Conformation analysis of D-xylose-containing O-glycopeptide sequences]

The preferred conformations of the sequences of O-glycopeptides containing a beta-D-xylosyl group linked to an L-seryl residue, found in the N-terminus of proteodermatan sulfate, show an almost extended peptide chain with the sugar unit in a specific position. The results of force-field calculations with the AMBER program have been confirmed, by n.m.r.-spectroscopic experiments, for a minimum conformation.     

Stereochemical analysis of ribosomal transpeptidation. Conformation of nascent peptide

Transpeptidation performed by the ribosome is considered as a nucleophilic Sn2 substitution reaction, passing through a tetrahedral intermediate. A stereochemically universal mechanism of the reaction is assumed to exist for all 20 amino acid residues, both in the attacked (donor) and in the attacking (acceptor) substrates. The angles of internal rotation around the bonds of the attacked carbonyl carbon and around the neighbouring bonds in the tetrahedral intermediate, as well as the stereoconfiguration of the intermediate, have been varied. All 54 combinations of the sterically allowed rotational isomers determined by the five torsional angles have been analysed by using Corey-Pauling-Koltun models and by direct calculations permitting the "extreme limits" in interatomic distances and +/- 7 degrees deviations in bond angles. Only one combination, i.e. one unique conformation of the tetrahedral intermediate, is found to be sterically compatible with all 400 possible pairs of the reacting amino acid residues and at the same time to be capable of cleaving into a planar trans-peptide group. The torsion angles phi and psi of this universally allowed intermediate and the peptide product resulting from its cleavage are similar to those in an alpha-helix. It is suggested that the ribosome generates the alpha-helical confirmation at the C-end of the nascent peptide.     

蛋白质序列的聚类结构分析

基于模糊邻近关系的粒度空间,对蛋白质序列进行聚类结构分析。利用MEGA软件计算选取的木聚糖酶序列间的比对距离,引入内积将其转化为模糊邻近关系(或矩阵),再应用算法求解其粒度空间,进行序列的聚类结构分析和最佳聚类确定研究。这些研究为蛋白质序列提供了定量分析的工具。     

Conformation analysis of modified tetrasaccharide sequences of the N-glycoprotein type--problem of the alpha-(1 to 6)-glycosidic bond

The conformational analysis of the recently synthesized tetrasaccharides alpha-D-Manp (1----3)-[alpha-D-Manp-(1----6)]-4-deoxy-beta-D-lyx-hexp+ ++-(1----4)-D-GlcNAc (2) and alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Talp -(1----4)-D-GlcNAc (3) will be described. The preferred solution conformation of 2 and 3 is a gt-conformation, which is nearly identical with the preferred conformation of the naturally occurring tetrasaccharide alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Manp -(1----4)-D-GlcNAc (1). The main structural feature is the backfolding of the alpha-(1----6)-linked D-Man to the reducing D-GlcNAc unit. Conformational analysis of the tetrasaccharides alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Manp -(1----4)-1,6- anhydro-beta-D-GlcNAc (4), alpha-D-Manp-(1----3)-alpha-D-Manp-(1----6)]-4-deoxy-beta-D- lyx-hexp-(1----4)- 1,6-anhydro-beta-D-GlcNAc (5), and alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Talp -(1----4)- 1,6-anhydro-beta-D-GlcNAc (6) gave additional proof for this backfolding. The substitution of the reducing unit leads to a smaller amount of gt- and a greater amount of gg-conformers. The method used for conformational analysis of 2-6 is a combination of n.m.r.-experiments and HSEA-calculations with the program GESA. Concerning the application of new 2D-techniques, the COLOC-experiment turned out to be extremely useful in sequencing oligosaccharides.     

Conformation of disulfide bridges in peptides with pepsin partial sequences.

On the basis of Raman spectra investigation of two model heterodetic cyclic peptides, containing partial sequences of pepsin fragments 45--50 and 206--210 of the chain, it was concluded that the disulfide bridge conformation in pepsin is determined not only by the size and conformation of the peptide loops created by disulfide bridges, but also by the peptide fragments located outside these loops.     

Conformational analysis of invariant peptide sequences in bacterial genomes

The functional significance of evolutionarily conserved motifs/patterns of short regions in proteins is well documented. Although a large number of sequences are conserved, only a small fraction of these are invariant across several organisms. Here, we have examined the structural features of the functionally important peptide sequences, which have been found invariant across diverse bacterial genera. Ramachandran angles (phi,psi) have been used to analyze the conformation, folding patterns and geometrical location (buried/exposed) of these invariant peptides in different crystal structures harboring these sequences. The analysis indicates that the peptides preferred a single conformation in different protein structures, with the exception of only a few longer peptides that exhibited some conformational variability. In addition, it is noticed that the variability of conformation occurs mainly due to flipping of peptide units about the virtual C(alpha)...C(alpha) bond. However, for a given invariant peptide, the folding patterns are found to be similar in almost all the cases. Over and above, such peptides are found to be buried in the protein core. Thus, we can safely conclude that these invariant peptides are structurally important for the proteins, since they acquire unique structures across different proteins and can act as structural determinants (SD) of the proteins. The location of these SD peptides on the protein chain indicated that most of them are clustered towards the N-terminal and middle region of the protein with the C-terminal region exhibiting low preference. Another feature that emerges out of this study is that some of these SD peptides can also play the roles of "fold boundaries" or "hinge nucleus" in the protein structure. The study indicates that these SD peptides may act as chain-reversal signatures, guiding the proteins to adopt appropriate folds. In some cases the invariant signature peptides may also act as folding nuclei (FN) of the proteins.     

Interfacial properties and structural analysis of the antimicrobial peptide NK-2.

The structure of the antimicrobial peptide NK-2 has been studied at the air-water interface and in different solutions using spectroscopic methods such as circular dichroism (CD) and infrared reflection absorption spectroscopy (IRRAS) as well as specular X-ray reflectivity (XR). NK-2 adopts an unordered structure in water, buffer, and in the presence of monomeric cationic and noncharged amphiphiles. However, it forms a stable alpha-helix in 2,2,2-trifluoroethanol (TFE) and in micellar solutions of anionic, cationic as well as nonionic amphiphiles, whereas only in sodium dodecyl sulfonate solutions the alpha-helical structure can also be found below the critical micellar concentration (cmc). The amphiphilic molecule NK-2 is surface active and forms a Gibbs monolayer at the air-buffer interface. In contrast, no adsorption was observed if NK-2 is dissolved in water. During the adsorption process in buffer solutions, NK-2 undergoes a conformational transition from random coil in bulk to alpha-helix at the interface. This change of the peptide's secondary structure is known to be associated with its antimicrobial activity. A comparison of the experimental IRRA spectra with the simulated spectra indicates that the adsorbed NK-2 alpha-helix lies flat at the interface. This is confirmed by XR measurements which show that the thickness of the NK-2 layer is approximately 17 A, which is the average diameter of a alpha-helix, indicating that only a monomolecular adsorption layer is formed.     

Molecular cloning and structural analysis of murine thymidine kinase genomic and cDNA sequences.

Two functional cytosolic thymidine kinase (tk) cDNA clones were isolated from a mouse L-cell library. An RNA blot analysis indicated that one of these clones contains a nearly full-length tk sequence and that LTK- cells contain little or no TK message. The nucleotide sequences of both clones were determined, and the functional mouse tk cDNA contains 1,156 base pairs. An analysis of the sequence implied that there is an untranslated 32-nucleotide region at the 5' end of the mRNA, followed by an open reading frame of 699 nucleotides. The 3' untranslated region is 422 nucleotides long. Thus, the gene codes for a protein containing 233 amino acids, with a molecular weight of 25,873. A comparison of the coding sequences of the mouse tk cDNA with the human and chicken tk genes revealed about 86 and 70% homology, respectively. We also isolated the tk gene from a mouse C57BL/10J cosmid library. The structural organization was determined by restriction mapping, Southern blotting, and heteroduplex analysis of the cloned sequences, in combination with a mouse tk cDNA. The tk gene spans approximately 11 kilobases and contains at least five introns. Southern blot analysis revealed that this gene is deleted in mouse LTK- cells, consistent with the inability of these cells to synthesize TK message. This analysis also showed that tk-related sequences are present in the genomes of several mouse strains, as well as in LTK- cells. These segments may represent pseudogenes.     

Conformation and dynamics of an Fab'-bound peptide by isotope-edited NMR spectroscopy.

The dynamics and conformation of the peptide antigen MHKDFLEKIGGL bound to the Fab' fragment of the monoclonal antipeptide antibody B13A2, raised against a peptide from myohemerythrin, have been investigated by isotope-edited NMR techniques. The peptides were labeled with 15N (98%) or 13C (99%) at the backbone of individual amino acid residues. Well-resolved amide proton and nitrogen backbone resonances were obtained and assigned for eight of the 12 residues of this bound peptide. Significant resonance line width and chemical shift differences were observed. The 15N and 1H line width variations are attributed to differential backbone mobilities among the bound peptide residues which are consistent with the previously mapped epitope of this peptide antigen. Local structural information was obtained from isotope-directed NOE studies. The approximate distances associated with the experimental NOEs were estimated on the basis of a theoretical NOE analysis involving the relative integrated intensities of the NOE and source peaks. In this way, the sequential NH-NH NOEs obtained for seven of the Fab'-bound peptide residues were shown to correspond to interproton separations of approximately 3 A or less. Such short distances indicate that the backbone dihedral angles of these residues are in the alpha rather than the beta region of phi,psi conformational space; the peptide most likely adopts a helical conformation from F5 to G11 within the antibody combining site. The significance of these results with respect to the type and extent of conformational information obtainable from studies of high molecular weight systems is discussed.     

Phylogenetic analysis of the sequences of gastrin-releasing peptide and its receptors: biological implications

The many biological activities of the hormone gastrin-releasing peptide (GRP), including stimulation of acid secretion and of tumour growth, are mediated by the gastrin-releasing peptide receptor (GRP-R). Here sequence comparisons are utilised to investigate the likely bioactive regions of the 125 amino acid GRP precursor and of GRP-R. Comparison of the sequences of the GRP precursor from 21 species revealed homology not only in the GRP region between amino acids 1 and 30, but also in C-terminal regions from amino acids 43 to 97. This observation is consistent with recent reports that peptides derived from the C-terminal region are biologically active. Comparison of the GRP-R sequence with the related receptors NMB-R and BRS-3 revealed that the family could be distinguished from other G-protein coupled receptors by the presence of the motif GVSVFTLTALS at the cytoplasmic end of transmembrane helix 3. Comparison of the sequences of the GRP-R from 21 species revealed that the most highly conserved regions occurred in transmembrane helices 2, 3, 5, 6 and 7, and in the third intracellular loop. These results will be important in guiding future structure-function studies of the GRP precursor and of GRP receptors.     

Conformation and lytic activity of eumenine mastoparan: a new antimicrobial peptide from wasp venom.

Eumenine mastoparan-AF (EMP-AF) is a novel membrane active tetradecapeptide recently isolated from the venom of solitary wasp, Anterhynchium flavomarginatum micado. It was reported previously that EMP-AF peptide presented low cytolytic activities in human erythrocytes and in RBL-2H3 mast cells. In the present work, we observed that this peptide is able to permeate anionic liposomes, and in less extension also the neutral ones. We present evidences showing that the permeation ability is well correlated with the amount of helical conformation assumed by the peptides in these environments. This peptide also showed a broad-spectrum inhibitory activity against Gram-positive and Gram-negative bacteria. The permeability of liposomes and the antibiotic effect showed a significant reduction when C-terminus was deamidated (in acidic form). The removal of the three first amino acid residues from the N-terminus rendered the peptide inactive both in liposomes and in bacteria. The results suggest that the mechanism of action involves a threshold in the accumulation of the peptide at level of cell membrane.     

Synthesis and structural analysis of 6-deoxy-6-hydroxyphosphinyl-D-fructopyranose derivatives

3,4-Di-O-benzyl-6-deoxy-6-diethoxyphosphinyl-1,2-O-isopropylidene-beta-D-fructofuranose (13) was prepared from the known 1,2-O-isopropylidene-6-O-tosyl-beta-D-fructofuranose in five steps. Reduction of 13 with sodium dihydrobis(2-methoxyethoxy)aluminate, followed by the action of hydrochloric acid and then hydrogen peroxide, afforded the 6-deoxy-6-hydroxyphosphinyl-D-fructopyranose derivative. This was converted into the 1,2,3,4,5-penta-O-acetyl-6-deoxy-6-methoxyphosphinyl-D-fructopyranoses, whose structure and conformation were established by 1H NMR spectroscopy.     

Conformation and packing analysis of polysaccharides and derivatives. I: Mannan

A previously described procedure for simultaneous optimization of bond lengths and angles was used to test different models for mannan I. Potential hydrogen bonds and the glycosidic angle were included in the optimization. A conformational model with bifurcated intramolecular hydrogen bonds of the type observed in the methyl cellobioside methanol complex showed the best agreement with available exprerimental data. The coordinates of this model were provided by computer calculations. The available X-ray data, however, were not sufficient for selecting this model; rather, ir data were necessary to furnish the needed information. The different conformational models tested all showed an almost constant virtual bond length O(1)–O(4) of the β-pyranose residue. This was in contrast to the previously obtained results for the α-pyranose residues.     

Analysis of protein crystallographic structural data: analysis of the orientation of peptide planes

A new dihedral angle parameter θ′, defined by C_i?1|N_i…C_i|N_i+1, involving pairs of backbone atoms in two adjacent peptide planes is used in this paper to analyse the relative orientation of the peptide planes in a protein chain. The equiangle contours for this parameter on the ?+ψ in an approximately linear way. Thus, θ′ serves as a good single parameter representation for chainfolding characterization by bringing out the relative orientation of successive peptide planes. Unlike the earlier proposed θ which gives the gross folding features, θ′ can be applied for nonregular helical features. Its utility in the detection of bend regions in protein chains is compared with that of θ through theoretically calculated tables as well as examples from actual proteins.     

BALB/c myeloma retroviruses: peptide mapping and immunological analysis of the pp12 structural protein.

We have compared the pp12 structural protein of the MO-21 and FL-1 BALB/c myeloma retroviruses with the pp12 of several prototype retroviruses. Chymotryptic peptide maps of 125I-labeled, immune-precipitated pp12 proteins revealed that the MO-21 and FL-1 proteins can be distinguished from one another. The MO-21 pp12 most closely resembled the NIH-xenotrophic virus pp12, and the FL-1 pp12 most closely resembled the pp12 of BV-2 and WN 1802 B. Competition radioimmunoassay studies showed that the MO-21 and FL-1 pp12 proteins are also antigenically distinct from one another and that both contain pp12 antigenic determinants of a xenotropic virus. These data support our proposal that these two BALB/c viruses contain a gag gene that was generated by recombination between endogenous eco- and xenotropic viral sequences.     

Chiral recognition of diastereomeric 6-cedrols by vibrational circular dichroism

The use of vibrational circular dichroism spectroscopy for the chiral recognition of the two epimers of 6-cedrol, tricyclic sesquiterpenes, which contains oxygen as the heaviest atom, is shown. Bands in the 1500-850 cm(-1) region of the spectra were analyzed to calculate the anisotropy factors (g), which provided the regions of maximum circular dichroism effect for each epimer.