Structural requirements of N-glycosylation of proteins. Studies with proline peptides as conformational probes

Conformational aspects of N-glycosylation have been investigated with a series of proline-containing peptides as molecular probes. The results demonstrate that, depending on the position of the imino acid in the peptide chain, dramatic alterations of glycosylation rates are produced, pointing to a critical contribution of the amino acids framing the 'marker sequence' triplet Asn-Xaa-Thr(Ser) on the formation of a potential sugar-attachment site. No glycosyl transfer at all was detectable to those peptides containing a proline residue either in position Xaa or in the next position beyond the threonine of the Asn-sequon on the C-terminal side, whereas the hexapeptide Pro-Asn-Gly-Thr-Ala-Val was glycosylated at a high rate. (Emboldened residues denote the 'marker sequence' that is identical in all the peptides; italicized residues distinguish the positions of proline in the various peptides.) Studies with space-filling models reveal that the lack of glycosyl-acceptor capabilities of Ala(Pro)-Asn-Gly-Thr-Pro-Val might be directly related to their inability to adopt and/or stabilize a turn or loop conformation which permits the catalytically essential interaction between the hydroxy amino acid and the asparagine residue within the 'marker sequence' [Bause & Legler (1981) Biochem. J. 195, 639-644]. This conclusion is supported by circular-dichroism spectroscopic data, which suggest structure-forming potentials in this type of non-acceptor peptides dominating over those that favour the induction of an appropriate sugar-attachment site in the acceptor peptides. The lack of acceptor properties of Tyr-Asn-Pro-Thr-Ser-Val indicates that even small modifications in the 'recognition' pattern are not tolerated by the N-glycosyltransferases.     

Conformational analysis of cyclic peptides in solution

The strategy and tactics of conformational analysis of cyclic peptides in solution is demonstrated by the example of cyclo(-D-Pro-Phe-Thr-Phe-Trp-Phe-). Spin-locked experiments like rotating frame nuclear Overhauser enhancement spectroscopy (ROESY), ROTO, and TOCSY are successfully applied to assign all proton signals and to obtain distance information. A crude conformational model was built using the nmr data. This starting model was refined by restrained molecular dynamics (MD) calculations using ROE derived distances and fixed bond angles as determined from homo- and heteronuclear coupling constants. To mimic the solvent and to reduce artifacts in an in vacuo calculation the charges of the solvent-exposed NH protons were gradually reduced according to the temperature gradients. The thus obtained "conformation" (mean of a 40 ps MD trajectory) shows very close similarity to x-ray structures in an orthorhombic and in two monoclinic crystal modifications of the same compound. The main difference is the breaking of an intermolecular hydrogen bond of the threonine hydroxyl group on dissolution of the crystal and forming an intramolecular hydrogen bond in solution.     

Conformational analysis of two cyclic disulfide peptides

Complete nmr and CD studies of two cyclic tetrapeptides with disulfide bonds, Ac-L-Pen-L-Pro-D-Val-L-Cys-NH2 (1) and Ac-L-Cys-L-Pro-D-Val-L-Cys-NH2 (2) bonds have been carried out in different solvents to investigate the formation and stabilization of beta-turn structures and to determine the stereochemistry of the disulfide linkage. Both peptides have three-dimensional structures with a type II beta-turn, as derived from quantitative nuclear Overhauser effect data. The combined use of CD and nmr indicates that the dihedral angle of the disulfide bridge is different in the two peptides, although the chirality is maintained.     

Studies on cyclic peptides. II. Synthesis of cyclic peptides containing sarcosine.

Cyclic peptides containing sarcosine, cyclo-(Pro-Sar-Gly)₂, cyclo-(Sar-Sar-Gly)₂, cyclo-(Sar₄), and cyclo-(Sar₆) have been synthesized by the cyclization of the p-nitrophenyl ester of linear peptides. The tert-butoxycarbonyl group was used as the N^α-protecting group, which was removed by acid. Benzyl ester was used to protect the C-terminal. tert-butoxycarbonylpeptide was obtained by the stepwise elongation of the peptide bond by the carbodiimide method. Deblocking and cyclization of the linear peptides gave the cyclic peptides.     

Solution stability of linear vs. cyclic RGD peptides.

Arg-Gly-Asp (RGD) peptides contain an aspartic acid residue that is highly susceptible to chemical degradation and leads to the loss of biological activity. Our hypothesis is that cyclization of RGD peptides via disulphide bond linkage can induce structural rigidity, thereby preventing degradation mediated by the aspartic acid residue. In this paper, we compared the solution stability of a linear peptide (Arg-Gly-Asp-Phe-OH; 1) and a cyclic peptide (cyclo-(1, 6)-Ac-Cys-Arg-Gly-Asp-Phe-Pen-NH2; 2) as a function of pH and buffer concentration. The decomposition of both peptides was studied in buffers ranging from pH 2-12 at 50 degrees C. Reversed-phase HPLC was used as the main tool in determining the degradation rates and pathways of both peptides. Fast atom bombardment mass spectrometry (FAB-MS), electrospray ionization mass spectrometry (ESI-MS), matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry, liquid chromatography-mass spectrometry (LC-MS), and one- and two-dimensional nuclear magnetic resonance spectroscopy (NMR) were used to characterize peptides 1 and 2 and their degradation products. In addition, co-elution with authentic samples was used to identify degradation products. Both peptides displayed pseudo-first-order kinetics at all pH values studied. The cyclic peptide 2 appeared to be 30-fold more stable than the linear peptide 1 at pH 7. The degradation mechanisms of linear (1) and cyclic (2) peptides primarily involved the aspartic acid residue. However, above pH 8 the stability of the cyclic peptide decreased dramatically due to disulphide bond degradation. Both peptides also exhibited a change in degradation mechanism upon an increase in pH. The increase in stability of cyclic peptide 2 compared to linear peptide 1, especially at neutral pH, may be due to decreased structural flexibility imposed by the ring. This rigidity would prevent the Asp side chain carboxylic acid from orientating itself in the appropriate position for attack on the peptide backbone.     

Stereochemical studies on cyclic peptides. Part X. Conformational analysis of hydrogen bonded cyclic pentapeptides.

Conformational aspects of 4 leads to 1 hydrogen bonded cyclic pentapeptides are considered in this paper from the point of view of "contact criteria" and potential energy calculations. Three types of such hydrogen bonded conformations, designated A1, A2 and B, are possible, involving some amount of strain on the bond angles. The energy of hydrogen bonded cyclopentaglycyl is somewhat less than that of the five-fold symmetrical conformation. The stereochemical feasibility of introducing L- and D-alanyl resudues in these structures has also been studied and the possible types for different sequences of alanyl residues have been determined. The results are discussed further in the light of the limited data available from crystal structure and nuclear magnetic resonance studies on cyclic pentapeptides.     

Differential self-assembly behaviors of cyclic and linear peptides

Here we ask the fundamental questions about the effect of peptide topology on self-assembly. The study revealed that the self-assembling behaviors of cyclic and linear peptides are significantly different in several respects, in addition to sharing several similarities. Their clear differences included the morphological dissimilarities of the self-assembled nanostructures and their thermal stability. The similarities include their analogous critical aggregation concentration values and cytotoxicity profiles, which are in fact closely related. We believe that understanding topology-dependent self-assembly behavior of peptides is important for developing tailor-made self-assembled peptide nanostructures.     

Nuclear Overhauser effects in aqueous solution as dynamic probes in short linear peptides

The possibility of obtaining interresidue NOEs from short linear peptides in aqueous solution has been investigated from an experimental point of view using peptides of various lengths (namely GGRA, LHRH and RNase S-peptide). It is shown that, provided that long (approximately 800 ms) NOESY mixing times are used, complete sets of sequential alpha N NOEs are obtainable. From the intensities and signs of the observed NOEs, the relative mobilities of different parts of the polypeptide chain can be determined.     

Conformational study of linear and cyclic peptides corresponding to the 276-284 epitope region of HSV gD-1

The results of conformational analysis of linear and cyclic peptides from the 276SALLEDPVG(284) sequence of glycoprotein D of Herpes simplex virus are presented. The epitope peptides were synthesized by SPPS and on resin cyclization was applied for preparation of cyclic compounds. Circular dichroism spectroscopy, Fourier-transform infrared spectroscopy and nuclear magnetic resonance (NMR) were used to determine of the solution structure of both linear and cyclic peptides. The results indicated that the cyclopeptides containing the core of the epitope (DPVG) as a part of the cycle have more stable beta-turn structure than the linear peptides or the cyclic analogues, where the core motif is not a part of the cycle. NMR study of H-SALLc(EDPVGK)-NH(2) confirm presence of a type I beta-turn structure which includes the DPVG epitope core.     

Studies of phosphorescent probes for proteins

1. Certain capabilities and limitations of using bound phosphorescent chromophores to study protein structure were investigated. Carbonic anhydrase inhibitors with three different arrangements of singlet and triplet energy levels relative to those of tryptophan were used to determine their ability to transfer triplet energy. 2. Ligands representing each of the three spectroscopic energy level arrangements were found to exhibit triplet-triplet energy transfer with a tryptophan residue at the active site of carbonic anhydrase. This greatly increases the number of ligands which may be useful as phosphorescent probes. 3. The efficiency of energy transfer occurs to varying degrees depending upon the inhibitor. This is a potential source of data for determining the position of the ligand in the binding site.     

Studies on cyclic peptides. I. Cyclodisarcosyl-metal salts complexes

The complexes of cyclodisarcosyl (N,N′-dimethyldiketopiperazine) have been synthesized with copper perchlorate, lithium perchlorate, barium perchlorate, silver perchlorate, silver nitrate, and boron trifluoride. It has been shown by infrared absorption spectra that the metal cations coordinate to carbonyl groups of the peptide to form insoluble complexes. It has been suggested that the complexation takes place by ion–dipole interaction. Since a linear analog of the peptide formed no insoluble complex with the metal salts, conformation of cyclodisarcosyl seems to play an important role in the complex formation. Examination of the molecular model and X-ray analysis suggest that the copper perchlorate complex of the cyclic peptide has a “2:1-sandwich” structure.     

Conformational propensities and residual structures in unfolded peptides and proteins

Ample evidence gathered over the last ten years indicates that unfolded and naturally disordered proteins and peptides can show local order in that short segments can adopt turn or polyproline II-like conformations. These findings show that unfolded states cannot be described by the so-called random coil model which assumes that individual amino acid residues sample the entire sterically accessible parts of the Ramachandran with very similar probabilities. This article reviews the experimental evidence for the notion that amino acid residues have different propensities for polyproline II, β-strand, helical and turn conformations in water. These propensities are changed by interactions with nearest neighbours. We show that for a substantial number of residues the conformational propensities in the unfolded state correlate with values for helix propagation and the Chou-Fasman propensities for β-strands. Based on the presented results we hypothesize that the conformational distributions of a representative set of short peptides could be used for predicting structural distributions of disordered peptides and proteins in the future.     

Stereochemical studies on cyclic peptides. IX. Conformational studies on cyclic tetrapeptides containing alternating cis and trans peptide units

Conformational analyses of cyclic tetrapeptides consisting of alternating cis and trans peptide units have been made using contact criteria and energy calculations. This study has been restricted to those structures having a symmetry element in the backbone ring, such as a twofold axis (d) or a center of inversion (i). There are five main results. (1) There are two distinct types of conformations, which are stereochemically favorable corresponding to each of twofold and inversion-symmetrical structures, designated as d₁, d₂ (for twofold symmetrical) and i₁, i₂ (for inversion-symmetrical). Among these, the i₁ type has the lowest energy when glycyl residues occur at all four α-carbon atoms. (2) With the glycyl residue at all four α-carbon atoms, methyl substitution at the cis peptide nitrogen atoms is possible in all the four types, whereas the substitution at trans peptide nitrogen atoms is possible only for the i₁ type. Thus only in the i₁ type can all the nitrogen atoms be methylated simultaneously. The conformation of the molecule in the crystal structure of cyclotetrasarcosyl belongs to the i₁ type. (3) When alanyl residues occur at all four α-carbon atoms, the possible symmetrical type is dependent on the enantiomorphic form and the actual sequence of the alanyl residues. (4) The methyl substitution at peptide nitrogen atoms for cyclic tetrapeptides having alanyl residues causes more stereochemical restriction in the allowed conformations than with glycyl residues. (5) The prolyl residue can be incorporated favorably at the cis-trans junction of both d and i types of structures. The results of the present study are compared with the data on cyclic tetrapeptides available from the crystal structure and nmr studies. The results show an overall agreement both regarding the type of symmetry and the conformational parameters.     

Backbone modifications in cyclic peptides. Conformational analysis of a cyclic pseudopentapeptide containing a thiomethylene ether amide bond replacement

NMR and X-ray crystallographic studies have shown that cyclic pentapeptides of the general structure cyclo(D-Xxx-Pro-Gly-Pro-Gly) possess beta- and gamma-turn intramolecular hydrogen bonds. As part of our continuing series surveying the compatibility of various amide bond replacements on peptide structure, we have synthesized cyclo(D-Phe-Pro psi[CH2S]Gly-Pro-Gly). The pseudopeptide was prepared by solid phase methods and cleaved from the resin by a new procedure involving phase transfer catalysis using K2CO3 and tetrabutylammonium hydrogen sulfate. Cyclization was carried out with the use of DPPA, HOBt, and DMAP to afford the product in 69% yield. The conformational behavior of the pseudopeptide was analyzed by 1H and 13C (1D and 2D) NMR techniques. The backbone modification replaced the amide bond that is involved in a gamma-turn intramolecular hydrogen bond in the all-amide structure. In CDCl3, the pseudopeptide adopted the same all-trans conformation as its parent, although the remaining beta-turn hydrogen bond was weaker according to delta delta/delta TNH measurements. In DMSO-d6, the all-trans conformer and a second conformer were observed in a ratio of 55:45. These conformers, which slowly interconverted on the NMR time scale, could be separately assigned; peaks due to chemical exchange were readily distinguishable by the ROESY technique as reported earlier by others. 13C and ROESY experiments suggested the minor conformer contained one cis amide bond at the Gly1-Pro2 position. Thus, both the location and type of amide surrogate are important determinants affecting the compatibility of the replacement with a particular conformational feature.     

Azobenzene as photoresponsive conformational switch in cyclic peptides.

Over the last decades azobenzene has been the most widely used optical trigger for the synthesis of photoresponsive systems ranging from poly-alpha-amino acids to innovative materials with light-controlled mechanical and optical properties. More recently, its use in form of appropriate derivatives allowed to generate cyclic peptide structures of constraint conformational space and thus to exploit its reversible photoisomerization to induce well defined transitions between different conformational states. These can be characterized in detail in both photostationary states making such systems ideal substrates for ultrafast spectroscopic analysis of conformational transitions. Moreover, the changes in biophysical properties that occur as a consequence of the different conformational states can be exploited for a photo-control of a large variety of molecular recognition processes.     

Synthetic peptides as probes of plant cell signalling

Synthetic peptides have found increasing use in dissecting cell signalling pathways and have been employed as synthetic antigens, protein kinase and protease substrates. Recently, it has become evident that relatively short (10–30mer) peptides are able to mimic that part of the signalling protein to which their sequence corresponds. In particular, peptides corresponding to the C-terminus of Zea mays auxin binding protein, ZmABP1, were able to modulate ion channel function within Vicia guard cells. In this report, GTPS binding to NaCl-washed Zea microsomal membranes is shown to be stimulated by peptide A6.2, corresponding to the C-terminal 16 residues of ZmABP1, only when the membranes are reconstituted with soluble Zea protein fractions containing GP1 and G₀ homologues.     

Marine bacteria associated with sponge as source of cyclic peptides

Two bacteria associated with the marine sponge Ircinia variabilis were isolated using commercial and experimental media. The use of media containing marine derived proteins improved the growth of both isolated bacteria, showing that marine bacteria need of marine derived proteins for a better growth. The composition of free and total fatty acids of both strains cultivated under different carbon source was investigated. Several diketopiperazines were isolated from both bacteria and the hypothesis of their role in the bacterial-spongy interaction is discussed.