N-methylleucine gramicidin-S and (di-N-methylleucine) gramicidin-S conformations with cis L-Orn-L-N-MeLeu peptide bonds.

Conformations containing all trans peptide bonds have previously been proposed for N-methylleucine gramicidin-S and (di-N-methylleucine) gramicidin-S based on an evaluation of proton nuclear magnetic resonance parameters in a series of solvents. These gramicidin-S derivatives exhibit full biological activity despite the fact that the proposed solution conformations differ in backbone topology and relative orientation of the Phe and Orn side chains compared to gramicidin-S. The present authors discuss conformations for N-methylleucine gramicidin-S and (di-N-methylleucine) gramicidin-S which incorporate cis peptide bonds at L -Orn-L -N-MeLeu, where the gramicidin-S backbone is essentially retained, and the relative orientation of the Pro, Orn, Val, and Phe side chains correspond to those observed for gramicidin-S. A novel hydrogen-bond arrangement involving one carbonyl group interacting with two peptide protons (1 ←4 and 1 ←5 types) is proposed to stabilize the backbone conformation in the gramicidin-S derivatives. A recent report on the cyclic heptapeptide antibiotic, Ilamycin B₁, shows the presence of cis peptide bonds at N-CH₃ amino acids, as well as the novel hydrogen-bond arrangement presented above.     

Conformation of malformin A: a proton magnetic resonance and a circular dichroism study

Malformin A is a cyclic pentapeptide with an intramolecular disulfide bridge. The conformation in solution of this molecule has been studied by NMR and CD. The 270 MHz Proton spectrum in dimethyl sulfoxide is well resolved and the peaks corresponding to the five residues have been assigned. From the temperature dependence of chemical shifts of the peptide protons and from the exchange rate of these protons, it is concluded that the NH proton of one Cys is shielded from the solvent. This observation and H? N? _αC? H angles, estimated from the corresponding coupling constants, a proposed conformation of the peptide backbone. From the H? _βC? _αC? H coupling constants, a P chirality for the disulfide bridge is proposed. Such a conformation is confirmed by the circular dichroism spectrum which shows a negative band at λ > 250 nm. It is concluded that the conformation of malformin A is rigid and that the disulfide bridge is exposed to interact with biological receptors.     

Use of amide 1H-nmr titration shifts for studies of polypeptide conformation

This paper shows that backbone amide proton titration shifts in polypeptide chains are a very sensitive manifestation of intramolecular hydrogen bonding between carboxylate groups and backbone amide protons. The population of specific hydrogen-bonded structures in the ensemble of species that constitutes the conformation of a flexible nonglobular linear peptide can be determined from the extent of the titration shifts. As an illustration, an investigation of the molecular conformation of the linear peptide H-Gly-Gly-L -Glu-L -Ala-OH is described. The proposed use of amide proton titration shifts for investigating polypeptide conformation is based on 360-MHz ¹H-nmr studies of selected linear oligopeptides in H₂O solutions. It was found that only a very limited number of amide protons in a polypeptide chain show sizable intrinsic intration shifts arising from through-bond interactions with ionizable groups. These are the amide proton of the C-terminal amino acid residue, the amide protons of Asp and the residues following Asp, and possibly the amide proton of the residue next to the N-terminus. Since the intrinsic titration shifts are upfield, the downfield titration shifts arising from conformation-dependent through-space interactions, in particular hydrogen bonding between the amide protons and carboxylate groups, can readily be identified.     

High resolution nuclear magnetic resonance spectroscopy of glycosphingolipids. I: 360 MHz 1H and 90.5 MHz 13C NMR analysis of galactosylceramides

The high resolution ¹H and ¹³C nuclear magnetic resonance (NMR) spectra of galactosylceramides containing n-fatty acids and α-hydroxy fatty acids were recorded in dimethylsulfoxide solution with and without addition of D₂O. From the coupling constants of the sugar ring protons, a ⁴C₁ conformation can be deduced. In contrast to the conformation in aqueous solution, the C⁶ hydroxymethylene group is freely rotating around the C⁶C⁵ bond. In the ceramide residue all signals produced by protons linked to carbons bearing electronegative substituents could be attributed. The large difference in coupling constants of the methylene protons of C^1′ to the C^2′ methine proton of the sphingosine indicates a restricted rotation around the C^1′C^2′ bond. The assignments of the hydroxy and amino protons follow from the decoupling of the corresponding methine protons.     

Nuclear magnetic resonance investigation of lysine–oligopeptides and a complex with d(pA)3pGpC(pT)3

We report proton magnetic resonance studies of a series of lysine oligopeptides in H₂O solution. At pH 5 the protonated ε-amino groups are seen as broad resonances; the peptide NH proton resonances are split by spin–spin coupling with the C^α-H proton, and appear at positions which depend on position in the chain and on chain length. Assignments were made by the europium shift method, and we observed the expected effect of catalysis by the terminal —NH₃⁺ of exchange of the adjacent peptide NH. Coupling constants and the temperature coefficient of chemical shift values were consistent with a non-hydrogen-bonded structure for the oligolysines. The rate and mechanism of NH hydrogen exchange were investigated by line-broadening measurements of the peptide protons as a function of pH. Exchange was found to be OH^? catalyzed, with large differences in the rate depending on position in the chain. Preliminary studies of the complex between double-helical d(pA)₃pGpC(pT)₃ and tetra(L -lysine) were performed using ¹H- and ³¹P-nmr techniques. Pmr spectra of the complex at pH values ranging from 3.98 to 8.15 showed very complicated patterns. Downfield shifts and reduction in exchange rates were observed for several tetra(L -lysine) protons. ³¹P-nmr spectra of the complex reveal an upfield shift of 1 ppm for 3′-5′ phosphate diester resonances on complexation. ³¹P T₁ relaxation times change little on complex formation at low temperature but are altered at higher temperature.     

A Proton Magnetic Resonance and a Circular Dichroism Study of the Solvent Dependent Conformation of the Synthetic Tubulin Fragment Ac Tubulin,Alpha (430–441) Amide and its Interaction with Substance-P

Abstract

Proton magnetic resonance techniques were used to study the conformation of the synthetic tubulin fragment Ac-tubulin (430–441) amide in H₂0 and 80% CD₃OH/20% D₂0 solutions, using water suppression techniques. Proton assignments are based on two-dimensional COSY experiments combined with one-dimensional spin decoupling.

A comparison of the NH proton shifts between the two solvents, namely ?(CD₃OH/H₂0-H₂O) shows a small solvent effect for the Lys¹ to Val⁶ region of the molecule, whereas for Gly⁷ to Glu¹² the solvent effect is much larger. The smaller effects in the region of Lys¹ to Val⁶ may be due to some hydrogen bonding as these protons are shielded from the solvent These conclusions are in agreement with the circular dichroism results in 80% methano1/20% water where the a helix is present to the extent of 30%, whereas the peptide is completely unstructured in water with some aggregation.

The temperature dependence of the NH proton shifts was also carried out. In water these shifts are of the order of7-9 × 10^?3 ppm/K indicating that most of the protons are not involved in hydrogen bonding. In CD₃0H/H₂0, these values range from about 4–6 × 10^?3 ppm/K, which are compatible with the presence of hydrogen bonds.

Finally, binding studies were carried out between the tubulin peptide and the undecapeptide neurotransmitter substance P. The largest shifts are for the Tyr³ NH proton of the tubulin fragment, whereas for substance P it is for the Lys³, Gin⁵ and Leu¹⁰ NH protons, indicating a change in conformation of both peptides on interaction.     

The effects of chain length on the secondary structure of oligoadenylates

The oligoadenylates (Ap)_2–4A have been studied by proton magnetic resonance (pmr) spectroscopy. All the exterior base protons and a number of the interior base proton resonance have been assigned. The results of this work showed that the adenine bases in these oligoadenylates are intramolecularly stacked at 20°C with their bases oriented preferentially in the anti conformation about their respective glycosidic bonds. The oligomers were found to associate extensively even at concentrations of 0.02 M, primarily via “end-to-end” stacking. With increasing temperature, the oligomer bases destack, but it is argued that this unfolding process cannot be described in terms of a two-state stacked versus unstacked model. Instead, the temperature dependences of the base proton chemical shifts support a base-oscillation model. The relationship between this model and the two-state model is discussed. Finally, on the basis of the chain-length dependence of the proton chemical shifts of the various adenine bases, it was concluded that subtle variations in the secondary structure of these oligomers exist with increasing chain length. Evidence is presented to show that the effects of distant base shielding are considerably smaller than what was previously estimated. The observed departures from the “extended dimer” model are attributed to differences in the relative orientations of the bases with respect to their neighbors in the oligomer.     

Assignment of the 270 MHz nuclear magnetic resonance spectrum of somatostatin in water

The proton nuclear magnetic resonance spectrum of the 14 residue peptide hormone somatostatin in D₂O at 270 MHz has been assigned by comparing the spectra of synthetic analogs with those of the native peptide. Extensive difference double resonance studies of all somatostatins and pH titrations confirmed all assignments. ³J_NHCH values and conventional NMR hydrogen bonding studies confirm the existence of preferred secondary conformations but not with a predominant conformation possessing a β-turn in either of the sequences 7–8–9–10 or 8–9–10–11. More extensive data treatment is needed before the actual conformation(s) of somatostatin is elucidated, but several NMR criteria for conformations are proposed.     

Synthesis and conformational analysis of aib-containing peptide modelling for N-glycosylation site in N-glycoprotein

A tetrapetide containing an Aib residue, Boc-Asn-Aib-Thr-Aib-OMe, was synthesized as a peptide model for the N-glycosylation site in N-glycoproteins. Backbone conformation of the peptide and possible intramolecular interaction between the Asn and Thr side chains were elucidated by means of n.m.r. spectroscopy. Temperature dependence of NH proton chemical shift and NOE experiments showed that Boc-Asn-Aib-Thr-Aib-OMe has a tendency to form a β-turn structure with a hydrogen bond involving Thr and Aib⁴ NH groups. Incorporation of Aib residues in the peptide model promotes folding of the peptide backbone. With folded backbone conformation, carboxyamide protons of the Asn residue are not involved in hydrogen bond network, while the OH group of the Thr residue is a candidate for a hydrogen bond in DMSO-d₆ solution.     

Stereospecific assignments of 1H-nmr methyl lines and conformation of valyl residues in the lac repressor headpiece

Two-dimensional ¹H-nmr methods are described to obtain information on the sidechain conformation of valyl residues of the lac repressor headpiece and to assign the resonances of their methyl groups stereospecifically. The spin–spin coupling constants (J_αβ) between C^αand C^β protons are obtained from two-dimensional correlated spectroscopy experiments. Large values for J_αβ(10–12 Hz) corresponding to trans orientations for these protons (g⁺ conformation) are found for all valyl residues in α-helical segments. For these valyl residues, the distance between one methyl group (γ₁)and the valyl amide proton is much shorter than for the other methyl group, so that stereospecific resonance assignments follow from relative intensities of the corresponding cross peaks in a two-dimensional nuclear Overhauser enhancement spectrum. Thus, streospecific assignments could be made for the methyl groups of Val 9, 20, 23, and 38 (of a total of eight valyl residues).     

Crystal structure and solution conformation of 1-N-acetyl-β-D-glucopyranosyl amine: A model for the glycopeptide linkage

The crystal structure of the title compound, a model for the glycosyl linkage between the asparagine side chain and N-acetyl glucosamine in glycoproteins, has been determined and compared to other model structures. The pyranose ring in the crystal is in the ⁴C₁ chair conformation and the amide functions at C₁ and at C₂ are both oriented such that the amide protons are nearly trans to their respective sugar-ring protons. Coupling constants determined from the fully assigned proton nmr spectrum in aqueous solution are consistent with the conformation in the crystal.     

Conformational analysis of thyrotropin releasing factor by proton magnetic resonance spectroscopy

The proton magnetic resonance spectrum of thyrotropin releasing factor (TRF) in solution in deuterium oxide and deuterated dimethylsulfoxide (DMSO–d₆) has been analyzed. Two forms differing in cis–trans isomerism about the His-Pro peptide bond are observed. From the temperature dependence of chemical shift of the amide protons, it is concluded that TRF in DMSO–d₆ does not contain intramolecular hydrogen bonds. Measurement of NH? C_αH coupling constant provides an estimate of the histidine dihedral angle ?. Structural information about the histidine side-chain is deduced from C_αH? C_βH coupling constants and from the nonequivalence of the two prolyl δ-protons. In DMSO–d₆, there is evidence for a tautomeric equilibrium corresponding to an exchange of imidazole proton between the two nitrogen atoms N-δ and N-ε. In water, the N-εH tautomer is found to be the predominant tautomeric form of the imidazole ring. These results in combination with energy calculation, vibrational analysis, and carbon nmr studies allow the determination of the conformationof TRF.     

Total assignments, including four aromatic residues, and sequence confirmation of the decapeptide tyrocidine A using difference double resonance. Qualitative nuclear overhauser effect criteria for beta turn and antiparallel beta-pleated sheet conformations.

The complete assignments of all the proton magnetic resonance signals from each NH-CalphaH-CbetaH2 moiety in a complex peptide containing several residues of the same type has not yet been achieved without specific or stereospecific isotopic enrichment. We report the sequencing and proton magnetic resonance spectral assignments, including those of 4 aromatic residues, of tyrocidine A, an analog of the decapeptide gramicidin S. Two complementary methods, proton-proton nuclear Overhauser enhancements and scalar decoupling, evaluated by two distinct forms of difference double resonance, were used. All chemical shifts, scalar coupling constants, and [1H:1H] nuclear Overhauser enhancements for the backbone protons are reported. The [1H:1H] nuclear Overhauser enhancements are consistent with tyrocidine A possessing a beta-I turn/beta-II' turn/antiparallel beta-pleated sheet conformation. In addition to the previously proposed nuclear Overhauser enhancement criteria for beta turns and antiparallel beta sheets, another criterion for identifying the antiparallel beta sheet is demonstrated; namely, the nuclear Overhauser enhancement between 2 CalphaH protons of the central resisdues, in this case the Phe7CalphaH and Orn2CalphaH.     

Proton and phosphorus NMR studies of d-CpG(pCpG)n duplexes in solution. Helix-coil transition and complex formation with actinomycin-D.

The Watson–Crick imino and amino exchangeable protons, the nonexchangeable base and sugar protons, and the backbone phosphates for d-CpG(pCpG)_n, n = 1 and 2, have been monitored by high-resolution nmr spectroscopy in aqueous solution over the temperature range 0°–90°C. The temperature dependence of the chemical shifts of the tetramer and hexamer resonances is consistent with the formation of stable duplexes at low temperature in solution. Comparison of the spectral characteristics of the tetranucleotide with those of the hexanucleotide with temperature permits the differentiation and assignment of the cytosine proton resonances on base pairs located at the end of the helix from those in an interior position. There is fraying at the terminal base pairs in the tetranucleotide and hexanucleotide duplexes. The Watson–Crick ring imino protons exchange at a faster rate than the Watson–Crick side-chain amino protons, with exchange occurring by transient opening of the double helix. The structure of the d-CpG(pCpG)_n double helices has been probed by proton relaxation time measurements, sugar proton coupling constants, and the proton chemical shift changes associated with the helix–coil transition. The experimental data support a structural model in solution, which incorporates an anti conformation about the glycosyl bonds, C(3) exo sugar ring pucker, and base overlap geometries similar to the B-DNA helix. Rotational correlation times of 1.7 and 0.9 × 10^?9 sec have been computed for the hexanucleotide and tetranucleotide duplexes in 0.1 M salt, D₂O, pH 6.25 at 27°C. The well-resolved ³¹P resonances for the internucleotide phosphates of the tetramer and hexamer sequences at superconducting fields shift upfield by 0.2–0.5 ppm on helix formation. These shifts reflect a conformational change about the ω,ω′ phosphodiester bonds from gauche-gauche in the duplex structure to a distribution of gauche-trans states in the coil structure. Significant differences are observed in the transition width and midpoint of the chemical shift versus temperature profiles plotted in differentiated form for the various base and sugar proton and internucleotide phosphorous resonances monitoring the d-CpG(pCpG)_n helix–coil transition. The twofold symmetry of the d-CpGpCpG duplex is removed on complex formation with the antibiotic actinomycin-D. Two phosphorous resonances are shifted downfield by ～2.6 ppm and ～1.6 ppm on formation of the 1:2 Act-D:d-CpGpCpG complex in solution. Model studies on binding of the antibiotic to dinucleotides of varying sequence indicate that intercalation of the actinomycin-D occurs at the GpC site in the d-CpGpCpG duplex and that the magnitude of the downfield shifts reflects strain at the O-P-O backbone angles and hydrogen bonding between the phenoxazone and the phosphate oxygens. Actinomycin-D is known to bind to nucleic acids that exhibit a B-DNA conformation; this suggests that the d-CpG(pCpG)_n duplexes exhibit a B-DNA conformation in solution.     

Conformation of tetranactin in solution.

The conformation of tetranactin, an ionophore, in chloroform was investigated by infrared and Raman spectra and by proton and ¹³C magnetic resonances. The infrared spectra show that the structure of its K⁺ complex in the solution is quite similar to that in crystals. The proton spin–spin coupling constants are explained well by assuming that the crystalline structure is retained in solution. The spin–lattice relaxation times of the ¹³C nuclei of the K⁺ complex indicate that its framework is rigid. The correlation time of the overall reorientation of the molecule was calculated to be 9 X 10^?11 sec. On the other hand, the conformation of the complexed form in chloroform differs from that in crystals. Despite the geometrical nonequivalence of the four subunits in the crystalline state, the nuclear magnetic resonance spectra show their magnetic equivalence in the solution. The proton spin–spin coupling constants have values that are averaged by rapid internal rotation. The spin–lattice relaxation times of the ¹³C nuclei in its framework are unexplained by the overall reorientation of the molecule, and reveal the existence of internal motion in the framework. The rate of the local motion of the framework is between 10²–10¹⁰ sec^?1. By comparison of the infrared spectra, it can be said that the mean conformation of the fluctuated framework of the uncomplexed tetranactin in the solution is similar to that of nonactin in the crystalline form, which has an S₄ symmetry axis through the center of the macrocyclic ring.     

Block copolymers of amino acids. II. Physicochemical data on copolymers containing L-alanine or L-phenylalanine

The water-soluble block copolymers, whose syntheses were described in the preceding paper, were subjected to ultracentrifugation, viscosity, optical rotatory dispersion circular dichroism, fluorescence, proton magnetic resonance, and infrared measurements. Discrepancies between M _n and M _w, and line broadening in the proton magnetic resonance spectra (attributed to the formation of rigid structures), support the conclusion (drawn in the preceding paper) that the block copolymers are aggregated in aqueous solution. It is shown that similar block copolymers reported in the literature are also aggregated.     

Mixed micelle formation between gramicidin-S and a nonionic detergent: a nuclear magnetic resonance model study of peptide/detergent aggregation

The interaction of the cyclic decapeptide antibiotic gramicidin-S (GrS) with the nonionic detergent octaethylene glycol mono-n-dodecyl ether was studied by NMR spectroscopy. Detergent binding led to a slightly altered average conformation in the d-Phe side chains of the peptide. The changing diamagnetic shielding of nearby protons resulted in chemical shift variations, the largest effect being observed for the d-Phe C _α proton. The continuous upfield shift of this proton resonance, indicating rapid exchange of the peptide between detergent-associated and unassociated states, was employed for an evaluation of the detergent/peptide aggregation equilibria. The nonlinear binding plot thus obtained was attributed to essentially different aggregational states, depending on the detergent/peptide ratio. The almost linear dependence of the spin-lattice relaxation rate and of the hydrogen-deuterium exchange rate on the fraction of detergent-associated GrS could be reconciled with a simple model, comprising binding of detergent monomers and cooperative binding of micelles at low and high detergent/peptide molar ratios, respectively. Thus, GrS provides a useful model for a study of backbone dynamics and water penetration in detergent- and membrane-bound peptides and proteins. The results will also be discussed with reference to the interaction of GrS with biological membranes. Received: 22 June 1998 / Revised version: 5 October 1998 / Accepted: 9 October 1998     

High resolution nuclear magnetic resonance studies of the active site of chymotrypsin. I. The hydrogen bonded protons of the "charge relay" system

High resolution proton nuclear magnetic resonance has been used to observe protons at the active site of chymotrypsin A_δ and at the same region of chymotrypsinogen A. A single resonance with the intensity of one proton is located in the low field region of the nuclear magnetic resonance spectrum. This resonance is observed in H₂O solutions but not in ²H₂O. On going from low to high pH the resonance titrates upfield 3 parts per million in both proteins and has a pK of 7.5. The titration can be prevented by alkylating His57 with either of two active site directed chloromethyl ketones. Using these data the proton resonance has been assigned to a proton in a hydrogen bond between His57 and Asp102. Further confirmation of this assignment lies in the observation of a similar resonance in this same low field region of the nuclear magnetic resonance spectrum of trypsin, trypsinogen, subtilisin BPN′ and α-lytic protease all of which have the Asp-His-Ser triad at their active sites.This proton resonance in chymotrypsin A_δ was used as a probe to monitor the charge state of the active site upon formation of a stable acyl-enzyme analogue N²(N-acetylalanyl)-N¹benzoylcarbazoyl-chymotrypsin A_δ. In this derivative the His-Asp proton resonance titrates from the same low pH end point as in the native enzyme, ?18 parts per million, to a new high pH end point of ?14.4 parts per million (versus ?15.0 parts per million in the native enzyme). The difference of 0.6 parts per million in the high pH end points between the native and acyl enzyme is interpreted as supporting the suggestion that a hydrogen bond exists between Ser195 and His57 in the native enzyme and zymogen.We conclude from these studies that the charge relay system from Asp102 across His57 to Ser195 is intact in chymotrypsin A_δ and chymotrypsinogen A, and that, in the native enzyme, it slightly polarizes Ser195.     

Nuclear magnetic resonance studies on oligo-deoxyribonucleotides containing thedam methylation site GATC. Adenine methylation of d(GGATCC) does not change the helix geometry

We have studied the conformation of two hexanucleotides d(GGATCC) and d(GGm⁶ATCC) using proton nuclear magnetic resonance. Nuclear Overhauser effect measurements show that d(GGATCC) assumes a normal right handed B helix. The single and double strand resonances are in fast exchange on a proton nuclear magnetic resonance time scale. For d(GGm⁶ATCC), up to the Tm separate resonances are observed for each state, indicating slow exchange, though above the Tm it becomes more rapid. The orientation of the adenosine methyl-amino group, preferentiallycis to N¹, hinders base pair formation.The connectivities of the resonances of the two states were established by saturation transfer experiments. At 0°C irradiation of the m⁶ A-T imino proton gives an nuclear Overhauser effect to AH² showing that base pairing is Watson-Crick. Intra and interresidue nuclear Overhauser effects starting from the 3′ terminus show that the helix is right handed and in the B-form.The results on the two oligomers demonstrate that adenosine methylation induces little or no change in the conformation of the helix, but reduces the Tm from 45° to 32°C and slows the opening and closing of the m₆A.T base pair by a factor of about 100.     

Comparative study of conformational behaviour of leucine and methionine enkephalinamides by1H-nuclear magnetic resonance spectroscopy

The conformational proclivity of leucine and methionine enkephalinamides in deuterated dimethyl sulphoxide has been investigated using proton magnetic resonance at 500 MHz. The resonances from the spin system of the various amino acid residues have been assigned from the 2-dimensional correlated spectroscopy spectra. The temperature variation of the amide proton shifts indicates that none of the amide proton is intramolecularly hydrogen-bonded or solvent-shielded. The analysis of vicinal coupling constants,³J_{HN.C 2}H,along with temperature coefficients and the absence of characteristic nuclear Overhauser effect cross peaks between the NH protons reveal that there is no evidence of the chain folding in these molecules. However, the observation of nuclear Overhauser effect cross peaks between the NH and the C^αH of the preceding residue indicates preference for extended backbone conformation with preferred side chain orientations particularly of Tyr and Phe in both [Leu⁵]- and [Met⁵]-enkephalinamides.