Conformational solution studies of the anti-microbial temporin A retro-analogues by using NMR spectroscopy.

Temporin A (TA) is a small, basic and highly hydrophobic peptide, isolated from the skin of the European red frog, Rana temporaria. The TA (FLPLIGRVLSGIL-NH2) displays a broad spectrum of anti-microbial activity against Gram-positive bacteria and fungi Candida albicans. In this study we investigate the solution structure of two TA retro-analogues, (6-1)(7-13)-TA (GILPLFRVLSGIL-NH2) and retro-TA (LIGSLVRGILPLF-NH2) by using nuclear magnetic resonance (NMR). The 3D solution structure of the analogues was established by using inter-proton distances and vicinal coupling constants in the Simulated Annealing (SA) calculations (XPLOR program). The NMR conformational studies show the existence of the helical structure in the middle part of the (6-1)(7-13)-TA peptide and an unordered structure of the retro-TA analogue under the D3-TFE/H2O (3:7, v/v) conditions. Our investigations have shown that the hydrophobic cluster at N-terminus with the Pro amino acid residue in position 3 or 4, the helical structure and the amphipathic character of the peptide are responsible for the anti-microbial activity of the TA analogues.     

Mimicking the membrane-mediated conformation of dynorphin A-(1-13)-peptide: circular dichroism and nuclear magnetic resonance studies in methanolic solution.

The structural requirements for the binding of dynorphin to the kappa-opioid receptor are of profound clinical interest in the search for a powerful nonaddictive analgesic. These requirements are thought to be met by the membrane-mediated conformation of the opioid peptide dynorphin A-(1-13)-peptide, Tyr1-Gly2-Gly3-Phe4-Leu5-Arg6-Arg7-Ile8-Arg9-Pro10- Lys11-Leu12-Lys13. Schwyzer has proposed an essentially alpha-helical membrane-mediated conformation of the 13 amino acid peptide [Schwyzer, R. (1986) Biochemistry 25, 4281-4286]. In the present study, circular dichroism (CD) studies on dynorphin A-(1-13)-peptide bound to an anionic phospholipid signified negligible helical content of the peptide. CD studies also demonstrated that the aqueous-membraneous interphase may be mimicked by methanol. The 500- and 620-MHz 1H nuclear magnetic resonance (NMR) spectra of dynorphin A-(1-13)-peptide in methanolic solution were sequence-specifically assigned with the aid of correlated spectroscopy (COSY), double-quantum filtered phase-sensitive COSY (DQF-COSY), relayed COSY (RELAY), and nuclear Overhauser enhancement spectroscopy (NOESY). 2-D CAMELSPIN/ROESY experiments indicated that at least the part of the molecule from Arg7 to Arg9 was in an extended or beta-strand conformation, which agreed with deuterium-exchange and temperature-dependence studies of the amide protons and analysis of the vicinal spin-spin coupling constants 3JHN alpha. The results clearly demonstrated the absence of extensive alpha-helix formation. chi 1 rotamer analysis of the 3J alpha beta demonstrated no preferred side-chain conformations.     

NMR conformational analysis of proadrenomedullin N-terminal 20 peptide, a proangiogenic factor involved in tumor growth

The preferred conformation of Proadrenomedullin N-Terminal 20 Peptide (PAMP; ARLDVASEFRKKWNKWALSR-amide) has been determined using 1H and 13C two-dimensional nuclear magnetic resonance (NMR) spectroscopy and molecular modeling. PAMP is a peptide that has various physiological functions, including its role as a proangiogenic factor in facilitating tumor growth and its inhibitory effect on catecholamine secretion at nicotinic receptors. The preferred conformation of PAMP was determined in a helix-inducing trifluoroethanol and water (TFE/H2O) solution, and in a membrane-mimetic sodium dodecylsulfate-d25 (SDS) micellar solution. The secondary structure consists of an alpha-helix for residues Arg2 to Arg20 in TFE/H2O solution and an alpha-helix for residues Arg2 to Ala17 in SDS solution. We postulate that the polar charged residues Arg2, Lys12, and Arg20 are responsible for the initial interaction of the peptide with the micelle, and that this is followed by the binding of the hydrophobic residues Leu3, Val5, Phe9, Trp13, and Trp16 to the micellar core. The three C-terminal amino acid residues adopt an extended structure in SDS, suggesting that they are important in receptor recognition and binding. This is supported by truncation studies done by Mahata et al. (Hypertension, 1998, Vol. 32, pp. 907-916), which show the importance of the C-terminal in physiological activity. Furthermore, Belloni et al. (Hypertension, 1999, Vol. 33, pp. 1185-1189), and Martinez et al. (Cancer Research, 2004, Vol. 64, pp. 6489-6494) suggested that the N-terminal was also important in PAMP activity. However, no differences in conformational preference of the N-terminal were observed between the two solvent systems.     

Estimated conformation, orientation, and accumulation of dynorphin A-(1-13)-tridecapeptide on the surface of neutral lipid membranes

Equilibrium thermodynamic and kinetic estimations were used to confirm the rather unusual conformation, orientation, and accumulation of dynorphin A-(1-13)-tridecapeptide (dynorphin1-13) on the surface of neutral lipid membranes, as observed by Erne et al. [Erne, D., Sargent, D. F., & Schwyzer, R. (1985) Biochemistry 24, 4261-4263]. I started from the premise that the most stable conformation of molecularly disperse peptides in contact with the hydrophobic phase of a membrane is helical [Henderson, R. (1979) Soc. Gen. Physiol. Ser. 33, 3-15]. Calculation of the Gibbs free energy difference for the transfer of increasing numbers m of N-terminal residues of dynorphin1-13 from their random-coil conformation in water to their alpha-helical conformation in a hydrophobic phase, with the values provided by Von Heijne and Blomberg [Von Heijne, G., & Blomberg, C. (1979) Eur. J. Biochem. 97, 175-181], showed an energy minimum at m = 9 that corresponded to the observed apparent association constant of 9 X 10(4) L/mol. This confirmed our experimental observations. The orientation of dynorphin1-13 in the interphase was estimated by calculation of the molecular amphiphilic moment A. This force vector was defined in analogy to the "helical" and "structural" hydrophobic moments of Eisenberg et al. [Eisenberg, D., Weiss, R. M., & Terwilliger, T. C. (1982) Nature (London) 299, 371-374]. It takes into account the segregation of hydrophobic and hydrophilic residues with respect to the center of the alpha-helix. A peptide located in a hydrophobic-hydrophilic gradient experiences a torque that tends to orient A in a direction perpendicular to the surfaces of equal hydrophobicity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solution conformation of the type I collagen alpha-1 chain N-telopeptide studied by 1H NMR spectroscopy

The solution conformation of the type I collagen alpha-1 chain N-telopeptide has been studied by CD and 1H NMR spectroscopy at 600 MHz in CD3OH/H2O (60/40 v/v) and H2O solutions. The 19 amino acids form the N-terminal end of the alpha-1 polypeptide chain. By the combined application of several two-dimensional, phase-sensitive NMR techniques (COSY, RELAY, ROESY), a complete assignment of all proton resonances was achieved, and the conformation of the backbone could be established on the basis of the coupling constant and NOE data. In CD3OH/H2O solutions the spectroscopic evidence clearly indicates that two sections of the molecule (pE1-Y6 and T11-M19) are extended and that the D7-S10 segment forms a beta-turn, stabilized by a hydrogen bond between NH(S10) and CO(D7). The data suggest that the turn is of the type I kind (minor) and that it coexists with an extended structure (major conformer). Interactions between the two extended parts of the peptide were not observed, thus excluding the existence of a beta-sheet. In H2O solution the conformation is significantly different, with no beta-turn, but a completely extended structure is observed.     

Secondary structure of pig skin proteodermatan sulfate: a perspective from Raman spectroscopic studies in aqueous solution

Raman spectroscopic studies of pig skin proteodermatan sulfate in H2O are indicative of a well defined secondary structure consisting of alpha-helical, beta-turn, and possibly "random" structures. The above conclusion is surprisingly close to the secondary structure of the "core" protein of pig skin proteodermatan sulfate proposed in the previous paper (V. Renugopalakrishnan et al., Biopolymers 28, 1923-1933, 1989) from FT-IR and CD spectroscopic studies in aqueous solution.     

用~1H NMR和FT－IR研究hF－GRP及其类似物TF14与DMPC脂质体的相互作用

用１ＨＮＭＲ和ＦＴ－ＩＲ技术研究了肽激素ｈＦ－ＧＲＰ及其类似物ＴＦ１４与ＤＭＰＣ脂质体的相互作用及其构象变化,１Ｈ－ＮＭＲ实验结果提示这两个肽与ＤＭＰＣ之间存在静电相互作用,这使其可从水相结合到脂相中。ＦＴ－ＩＲ实验结果显示它们的构象从水相到脂相确实发生了某些调整变化,ｈＦ－ＧＲＰ趋于更加固定和弯曲,ＴＦ１４趋于更加伸展。以上工作为进一步从事ｈＦ－ＧＲＰ和ＴＦ１４受体结合的研究奠定了基础。     

1H NMR assignment and melting temperature study of cis-syn and trans-syn thymine dimer containing duplexes of d(CGTATTATGC).d(GCATAATACG)

The preparation and spectroscopic characterization of duplex decamers containing site-specific cis-syn and trans-syn thymine dimers are described. Three duplex decamers, d(CGTATTATGC).d(GCATAATACG), d(CGTAT[c,s]TATGC).d(GCATAATACG), and d(CGTAT[t,s]TATGC).d(GCATAATACG), were prepared by solid-phase phosphoramidite synthesis utilizing cis-syn and trans-syn cyclobutane thymine dimer building blocks (Taylor et al., 1987; Taylor & Brockie, 1988). NMR spectra (500 MHz 2D 1H and 202 MHz 1D 31P) were obtained in "100%" D2O at 10 degrees C, and 1D exchangeable 1H spectra were obtained in 10% D2O at 10 degrees C. 1H NMR assignments for H5, H6, H8, CH3, H1', H2', and H2" were made on the basis of standard sequential NOE assignment strategies and verified in part by DQF COSY data. Comparison of the chemical shift data suggests that the helix structure is perturbed more to the 3'-side of the cis-syn dimer and more to the 5'-side of the trans-syn dimer. Thermodynamic parameters for the helix in equilibrium coil equilibrium were obtained by two-state, all or none, analysis of the melting behavior of the duplexes. Analysis of the temperature dependence of the T5CH3 1H NMR signal gave delta H = 44 +/- 4 kcal and delta S = 132 +/- 13 eu for the trans-syn duplex. Analysis of the concentration and temperature dependence of UV spectra gave delta H = 64 +/- 6 kcal and delta S = 178 +/- 18 eu for the parent duplex and delta H = 66 +/- 7 kcal and delta S = 189 +/- 19 eu for cis-syn duplex. It was concluded that photodimerization of the dTpdT unit to give the cis-syn product causes little perturbation of the DNA whereas dimerization to give the trans-syn product causes much greater perturbation, possibly in the form of a kink or dislocation at the 5'-side of the dimer.     

用~1H NMR和FT－IR研究hF－GRP及其类似物TF14与DMPC脂质体的相互作用

用＾１ＨＮＭＲ和ＦＴ－ＩＲ技术研究了肽激素ｈＦ－ＧＲＰ及其类似物ＴＦ１４与ＤＭＰＣ脂质体的相互作用及其构象变化,＾１Ｈ－ＮＭＲ实验结果提示这两个肽与ＤＭＰＣ之间存在静电相互作用,这使其可从水相结合到脂相中。     

Sequence-specific 1H assignment and secondary structure of the bacteriocin AS-48 cyclic peptide

The bacteriocin AS-48 is a cationic peptide (7149 Da) having a broad antimicrobial spectrum, encoded by the 68 kb conjugative plasmid pMB2 from Enterococcus faecalis S-48. It is a unique peptide since it has a cyclic structure, which is achieved by the formation of a tail–head peptide bond after ribosomal synthesis (Gálvez et al., 1989; Martínez-Bueno et al., 1994; Samyn et al., 1994). Preliminary CD and calorimetric studies (data not shown) pointed towards a highly helical and very stable three dimensional structure.All the information gathered until now indicates that the target of AS-48 is the cytoplasmic membrane in which it opens channels or pores, leading to dissipation of the proton motive force and cell death, which in some cases is also followed by bacterial lysis (Gálvez et al., 1991). This peptide is a suitable tool for studying protein–membrane interactions, and it also offers promising perspectives for biotechnological applications.Knowledge of the 3D structure of AS-48 is a first step in the conduct of further structure–function studies. Here we report the complete¹ H NMR assignment of its proton resonances together with the resulting secondary structure pattern as prerequisites for the determination of a high-resolution 3D solution structure.     

Beware of proteins in DMSO

The effect on the secondary structure of representative alpha-helical, beta-sheet and disordered proteins by varying concentrations of dimethyl sulphoxide (DMSO) in 2H2O has been investigated by Fourier transform infrared spectroscopy. Significant perturbations of protein secondary structure are induced by DMSO and DMSO/2H2O mixtures. For highly structured proteins, such as myoglobin and concanavalin A, the infrared spectra point to a progressive destabilisation of the secondary structure until at moderate DMSO concentrations (around 0.33 mol fraction) intermolecular beta-sheet formation and aggregation are induced, as indicated by the appearance of a strong band at 1621 cm-1. This is a direct consequence of the disruption of intramolecular peptide group interactions by DMSO (partial unfolding). At higher DMSO concentrations (above 0.75 mol fraction), such aggregates are dissociated by disruption of the intermolecular C = O...2H-N deuterium bonds. The presence of a single amide I band at 1662 cm-1 corresponding to free amide C = O groups indicates that at high concentrations and in pure DMSO the proteins are completely unfolded, lacking any secondary structure. While low concentrations of DMSO showed no detectable effect upon the gross secondary structure of myoglobin and concanavalin A, the thermal stability of both proteins was markedly reduced. In alpha-casein, a highly unstructured protein, the situation is one of direct competition. The amide I maximum in 2H2O, at 1645 cm-1, is typical of unordered proteins with C = O groups deuterium-bonded predominantly to 2H2O. Addition of DMSO disrupts such interactions by competing with the peptide C = O group for the deuterium bond donor capacity of the 2H2O, and so progressively increases the amide I maximum until it stabilizes at 1663 cm-1, a position indicative of free C = O groups.     

Interaction of the Saccharomyces cerevisiae alpha-factor with phospholipid vesicles as revealed by proton and phosphorus NMR

Proton and phosphorus-31 nuclear magnetic resonance (1H and 31P NMR) studies of the interaction between a tridecapeptide pheromone, the alpha-factor of Saccharomyces cerevisiae, and sonicated lipid vesicles are reported. 31P NMR studies demonstrate that there is interaction of the peptide with the phosphorus headgroups, and quasielastic light scattering (QLS) studies indicate that lipid vesicles increase in size upon addition of peptide. Previous solution (aqueous and DMSO) studies from this laboratory indicate that alpha-factor is highly flexible with only one long-lived identifiable structural feature, a type II beta-turn spanning the central portion of the peptide. Two-dimensional (2D) 1H nuclear Overhauser effect spectroscopy (NOESY) studies demonstrate a marked ordering of the peptide upon interaction with lipid, suggesting a compact N-terminus, in addition to a stabilized beta-turn. In contrast to our results in both solution and lipid environment, Wakamatsu et al. [Wakamatsu, K., Okada, A., Suzuki, M., Higashijima, T., Masui, Y., Sakakibara, S., & Miyazawa, T. (1986) Eur. J. Biochem. 154, 607-615] proposed a lipid environment conformation, on the basis of one-dimensional transferred NOE studies in D2O, which does not include the beta-turn.     

Molecular structure of tetanus neurotoxin as revealed by Fourier transform infrared and circular dichroic spectroscopy

Secondary structure contents of tetanus neurotoxin have been estimated at neutral and acidic pH using circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy. An analysis of the far-ultraviolet CD spectra of the neurotoxin dissolved in 50 mM citrate-phosphate buffer (pH 7.0) revealed 20.0 +/- 2.1% alpha-helix, 50.5 +/- 2.1% beta-pleated sheets, no beta-turns, and 29.5% random coils, which is at considerable variance with results from an earlier detailed study of tetanus neurotoxin's secondary structures (J.P. Robinson, L.A. Holladay, J.H. Hash and D. Puett, J. Biol. Chem. 257 (1982) 407). However, the alpha-helix content estimated in this study is consistent with the earlier studies of Robinson et al. (J.P. Robinson, L.A. Holladay, J.B. Picklesimer and D. Puett, Mol. Cell. Biochem. 5 (1974) 147; J.P. Robinson, J.B. Picklesimer and D. Puett, J. Biol. Chem. 250 (1975) 7435) and with the study by Lazarovici et al. (P. Lazarovici, P. Yanai and E. Yavin, J. Biol. Chem. 262 (1986) 2645), although other secondary structural features do not agree with those of the previous studies. Secondary structure estimation from Fourier transform infrared spectra in both amide I and amide III frequency regions revealed 22-23% alpha-helix, 49-51% beta-pleated sheets and 27-28% random coils, indicating a good correlation with the secondary structure content estimated from CD analysis. Lowering of the pH of the neurotoxin to 5.5 or 4.0 did not result in any noticeable change in the overall secondary structures. However, there were significant pH-induced variations observed in the individual curve-fitted FT-IR bands in the amide III frequency region. For example, the 1302 cm-1 band (relative area, 4.2%) observed at pH 7.0 was shifted to 1297 cm-1 (relative area, 2.2%) at pH 5.5, and the relative area of the band at 1316-1317 cm-1 (alpha-helix) increased by approx. 40%. This study suggests that contrary to earlier reports, tetanus neurotoxin is a beta-pleated sheet dominated structure, and although lower pH does not change the overall contents of the secondary structures, significant conformational alterations are observed.     

Homo- and heteronuclear NMR studies of the human retinoic acid receptor beta DNA-binding domain: sequential assignments and identification of secondary structure elements.

An 80 amino acid polypeptide corresponding to the DNA-binding domain (DBD) of the human retinoic acid receptor beta (hRAR-beta) has been studied by 1H homonuclear and 15N-1H heteronuclear two- and three-dimensional (2D and 3D) NMR spectroscopy. The polypeptide has two putative zinc fingers homologous to those of the receptors for steroid and thyroid hormones and vitamin D3. The backbone 1H resonances as well as over 90% of the side-chain 1H resonances have been assigned by 1H homonuclear 2D techniques except for the three N-terminal residues. The assignments have been confirmed further by means of 15N-1H heteronuclear 3D techniques, which also yielded the assignments of the 15N resonances. Additionally, stereospecific assignments of methyl groups of five valine residues were made. Sequential and medium-range NOE connectivities indicate several elements of secondary structure including two alpha-helices consisting of residues E26-Q37 and Q61-E70, a short antiparallel beta-sheet consisting of residues P7-F9 and S23-C25, four turns consisting of residues P7-V10, I36-N39, D47-C50, and F69-G72, and several regions of extended peptide conformation. Similarly, two helices are found in the glucocorticoid receptor (GR) DBD in solution [H?rd et al. (1990) Science 249, 157-160] and in crystal [Luisi et al. (1991) Nature 352, 497-505], and in the estrogen receptor (ER) DBD in solution [Schwabe et al. (1990) Nature 348, 458-461], although the exact positions and sizes of the helices differ somewhat. Furthermore, long-range NOEs suggest the existence of a hydrophobic core formed by the two helices.     

Epitope mapping of antigenic MUC1 peptides to breast cancer antibody fragment B27.29: a heteronuclear NMR study

MUC1 mucin is a breast cancer-associated transmembrane glycoprotein, of which the extracellular domain is formed by the repeating 20-amino acid sequence GVTSAPDTRPAPGSTAPPAH. In neoplastic breast tissue, the highly immunogenic sequence PDTRPAP (in bold above) is exposed. Antibodies raised directly against MUC1-expressing tumors offer unique access to this neoplastic state, as they represent immunologically relevant "reverse templates" of the tumor-associated mucin. In a previous study [Grinstead, J. S., et al. (2002) Biochemistry 41, 9946-9961], (1)H NMR methods were used to correlate the effects of cryptic glycosylation outside of the PDTRPAP core epitope sequence on the recognition and binding of Mab B27.29, a monoclonal antibody raised against breast tumor cells. In the study presented here, isotope-edited NMR methods, including (15)N and (13)C relaxation measurements, were used to probe the recognition and binding of the PDTRPAP epitope sequence to Fab B27.29. Two peptides were studied: a one-repeat MUC1 16mer peptide of the sequence GVTSAPDTRPAPGSTA and a two-repeat MUC1 40mer peptide of the sequence (VTSAPDTRPAPGSTAPPAHG)(2). (15)N and (13)C NMR relaxation parameters were measured for both peptides free in solution and bound to Fab B27.29. The (13)C(alpha) T(1) values best represent changes in the local correlation time of the peptide epitope upon binding antibody, and demonstrate that the PDTRPAP sequence is immobilized in the antibody-combining site. This result is also reflected in the appearance of the (15)N- and (13)C-edited HSQC spectra, where line broadening of the same peptide epitope resonances is observed. The PDTRPAP peptide epitope expands upon the peptide epitope identified previously in our group as PDTRP by homonuclear NMR experiments [Grinstead, J. S., et al. (2002) Biochemistry 41, 9946-9961], and illustrates the usefulness of the heteronuclear NMR experiments. The implications of these results are discussed within the context of MUC1 breast cancer vaccine design.     

Conformation of the cecropin-melittin hybrid, cecropin A(1-8)-melittin (1-18) antibacterial Peptide

Cecropin A (1-8)-Melittin (1-18) is a synthetic cecropin A-melittin hybrid peptide with leishmanicidal activity. The primary sequence of the peptide is as follows: KWKLPKKIGIGAVLKVLTTGLPALIS-NH2. 1H and 13C 2D NMR techniques were used to deduce the conformational parameters of chemical shift, 3JNHalpha coupling constants, temperature coefficients of NH chemical shifts and the pattern of intra and inter-residue nOe's. NMR studies were carried out in water (pH 6.0) and hexafluoroacetone (HFA). The peptide was found in a beta-pleated structure in water, and in HFA it adopts a right-handed alpha-helix conformation. Solution structures generated using restrained molecular dynamics simulations were refined by Mardigras to R factors ranging from 0.5 to 0.6.     

C-terminal retroviral-type zinc finger domain from the HIV-1 nucleocapsid protein is structurally similar to the N-terminal zinc finger domain

Two-dimensional NMR spectroscopic and computational methods were employed for the structure determination of an 18-residue peptide with the amino acid sequence of the C-terminal retroviral-type (r.t.) zinc finger domain from the nucleocapsid protein (NCP) of HIV-1 [Zn(HIV1-F2)]. Unlike results obtained for the first retroviral-type zinc finger peptide, Zn(HIV1-F1), [Summers et al. (1990) Biochemistry 29, 329], broad signals indicative of conformational lability were observed in the 1H NMR spectrum of Zn-(HIV1-F2) at 25 degrees C. The NMR signals narrowed upon cooling to -2 degrees C, enabling complete 1H NMR signal assignment via standard two-dimensional (2D) NMR methods. Distance restraints obtained from qualitative analysis of 2D nuclear Overhauser effect (NOESY) data were used to generate 30 distance geometry (DG) structures with penalties (penalty = sum of the squared differences between interatomic distances defined in the restraints file and in the DG structures) in the range 0.02-0.03 A2. All structures were qualitatively consistent with the experimental NOESY spectrum based on comparisons with 2D NOESY back-calculated spectra. Superposition of the backbone atoms (C, C alpha, N) for residues C(1)-C(14) gave pairwise RMSD values in the range 0.16-0.75 A. The folding of Zn(HIV1-F2) is very similar to that observed for Zn(HIV1-F1). Small differences observed between the two finger domains are localized to residues between His(9) and Cys(14), with residues M(11)-C(14) forming a 3(10) helical corner.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformational features of dynorphin A-(1-13), Laser Raman spectroscopic studies

Conformational features of dynorphin A-(1-13) were examined by laser Raman spectroscopy. Dynorphin A-(1-13) appears to have a mixture of extended beta-pleated sheet and "random" structure.     

Synthesis and conformational studies of N-glycosylated analogues of the HIV-1 principal neutralizing determinant.

The principal neutralizing determinant (PND) of HIV-1 is found in the V3 loop of the envelope glycoprotein. Antibodies elicited by peptides from this region, containing the GlyProGlyArgAlaPhe (GPGRAF) sequence, were able to neutralize diverse HIV-1 isolates [Javaherian et al. (1990) Science 250, 1590-1593]. The GPGR tetrapeptide was predicted to adopt a type II beta-turn conformation. Earlier, we showed that glycosylation of synthetic T cell epitopic peptides at natural glycosylation sites stabilized beta-turns [Otv?s et al. (1991) Int. J. Pept. Protein Res. 38, 467-482]. To evaluate the secondary structure modifying effect of the introduction of an N-glycosylated asparagine residue and to find a correlation between conformation and a possible PND potential, a series of glycopeptide derivatives, N(sugar) GPGRAFY-NH2 (4a-f), have been prepared, together with the parent peptides GPGRAFY-NH2 (2) and NGPGRAFY-NH2 (3), by solid-phase peptide synthesis [sugars: (a) beta-D-glucopyranosyl (Glc); (b) beta-D-galactopyranosyl (Gal); (c) Glc-beta(1----4)-Glc; (d) 2-acetamido-2-deoxy-beta-D-glucopyranosyl (GlcNAc); (e) 2-acetamido-2-deoxy-beta-D-galactopyranosyl (GalNAc); (f) GlcNAc-beta(1----4)-GlcNAc; sugars are attached through a beta (1----N beta) linkage to asparagine (N).] Peptides 2-4 were characterized by amino acid analysis, reversed-phase HPLC, and fast atom bombardment mass spectrometry. Circular dichroism (CD) and Fourier-transform infrared (FT-IR) spectroscopic studies were performed in trifluoroethanol (TFE) and water (D2O was used in FT-IR experiments). Nonglycosylated peptides showed significantly different CD spectra in aqueous and TFE solution.(ABSTRACT TRUNCATED AT 250 WORDS)     

On the structures of flavoprotein D-amino acid oxidase purple intermediates. A resonance Raman study

Resonance Raman (RR) spectra were obtained in H2O or D2O solution for the purple intermediates of D-amino acid oxidase (DAO) with isotopically labeled substrates, i.e., [1-13C]-, [2-13C]-, [3-13C]-, [15N]-, and [3,3,3-D3]alanine; [carboxyl-13C]- and [15N]proline. RR spectra were also measured for the intermediates of DAO reconstituted with isotopically labeled FAD's, i.e., [4a-13C]-, [4,10a-13C2]-, [2-13C]-, [5-15N]-, and [1,3-15N2]FAD in D2O. The isotopic shift of the 1692 cm-1 band upon [15N]- or [2-13C]-substitution of alanine indicates that the band is due to the C = N stretching mode of an imino acid derived from D-alanine, i.e., alpha-iminopropionate. The 1658 cm-1 band with D-proline was also assigned to the C = N stretching mode of an imino acid derived from D-proline, i.e., delta 1-pyrrolidine-2-carboxylate, since the band shifts to 1633 cm-1 upon [15N]-substitution and its stretching frequency is generally found in this frequency region. Since the band shifts to low frequency in D2O, the imino acid should have a protonated imino group such as the C = N+1H form. The intense band at 1363 cm-1 with D-alanine was assigned to a mixing of the CO2- symmetric stretching and CH3 symmetric deformation modes in alpha-iminopropionate, based on the isotope effects. The 1359 cm-1 band with D-proline has probably contributions of CO2- symmetric stretching and CH2 wagging, considering the isotope effects with [carboxyl-13C]proline. The 1359 cm-1 band with D-proline was split into 1371 cm-1 and 1334 cm-1 bands in D2O. As this splitting of the 1359 cm-1 band with D-proline in D2O can not be interpreted only by the replacement of the C = N+1-H proton by deuterium, the carboxylate of the imino acid probably interacts with the enzyme through some proton(s) exchangeable by deuterium(s) in D2O. The bands around 1605 cm-1 which shift upon [4a-13C]- and [4,10a-13C2]-labeling of FAD are derived from a fully reduced flavin, because the isotopic shifts of the band are very different from those of the bands of oxidized or semiquinoid flavin observed near 1605 cm-1.(ABSTRACT TRUNCATED AT 400 WORDS)