups than in silent bats. Up-regulated cDNA was amplified to identify the target gene (TOE1) of the protein Egr-1. The observed expression pattern Toe1 was similar to that of egr-1. These findings suggest that the neuronal activity related to recognition of a distress call and an auditory feedback mechanism induces the expression of Egr-1. Co-expression of egr-1 with Toe1 may play a role in initial triggering of the genetic mechanism that could be involved in the consolidation or stabilization of distress call memories.

A Haplotype at the UCP1 Gene Locus Contributes to Genetic Risk for Type 2 Diabetes in Asian Indians (CURES-72)

Background: The gene encoding for uncoupling protein-1 (UCP1) is considered to be a candidate gene for type 2 diabetes because of its role in thermogenesis and energy expenditure. The objective of the study was to examine whether genetic variations in the UCP1 gene are associated with type 2 diabetes and its related traits in Asian Indians. Methods: The study subjects, 810 type 2 diabetic subjects and 990 normal glucose tolerant (NGT) subjects, were chosen from the Chennai Urban Rural Epidemiological Study (CURES), an ongoing population-based study in southern India. The polymorphisms were genotyped using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Linkage disequilibrium (LD) was estimated from the estimates of haplotypic frequencies. Results: The three polymorphisms, namely -3826A -> G, an A -> C transition in the 5'-untranslated region (UTR) and Met229Leu, were not associated with type 2 diabetes. However, the frequency of the A-C-Met (-3826A -> G-5'UTR A -> C-Met229Leu) haplotype was significantly higher among the type 2 diabetic subjects (2.67%) compared with the NGT subjects (1.45%, P < 0.01). The odds ratio for type 2 diabetes for the individuals carrying the haplotype A-C-Met was 1.82 (95% confidence interval, 1.29-2.78, P = 0.009). Conclusions: The haplotype, A-C-Met, in the UCP1 gene is significantly associated with the increased genetic risk for developing type 2 diabetes in Asian Indians.

Hydrophobic Peptide Channels and Encapsulated Water Wires

Peptide nanotubes with filled and empty pores and close-packed structures are formed in closely related pentapeptides. Enantiomorphic sequences, Boc-(D)Pro-Aib-Xxx-Aib-Val-OMe (Xxx = Leu, 1; Val, 2; Ala, 3; Phe, 4) and Boc-Pro-Aib-(D)Xxx-Aib-(D)Val-OMe ((XXX)-X-D = (D)Leu, 5; (D)Val, 6; (D)Ala, 7; (D)Phe, 8), yield molecular structures with a very similar backbone conformation but varied packing patterns in crystals. Peptides 1, 2, 5, and 6 show tubular structures with the molecules self-assembling along the crystallographic six-fold axis (c-axis) and revealing a honeycomb arrangement laterally (ab plane). Two forms of entrapped water wires have been characterized in 2: 2a with d(O center dot center dot center dot O) = 2.6 angstrom and 2b with d(O center dot center dot center dot O) = 3.5 angstrom. The latter is observed in 6 (6a) also. A polymorphic form of 6 (6b), grown from a solution of methanol-water, was observed to crystallize in a monoclinic system as a close-packed structure. Single-file water wire arrangements encapsulated inside hydrophobic channels formed by peptide nanotubes could be established by modeling the published structures in the cases of a cyclic peptide and a dipeptide. In all the entrapped water wires, each water molecule is involved in a hydrogen bond with a previous and succeeding water molecule. The O-H group of the water not involved in any hydrogen bond does not seem to be involved in an energetically significant interaction with the nanotube interior, a general feature of the one-dimensional water wires encapsulated in hydrophobic environements. Water wires in hydrophobic channels are contrasted with the single-file arrangements in amphipathic channels formed by aquaporins.