NH Temperature Coefficients Research Articles

Conformational properties and aggregation of homo-oligomeric β3 (R)-valine peptides in organic solvents.

The conformational characteristics of protected homo-oligomeric Boc-[β3 (R)Val]n -OMe, n = 1, 2, 3, 4, 6, 9, and 12 have been investigated in organic solvents using nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR) absorption spectroscopy and circular dichroism (CD) methods. The detailed 1 H NMR analysis of Boc-[β3 (R)Val]12 -OMe reveals that the peptide aggregates extensively in CDCl3 , but is disaggregated in 20%, (v/v) dimethyl sulfoxide (DMSO) in CDCl3 and in CD3 OH. Limited assignment of the N-terminus NH groups, together with solvent dependence of NH chemical shifts and temperature coefficients provides evidence for 14-helix conformation in the 12-residue peptide. FTIR analysis in CHCl3 establishes that the onset of folding and aggregation, as evidenced by NH stretching bands at 3375 cm-1 (intramolecular) and 3285 cm-1 (intermolecular), begins at the level of the tetrapeptide. The observed CD bands, 214 nm (negative) and 198 nm (positive), support 14-helix formation in the 9 and 12 residue sequences. The folding and aggregation tendencies of homo-oligomeric α-, β-, and γ- residues is compared in the model peptides Boc-[ωVal]n -NHMe, ω = α, β, and γ and n = 1, 2, and 3. Analysis of the FTIR spectra in CHCl3 , establish that the tendency to aggregate at the di and tripeptide level follows the order β > α∼γ, while the tendency to fold follows the order γ > β > α.

NMR analysis of G7‐18NATE, a nonphosphorylated cyclic peptide inhibitor of the Grb7 adapter protein

G7-18NATE is a nonphosphorylated, cyclic peptide that specifically inhibits the Grb7 adapter protein implicated in several pathways critical to cell proliferation and migration. It has been shown that G7-18NATE is able to compete with natural ligands for the Grb7 SH2 phosphotyrosine binding site, and to attenuate cell migration in a pancreatic cancer cell line. It is thus an important lead in the development of a selective inhibitor of Grb7 and potential novel anticancer therapeutics. The current study reports the solution properties of G7- 18NATE determined using NMR spectroscopy, in both water (pH 2-3) and phosphate buffer (pH 6.0), with 100 mM NaCl. The spectra reveal that G7-18NATE exists in two distinguishable conformational states on the NMR timescale, most likely due to cis-trans proline isomerization. In addition, the chemical shift data are consistent with a tendency of G7-18NATE to form a turn about the YDN motif, known to be important for binding, and suggest that this turn is stabilized in low salt and low pH conditions. Low NH temperature coefficients of Tyr-5 and Asn-7 amide protons may reflect their involvement in the formation of hydrogen bonds that stabilize such a turn. Overall, however, the peptide does not form a rigid structure, but exists in a highly flexible state in solution. Averaged 3JNH-H coupling constants and a lack of interresidue NOEs are characteristic of such peptide solution behavior. This suggests that there is scope for increasing the rigidity of the peptide that may enhance its binding affinity and specificity for Grb7.

Efficient synthesis of enantiopure pyrrolizidinone amino acid.

Enantiopure (3S,5R,8S)-3-[N-(Boc)amino]-1-azabicyclo[3.3.0]octan-2-one 8-carboxylic acid (1) was synthesized in nine steps and 16% overall yield from aspartate beta-aldehyde 7. Carbene-catalyzed acyloin condensation of 7, followed by acetylation and samarium iodide reduction, gave linear precursor (2S,7S)-alpha,omega-diamino-4-oxosuberate 11, which was converted to N-(Boc)aminopyrrolizidin-2-one carboxylic acid 1 by a reductive amination/lactam cyclization sequence. X-ray analysis of (3S,5R,8S)-methyl N-(Boc)aminopyrrolizidin-2-one carboxylate 21 showed that its internal backbone dihedral angles (psi = -149 degrees, phi = -49 degrees ) were in good agreement with the ideal values for a type II' beta-turn. Proton NMR experiments on N'-methyl-N-(Boc)aminopyrrolizidin-2-one carboxamide 23 demonstrated significantly different NH chemical displacements and temperature coefficients suggestive of solvent shielded and exposed hydrogens indicative of a turn conformation. Because pyrrolizidinone amino acids can serve as conformationally rigid dipeptide surrogates, this synthesis should facilitate their application in the exploration of conformation-activity relationships of various biologically active peptides.

NMR conformational analysis of cis and trans proline isomers in the neutrophil chemoattractant, N-acetyl-proline-glycine-proline.

Alkaline hydrolysis of corneal proteins in the alkali-injured eye releases N-acetyl-proline-glycine-proline (Ac-Pro-Gly-Pro-OH) among other peptides. It has been shown that this tripeptide is a neutrophil chemoattractant. Existing data suggest that the release of this peptide is the catalytic event for early neutrophil invasion of the cornea leading to corneal ulcers. In order to design inhibitors of this tripeptide chemoattractant that would block neutrophil invasion and diminish corneal ulcers, we studied the solution properties of this tripeptide by NMR spectroscopy and compared this peptide to Ac-Pro-Gly-OH (a weaker chemoattractant), and to Ac-Pro-OH (inactive). The NMR data were consistent with Ac-Pro-Gly-Pro-OH existing in solution as a mixture of four isomers with different cis and trans conformations about the two X-proline amide bonds. The isomer with two trans conformations (trans-trans) was the most dominant (41%) in aqueous solution. This was followed by the isomers with mixed cis and trans conformations (trans-cis, 26% and cis-trans, 20%). The isomer with two cis conformations (cis-cis) was the least favored (13%). The populations of these isomers were investigated in DMSO and they were similar to those reported in aqueous solutions except that the ordering of the trans-cis and cis-trans isomers were reversed. NMR NH temperature coefficients and nuclear Overhauser effect (NOE) measurements as well as CD spectroscopy were used to demonstrate that the four isomers exist primarily in an extended conformation with little hydrogen bonding. The available (NOE) information was used with molecular dynamics calculations to construct a dominant solution conformation for each isomer of the tripeptide. This information will serve as a model for the design of peptide and nonpeptide inhibitors of the chemoattractant.

Helical and coiled-coil-forming properties of peptides derived from and inhibiting human immunodeficiency virus type 1 integrase assessed by 1H-NMR--use of NH temperature coefficients to probe coiled-coil structures.

Human immunodeficiency virus type 1 integrase (HIV-1 IN) which catalyzes viral DNA integration into the host genome of infected cells represents an attractive target for AIDS therapy. We have previously demonstrated the ability of the IN-(147-175)-peptide derived from the catalytic core domain of HIV-1 IN to inhibit the enzyme activity in vitro. IN-(147-175)-peptide contains four heptad repeats and displays a high propensity for coiled-coil formation while its [P159]IN-(147-175)-peptide analog (Lys159-->Pro in the protein, Lys13-->Pro in the peptide) is unable to form a stable coiled-coil and is devoid of inhibitory activity [Sourgen, F., Maroun, R. G., Frère, V., Bouziane, M., Auclair, C., Troalen, F. & Fermandjian, S. (1996) Eur. J. Biochem. 240, 765-773]. Now, we report results from an NMR study on IN-(147-175)-peptide and [P159]IN-(147- 175)-peptide as well as on an optimized [E156, A163, A167]IN-(147-175)-peptide that is a better inhibitor of IN than IN-(147-175)-peptide. While in aqueous solution, IN-(147-175)-peptide and [P159]IN-(147-175)-peptide display only nascent helical features, [E156, A163, A167]IN-(147-175)-peptide exhibits 20% of helical content. In 20% trifluoroethanol/80% H2O, the helix content is the highest for [E156, A163, A167]IN-(147-175)-peptide (approximately 70%) and the lowest for [P159]IN-(147-175)-peptide (approximately 40%), due to a local helix break caused by the Pro residue. The NHs of residues in the two central helical heptads (a-g) of IN-(147-175)-peptide and [E156, A163, A167]IN-(147-175)-peptide display a regular periodic variation of their temperature coefficients in 20% trifluoroethanol. The b, c and f residues on the hydrophilic face of the amphipathic helix show high coefficients reflecting hydrogen bonded NHs, while the a and d residues on the hydrophobic face exhibit low coefficients, near random-coil values. The particular arrangement of the hydrophobic side-chains of a and d residues at the coiled-coil interface reduces the access of trifluoroethanol molecules to their amide groups. The inability of trifluoroethanol molecules to create interactions with the amide C=O groups, these being required to strengthen the intrahelical C=O...H-N hydrogen bonds, is the main cause for observation of heptadic a and d residues with low NH temperature coefficients. Such effects concern mostly the two central helical heptads of IN-(147-175)-peptide and [E156, A163, A167]IN-(147-175)-peptide implying that these ones are engaged in stable parallel coiled coils. Our results provide a link between the propensity of peptides for helix formation, their coiled-coil properties and their efficiency to inhibit IN.

Dissection of the basic subdomain of the c-Jun oncoprotein: a structural analysis of two peptide fragments by CD, Fourier-transform infrared and NMR.

In a previous paper, we reported on the structural properties of a 35-residue peptide corresponding to a modified basic subdomain (bSD) of the basic zipper protein c-Jun (residues 252-281) as determined by combined use of 1H-NMR, circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopies [Krebs, D., Dahmani, B., El Antri, S., Monnot, M., Convert, O,. Mauffret, O., Troalen, F. & Fermandjian, S. (1995) Eur. J. Biochem. 231, 370-380]. The fragments NP and CP (the N-terminal residues 1-19 and C-terminal residues 16-35 of bSD, respectively) proved to be particularly useful for the assignment of the 1H-NMR spectra of the full-length bSD, which has been achieved completely in aqueous solution and partially in trifluoroethanol. Here, we report on the structural properties of NP and CP in aqueous solution and under varying H2O/trifluoroethanol conditions, again using 1H-NMR, CD and FT-IR experiments. Both CD and FT-IR results established that the fragments are weakly structured in aqueous solution. Addition of trifluoroethanol to aqueous solutions of the peptides produced their stabilization into helix, following a profile sigmoidal for NP and nearly linear for CP. Quantitative NOEs, secondary Halpha chemical shifts, NH temperature coefficients and 3JalphaN coupling constants for the peptides in aqueous solutions provided indications for weak helix features (nascent helices) manifested within two sites (continuous dNN NOEs) in both NP and CP. For each peptide, an excellent agreement was observed between experiments and predictions with the AGADIR program for the location of these nascent helices in the sequences. Trifluoroethanol provoked both the alpha-helix stabilization within these sites and the alpha-helix propagation to adjacent amino acid residues. Finally, our results reflected the high flexibility and helix potential of the NP and CP fragments, these two properties seeming crucial for the accommodation of c-Jun to its specific DNA targets. The results demonstrated also the fragmentation's benefits in dissecting a protein or a complex peptide into smaller fragments and analyzing their structure individually.

Sequence-Specific Peptide-Carbohydrate Interactions in an Asparagine-Linked Glycopeptide.

Asparagine glycosylation (N-glycosylation) of the AsnXaa-Ser/Thr sequence is a cotranslational process that can affect protein structure and function.1,2 Little is known, however, about glycosylation’s influence on the structure of flexible N-glycopeptides.3-7 Fluorescence studies show that N-glycosylation can alter peptide backbone conformation.6 Circular dichroism also indicates that N-glycosylation can perturb R-helical structure.7 In this work, we use NMR to determine details about N-glycopeptide structure in an organic solvent. Significantly, glycopeptide Ac-Asn(â1-N-GlcNAc)Leu-ThrNH2 (1) shows long-range 1H-1H NOEs between the peptide and the N-acetylglucosamine (GlcNAc) sugar attached to Asn1. Besides these peptide-sugar NOEs, a comparison of JR,â coupling constants and NH temperature coefficients in glycopeptide 1 and aglycosyl peptide Ac-Asn-Leu-Thr-NH2 3 indicate that N-glycosylation stabilizes peptide conformation. Until recently, NMR studies showed that N-glycosylation caused little change in peptide structure.3,4 Peptide-sugar NOEs have not been observed in flexible N-glycopeptides. Our studies of a glycodecapeptide in H2O indicated nonrandom conformation near the Nglycosylation site but showed no intramolecular peptidesugar NOEs.3e We have now examined tripeptides containing one sugar, Ac-Asn(â1-N-GlcNAc)Leu-Thr-NH2 (1) and Ac-Gln(â1-N-GlcNAc)Leu-Thr-NH2 (2). The “unnatural” glycopeptide 2 has an Asn1-Gln1 mutation that allows one to determine if glycopeptide structure is sequence-specific. Glycopeptide 1 and aglycosyl peptide 3 contain an N-glycosylation consensus sequence. Comparison of 1 and 3 reveals glycosylation’s influence on peptide conformation. Glycopeptides 1 and 2 were synthesized from â-1amino-GlcNAc and the corresponding Asp and Glu peptides.8 The 1H resonances in 1-3 were assigned using TOCSY, DQF-COSY, and ROESY experiments.9 NMR data indicated that glycopeptide 1 was not structured in DMSO-d6 at 298 K. To narrow the conformational distribution and favor low-energy structures, NMR analysis of 1-3 was done at low temperature. ROESY experiments at 233 K in 30% DMSO/70% CD2Cl2 revealed that glycopeptide 1 (2.0 mM) had unique intramolecular peptide-sugar NOEs. The aglycosyl peptide 3 showed no long-range NOEs under identical conditions. As shown in Figure 1, the C-terminal trans amide NH (δ 7.01) has an NOE to GlcNAc H1 (δ 4.60) while the C-terminal cis amide NH (δ 7.45) has NOEs to GlcNAc H5 (δ 3.26) and a GlcNAc H6 (δ 3.33). Importantly, NMR spectra of glycopeptide 1 in 30% DMSO/ 70% CD2Cl2 at 233 K indicated no change in the NH resonances over a 5.0-0.05 mM concentration range. The concentration independence of the NH chemical shifts verified that the peptide-sugar NOEs were intramolecular. The peptide-sugar NOEs significantly limit glycopeptide conformation since, under these conditions, dipolar interactions are not observed for interproton distances >3.1 A.10 These intramolecular peptide-sugar interac(1) (a) Kornfeld, R.; Kornfeld, S. Ann. Rev. Biochem. 1985, 54, 631. (b) Gilmore, R. Cell 1993, 75, 589. (2) Varki, A. Glycobiology 1993, 3, 97. (3) For NMR of N-glycopeptides: (a) Ishii, H.; Inoue, Y.; Chujo, R. Int. J. Pept. Protein Res. 1984, 24, 421. (b) Ishii, H.; Inoue, Y.; Chujo, R. Polym. J. (Tokyo) 1985, 17, 693. (c) Wormald, M. R.; Wooten, E. W.; Bazzo, R.; Edge, C.; Feinstein, A.; Rademacher, T. W.; Dwek, R. A. Eur. J. Biochem. 1991, 198, 131. (d) Davis, J. T.; Hirani, S.; Bartlett, C.; Reid, B. R. J. Biol. Chem. 1994, 269, 3331. (4) N-glycosylation can alter Pro cis/trans and Cys thiol/disulfide equilibria: Rickert, K. W.; Imperiali, B. Chem. Biol. 1995, 2, 751. (5) O-Glycosylation influences peptide conformation: (a) Andreotti, A. H.; Kahne, D. J. Am. Chem. Soc. 1993, 115, 3352. (b) Liang, R.; Andreotti, A. H.; Kahne, D. J. Am. Chem. Soc. 1995, 117, 10395. (6) Imperiali, B.; Rickert, K. W. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 97. (7) (a) Otvos, L.; Thurin, J.; Kollat, E.; Urge, L.; Mantsch, H. H.; Hollosi, M. Int. J. Pept. Protein Res. 1991, 38, 476. (8) Cohen-Anisfeld, S. T.; Lansbury, P. T., Jr. J. Am Chem. Soc. 1993, 115, 10531. (9) (a) Davis, D. G.; Bax, A. J. Am. Chem. Soc. 1985, 107, 2820. (b) Piantini, U; Sorenson, O. W.; Ernst, R. R. J. Am. Chem. Soc. 1982, 104, 6800. (c) Bothner-By, A. A.; Stephens, R. L.; Lee, J.; Warren, C. D.; Jeanloz, R. W. J. Am. Chem. Soc. 1984, 106, 811. Figure 1. Contour plot of a region of the 500 MHz ROESY spectrum of Ac-Asn(â1-N-GlcNAc)-Leu-Thr-NH2 (1) (2.0 mM) recorded at 233 K in 30% DMSO-d6/70% CD2Cl2. The spin locking time was 200 ms. Key peptide-sugar NOEs are indicated. 4198 J. Org. Chem. 1996, 61, 4198-4199

NMR Analysis of a Conformational Transition in an Acyclic Peptide. Model System for Studying Helix Unfolding

The stabilization of helical structures in short apolar peptides is readily achieved by introduction of α,α-dialkylamino acids. The use of stereochemically constrained residues in conjunction with conformationally flexible segments permits the design of peptides that are poised to undergo structural transitions. The octapeptide Boc-Leu-Ac8c-Val-Gly-Gly-Leu-Ac8c-Val-OMe (Ac8c = 1-aminocyclooctane-1-carboxylic acid) incorporates residues with contradictory conformational tendencies. NMR analysis in CDCl3, using nuclear Overhauser effects and delineation of hydrogen-bonded NH groups establishes a 310-helical conformation. In a polar strongly solvating medium, like DMSO, the helix unfolds. Studies in CDCl3/DMSO mixtures provide clear evidence for a solvent dependent conformational transition. Amide NH chemical shifts and temperature coefficients at varying solvent composition allow a detailed structural analysis of the unfolding process. The intrinsic fragility of the octapeptide helix provides an opportunity to examine invasion of the helix backbone by water molecules. Studies in DMSO solution containing low concentrations of water establish that preferential water peptide interactions may indeed be present.

The basic subdomain of the c-Jun oncoprotein. A joint CD, Fourier-transform infrared and NMR study.

The structural properties of the basic subdomain of the basic zipper (bZIP) protein c-Jun were examined by joint means of 1H-NMR, CD and Fourier-transform infrared (FTIR) spectroscopies. The basic subdomain (residues 252-281 in c-Jun) is responsible for sequence-specific recognition of DNA. A modified basic subdomain bSD (residues 1-35) and its N-terminal part and C-terminal part fragments (NP, residues 1-19; and, CP, residues 16-35) were prepared by solid-phase synthesis and purified by HPLC. In aqueous solution, in the absence of DNA, bSD behaved mostly as an unstructured peptide characterized by only 5% alpha helix. However, upon mixing bSD and a specific DNA fragment, i.e. a CRE(cAMP-responsive element)-containing hexadecanucleotide, the alpha helix was stabilized to an extent of 20% at 20 degrees C or 35% at 2 degrees C. At the same time, no significant change could be detected in the DNA spectra. Addition of trifluoroethanol to an aqueous bSD sample resulted in an increase of the alpha-helix content so that about 60% of alpha helix was found at a ratio of 75% trifluoroethanol (20 degrees C). These effects were reflected in both CD and FTIR measurements. Changes shown by the CD spectra during the process suggested a mechanism dominated by a two-state helix/unordered transition. NMR data, namely alpha H chemical shifts, NOE cross-peaks and NH temperature coefficients provided indications for extended or nascent helix structures within four short stretches dispersed along the sequence for c-Jun bSD, contrasting with the unique and continuous stretch reported for Gcn4 (yeast general control protein 4) bSD in aqueous solution. Trifluoroethanol stabilized the alpha-helix structure mainly at these four sites. The malleability of the basic subdomain of c-Jun was emphasized in relation to its ability to fit the DNA helix in adopting an alpha-helix structure. The complex formation apparently requires substantial conformational change from the peptide and only little from the oligonucleotide.

The Basic Subdomain of the c‐Jun Oncoprotein

The structural properties of the basic subdomain of the basic zipper (bZIP) protein c‐Jun were examined by joint means of 1H‐NMR, CD and Fourier‐transform infrared (FTIR) spectroscopies. The basic subdomain (residues 252–281 in c‐Jun) is responsible for sequence‐specific recognition of DNA. A modified basic subdomain bSD (residues 1–35) and its N‐terminal part and C‐terminal part fragments (NP, residues 1–19; and, CP, residues 16–35) were prepared by solid‐phase synthesis and purified by HPLC. In aqueous solution, in the absence of DNA, bSD behaved mostly as an unstructured peptide characterized by only 5% a helix. However, upon mixing bSD and a specific DNA fragment, i.e. a CRE(cAMP‐responsive element)‐containing hexadecanucleotide, the α helix was stabilized to an extent of 20% at 20 °C or 35% at 2%C. At the same time, no significant change could be detected in the DNA spectra. Addition of trifluoroethanol to an aqueous bSD sample resulted in an increase of the α‐helix content so that about 60% of α helix was found at a ratio of 75% trifluoroethanol (20°C). These effects were reflected in both CD and FTIR measurements. Changes shown by the CD spectra during the process suggested a mechanism dominated by a two‐state helix/unordered transition. NMR data, namely αH chemical shifts, NOE cross‐peaks and NH temperature coefficients provided indications for extended or nascent helix structures within four short stretches dispersed along the sequence for c‐Jun bSD, contrasting with the unique and continuous stretch reported for Gcn4 (yeast general control protein 4) bSD in aqueous solution. Trifluoroethanol stabilized the α‐helix structure mainly at these four sites. The malleability of the basic subdomain of c‐Jun was emphasized in relation to its ability to fit the DNA helix in adopting an α‐helix structure. The complex formation apparently requires substantial conformational change from the peptide and only little from the oligonucleotide.

Solution three-dimensional structure of surfactin: a cyclic lipopeptide studied by 1H-NMR, distance geometry, and molecular dynamics.

The solution three-dimensional structure of the protonated [Leu7]-surfactin, an heptapeptide extracted from Bacillus subtilis, has been determined from two-dimensional 1H-nmr performed in 2H6-dimethylsulfoxide and combined with molecular modeling. Experimental data included 9 coupling constants, 61 nuclear Overhauser effect derived distances, NH temperature coefficients, and 13C relaxation times. Two distance geometry (DISMAN) protocols converged toward models of the structure and the best of them were refined by restrained and unrestrained molecular dynamics (GROMOS). Two structures in accord with the set of experimental constraints are presented. Both are characterized by a "horse saddle" topology for ring atoms on which are attached the two polar Glu and Asp side chains showing an orientation clearly opposite to that of the C11-13 aliphatic chain. Amphipathic and surface properties of surfactin are certainly related to the existence of such minor polar and a major hydrophobic domains. The particular "claw" configuration of acidic residues observed in surfactin gives important clues for the understanding of its cation binding and transporting ability.

Quantitative two-dimensional NMR study of dermenkephalin, a highly potent and selective delta-opioid peptide.

Dermenkephalin, H-Tyr-(D)Met-Phe-His-Leu-Met-Asp-NH2, a highly potent and selective delta-opioid peptide isolated from frog skin, was studied in DMSO-d6 solution by two-dimensional nmr spectroscopy, including the determination of NH temperature coefficients, the evaluation of 3J coupling constants from phase-sensitive correlated spectroscopy (COSY) and the volumes of nuclear Overhauser effect (NOE) correlations. The two-dimensional NOE spectroscopy (NOESY) spectrum of dermenkephalin revealed sequential, medium-, and long-range effects. To put this information on a quantitative basis, special attention was devoted to J cross-peak suppression, quantification of the NOE volumes and analysis of the overlaps, normalization of the NOEs against diagonal peaks and H beta beta' geminal interactions. Although most of the dihedral angles deduced from the 3JN alpha coupling constants together with several Ni alpha i and alpha iNi+1 NOEs pointed to a partially extended peptide backbone, several NiNi+1 NOEs and beta iNi+1 interactions argued in favor of a folded structure. Moreover, several long-range correlations of strong intensities were found that supported a close spatial proximity between the side chains of D-Met2 and Met6, Tyr1 and His4, Tyr1 and Asp7, and His4 and the C-terminal amide group. In Phe, the g- rotamer in the side chain is deduced from the 3J alpha beta coupling constants and alpha beta and N beta NOE correlations. Whereas the amide proton dependency was not indicative of stable hydrogen bonds, the nonuniform values of the temperature coefficient may reflect an equilibrium mixture of folded and extended conformers. The overall data should provide realistic starting models for energy minimization and modelization studies.

The conformation of a-factor is not influenced by the S-prenylation of Cys 12

Two-Dimensional NMR was used to examine the solution conformation of the lipopeptide a-factor, YIIKGVFWDPAC(S-farnesyl) OCH 3, from the yeast Saccharomyces cerevisiae and five analogues containing various S-alkylated cysteines in DMSO-d 6. NOESY data, NH temperature coefficients, and 3J αNH coupling constants indicate that the a-factor is a predominantly unstructured peptide in DMSO. Similar results were obtained for the other peptides indicating that S-prenylation of Cys 12 does not affect the conformation of these peptides.

Cell adhesion promoting peptide GVKGDKGNPGWPGAP from the collagen type IV triple helix: cis/trans proline-induced multiple proton NMR conformations and evidence for a KG/PG multiple turn repeat motif in the all-trans proline state

Peptide GVKGDKGNPGWPGAPY (called peptide IV-H1), derived from the protein sequence of human collagen type IV, triple-helix domain residues 1263-1277, represents an RGD-independent, cell-specific, adhesion, spreading, and motility promoting domain in type IV collagen. In this study, peptide IV-H1 has been investigated by 1H NMR (500 MHz) spectroscopy. Cis-trans proline isomerization at each of the three proline residues gives rise to a number of slowly exchanging (500-MHz NMR time scale) conformation states. At least five such states are observed, for example, for the well-resolved A14 beta H3 group, and K3, which is six residues sequentially removed from the nearest proline, i.e., P9, shows two sets. The presence of more than two sets of resonances for residues sequentially proximal to a proline, e.g., A14-cis-P15 and A14-trans-P15, and more than one set for a residue sequentially well-removed from a proline, e.g., K3, indicates long range conformation interactions and the presence of preferred structure in this short linear peptide. Many resonances belonging to these multiple species have been assigned by using mono-proline-substituted analogues. Conformational (isomer) state-specific 2D 1H NMR assignments for the combination of cis and trans proline states have been made via analysis of COSY-type, HOHAHA, and NOESY spectra. Peptide IV-H1 in the all-trans proline state ttt exists in relatively well-defined conformation populations showing numerous short- and long-range NOEs and long-lived backbone amide protons and reduced backbone NH temperature coefficients, suggesting hydrogen-bonding, and structurally informative 3J alpha N coupling constants. The NMR data indicate significant beta-turn populations centered at K3-G4, K5-G6, P9-G10, and P12-G13, and a C-terminal gamma-turn within the A14-P15-Y16 sequence. These NMR data are supported by circular dichroic studies which indicate the presence of 52% beta-turn, 10% helix, and 38% random coil structural populations. Since equally spaced KG and PG residues are found on both sides of peptide IV-H1 in the native collagen type IV sequence, this multiple turn repeat motif may continue through a longer segment of the protein. Synthetic peptide IV-H1 overlapping sequence "walk throughs" indicate that the primary biological activity is localized in the GNPGWPGAP double beta-turn domain, which contains the backbone constraining proline residues. This proline-domain conformation may suggest a collagen type IV receptor-specific, metastatic cell adhesion promoting binding domain.

The solution conformation of tubulin‐β(422–434)‐NH2 and its Nac‐DATADEQG‐NH2 fragment based on nmr

The solution conformation of tubulin-beta(422-434)-NH2 (YQQYQDATADEQG-NH2) and its Nac-DATADEQG-NH2 fragment has been studied by two-dimensional 1H-nmr spectroscopy in CD3OH/H2O (90/10 v/v) at neutral and low pH. The 13 amino acid peptide is a segment of the C-terminal region of tubulin, and is directly involved in the selective binding site with microtubule-associated proteins MAP-2 and the tau protein. Based on correlated spectroscopy, total correlation spectroscopy, and rotating frame nuclear Overhauser effect spectroscopy experiments, a complete assignment of all proton resonances was achieved, and the conformation of the backbone could be deduced from coupling constants, NH temperature coefficients, and nuclear Overhauser effects. The spectroscopic evidence indicates that the T8-Q12 section of both molecules forms one complete alpha-helical turn, stabilized by a NH (Q12)-C = O (T8) hydrogen bond. Furthermore, strong pH-dependent backfolding of the E11 side chain to its own NH proton was found. In addition, close proximity between the aromatic side chains of Y1, Y4, and the alpha-helical part, resulting in some substantial chemical shift changes when comparing the entire 13-mer with the octamer, could be explained in terms of a nonclassical kink in the DATA section. The conformational space is dominated by extended structures and the nonextended conformers are only a minor, yet spectroscopically clearly discernible entity. The presence of the alpha-helical region at the C-terminus of the 13-mer is important because binding studies of this peptide with MAP-2 indicate that the D10-E11-Q12-G13 fragment is critical for the binding interaction.

Evidence for β-turn structure in model peptides reproducing pro-ocytocin/neurophysin proteolytic processing site

The structural organization of small peptides reproducing the amino acid sequence of the common ocytocin/neurophysin precursor around the LysArg cleavage locus was investigated by a combination of spectroscopical techniques. In water both circular dichroism and [ 1H] NMR spectra indicated that these peptides adopted a random conformation. Evidence for folded structures was obtained when these compounds were placed in a membrane-like environment i.e. 40 mM SDS in phosphate buffer or trifluoroethanol. Whereas the CD spectra indicated the formation of various types of β-turn in rapid equilibrium, measurements of NH temperature coefficients and Nuclear Overhauser Effects by 400 and 500 MHz NMR revealed the existence of contacts and of a folded conformation. These observations are discussed in relation with previous hypothesis made on the secondary structure organization of the proteolytic processing site of polypeptide hormone precursors.

Hydrogen-deuterium isotope effects in 13C NMR experiments of peptides

In our recent NMR studies of linear peptides (l-3), ranging from 4 to 10 amino acids, we observed an interesting and unexpected line-splitting effect in the backbone 13C resonances. This effect was observed first in NAc-D-E-K-S-NH2 ( I ) , a fragment of the type I collagen (L1 chain N-telopeptide (4). A very similar effect was also found in two decapeptides related to the luteinizing hormone-releasing hormone (LHRH), namely the LHRH anti-sense (5) and reversed anti-sense peptide (2). In all these cases the C,, the backbone carbonyl, and the N-acetyl signals were each recorded as two lines separated by approximately 0.1 ppm whereas the signals of the side-chain carbons appeared as sharp lines. As there was independent and unambiguous evidence from the ‘H data that at least two of the three peptides mentioned were neither completely random nor extended, but rather an equilibrium between a type I p-turn with extended conformers ( I ) and a 3 iO-helical turn together with extended conformers (LHRH anti-sense (2))) we first considered the possibility of the presence of two conformers being the reason for the line doubling. However, a more recent study of the Cand N-terminal telopeptides of the type I collagen (u-2 chain (a hexapeptide and a nonapeptide, respectively), which provided up to four lines per a-carbon, revealed that this phenomenon cannot be conformation-related, since for both peptides the proton NMR data (3) clearly confirmed completely extended conformations. This conclusion was based on the absence of any particularly small or large 3 J Nn na _ COUphUg COnStantS, the lack Of NH ( i) -NH ( i + 1) nuclear Overhauser effects, and the absence of reduced NH temperature coefficients, all of which are in general associated with nonextended structures (6, 7). In our quest for the correct explanation of the phenomenon, we found a close relationship between the proton:deuteron ratio in solution and the observed peak intensities. All our experiments were carried out in so-called mixed solvents, containing a mixture of water and an organic solvent, such as trifluoroethanol or methanol, to mimic the physiological, nonaqueous conditions as found, for example, in the growing collagen fibrils. For most of our studies water/methanol solutions were used, typically 60 v% CD30H and 40 v% HzO. Half of the latter was added as D@ to

1H‐NMR of met‐enkephalin in AOT reverse micelles

1H‐NMR spectra of Met‐enkephalin dissolved in AOT/isooctane reverse micelles are reported at various temperatures. The NH temperature coefficients are compared with those obtained for the same peptide in normal SDS micelles, in bulk water, and in DMSO. The results show that the opioid molecule undergoes the greatest folding in the reverse micellar system. Shift reagents selectively dissolved in the water pools or in the hydrophobic matrix affect the Phe‐4 or the Tyr‐1 aromatic resonances, respectively. This suggests that the phenylalanyl sidechain is spatially close to the carboxyl group at the C‐terminus, whereas the tyrosyl ring points towards the outer region of the AOT micelles.

High-resolution proton nuclear magnetic resonance analysis of conformational properties of biosynthetic actinomycin analogues.

High-resolution proton nuclear magnetic resonance (1H NMR) has been used to study the conformation and dynamics of biosynthetic analogues of actinomycin D that have been substituted at one or both of the 3'-amino acids with azetidine-2-carboxylic acid, pipecolic acid, or 4-ketoproline for one or both of the prolines. The results of measurements made in organic solvents indicate that the amino acid residues on the alpha and beta pentapeptide lactone rings experience different magnetic environments in all the analogues studied. Analysis of the J alpha NH coupling constants and temperature coefficients for the valine and threonine amide protons indicates that substitution at the 3' position has little effect on the conformational and hydrogen-bonding properties of the amino acids at the 1' or 2' position. Examination of the low-field position of the H alpha proton of the 3'-amino acid reveals that the biosynthetic substitutions were made at a unique site on either the alpha or the beta pentapeptide ring and that only one conformation of proline, azetidine-2-carboxylic acid, or pipecolic acid is observed. In D2O, the dynamics of the prolines appear to differ; one remains in a preferred conformation while the other fluctuates at a rate that is intermediate on the NMR time scale. A possible relationship between the conformational and dynamic properties and the DNA binding and kinetic properties is discussed.

Assignment and analysis of the 500 MHz 1H NMR spectra of somatostatin and the acyclic precursor (S3,14‐Acm)‐somatostatin in dimethyl sulphoxide

AbstractThe 500 MHz 1H NMR spectra of somatostatin and the precursor, di‐S3,14‐acetamidomethyl dihydrosomatostatin, in dimethyl sulphoxide solution have been assigned. Chemical shifts, coupling constants and NH shifts vs temperature coefficients are tabulated. The data are consistent with a stable β‐turn/β‐sheet conformation for the precurso, while somatostatin itself appears to be more conformationally mobile, with an average conformation significantly different from that in aqueous solution.