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.     

Conformation of the glycotripeptide repeating unit of antifreeze glycoprotein of polar fish as determined from the fully assigned proton n.m.r. spectrum

With its simple glycotripeptide repeating structure the antifreeze glycoprotein of polar fish may be an especially simple conformational mode for mucin glycoproteins with similar but more complex structures. The fully assigned proton n.m.r. spectrum confirms the anomeric configurations of the hexapyranosidic sugars of the side chains and the coupling constants of the alpha GalNAc and the beta Gal residues show both to be in the expected 4C1 chair conformation. The assignment of a single resonance for each proton of the (Ala-Thr-Ala)n repeat unit coupled with the observation of long range nuclear Overhauser effects (n.O.e.) implies a three-fold repeating conformation. The resonances of the two alanines are distinct and can be assigned to their correct positions in the peptide sequence by n.O.e. observed at the amide proton resonances on saturation of the alpha proton signals. The amide proton coupling constants of all three peptide residues are similar and imply a limited range of peptide backbone torsion angles, phi CN. The large n.O.e. which has been observed between the amide proton and the alpha proton of the residue preceding it in the sequence implies large positive values for the peptide dihedral angle, psi CC. Limits are placed on possible values of side chain dihedral angles by the observation of the coupling constant between the alpha and beta protons of the threonyl residue. The observation of n.O.e. between the anomeric proton of GalNAc and the threonyl side chain protons gives information on the conformation of the alpha glycosidic linkage between the disaccharide and the peptide. n.O.e. observed between the protons of the beta glycosidic linkage indicates the conformation of the disaccharide and the large amide proton coupling constant of the GalNAc residue shows a trans proton relationship. The spectroscopically derived data have been combined with conformational energy calculations to give a conformational model for antifreeze glycoprotein in which the hydrophobic surfaces of the disaccharide side chains are wrapped closely against a three-fold left handed helical peptide backbone. The hydrophilic sides of the disaccharides are aligned so that they may bind to the ice crystal face, which is perpendicular to the fast growth axis inhibiting normal crystal growth.     

Nuclear magnetic resonance studies on the structure of the tetrapeptide tuftsin, L-threonyl-L-lysyl-L-prolyl-L-arginine, and its pentapeptide analogue L-threonyl-L-lysyl-L-prolyl-L-prolyl-L-arginine.

Nuclear magnetic resonance spectroscopy has been used to investigate the solution conformation of tuftsin, threonyllysylprolylarginine, as well as a pentapeptide inhibitor of tuftsin, threonyllysylprolylprolylarginine. Both proton and carbon-13 studies were performed. In water, neither peptide gives evidence of a preferred conformation. In dimethyl-d6 sulfoxide, tuftsin appears to prefer a particular conformation, but the inhibitor does not. The conformation of tuftsin is one in which the amide NH proton of arginine is solvent shielded. The conformation does not, however, appear to be such that a normal 4 leads to 1 beta turn exists.     

1H-NMR conformational study of a synthetic peptide derived from the consensus sequence of annexins.

The solution conformation of a synthetic 18 amino acid peptide derived from a consensus sequence of Annexins has been investigated by 1H NMR. Full sequential assignment has been achieved. Conformational properties of the peptide were deduced from the analysis of J(NH-CH alpha) coupling constants, amide proton exchange, 2D NOESY connectivities and computer modeling.     

Two-dimensional NMR studies of the antimicrobial peptide NP-5

Nearly complete proton resonance assignment of the rabbit antimicrobial peptide NP-5 has been made from two-dimensional NMR data taken at a single temperature. The assignment procedure involved acquisition of phase-sensitive double-quantum-filtered correlation spectra, relayed coherence-transfer spectra, total correlation (homonuclear Hartmann-Hahn) spectra, double- and triple-quantum spectra, and nuclear Overhauser effect spectra. The combination of these complementary experiments simplified and accelerated resonance assignment of the peptide. Individual assignments were made at 20 degrees C for all amide and C alpha protons in the peptide, and for all nonlabile side-chain protons on 26 of the 33 amino acid residues in NP-5. Analysis of the proton-proton nuclear Overhauser effect connectivities, the slowly exchanging amide protons, and the proton chemical shifts in NP-5 indicates that the peptide has a stable, ordered structure in solution. These data also indicate that residues 19-29 in NP-5 are involved in an antiparallel beta-sheet that has a hairpin conformation.     

Effects of glycosylation on the structure and dynamics of eel calcitonin in micelles and lipid bilayers determined by nuclear magnetic resonance spectroscopy.

The three-dimensional structures of eel calcitonin (CT) and two glycosylated CT derivatives, [Asn(GlcNAc)3]-CT (CT-GlcNAc) and [Asn(Man6-GlcNAc2)3]-CT (CT-M6), in micelles were determined by solution NMR spectroscopy. The topologies of these peptides associated with oriented lipid bilayers were determined with solid-state NMR. All of the peptides were found to have an identical conformation in micelles characterized by an amphipathic alpha-helix consisting of residues Ser5 through Leu19 followed by an unstructured region at the C-terminus. The overall conformation of the peptide moiety was not affected by the glycosylation. Nevertheless, comparison of the relative exchange rates of the Leu12 amide proton might suggest the possibility that fluctuations of the alpha-helix are reduced by glycosylation. The presence of NOEs between the carbohydrate and the peptide moieties of CT-GlcNAc and CT-M6 and the amide proton chemical shift data suggested that the carbohydrate interacted with the peptide, and this might account for the conformational stabilization of the alpha-helix. Both the unmodified CT and the glycosylated CT were found to have orientations with their helix axes parallel to the plane of the lipid bilayers by solid-state NMR spectroscopy.     

Conformational energy calculations on glycosylated turns in glycoproteins

Analysis of the amino acid sequence of glycoproteins has suggested the β-turn as a likely site of glycosylation in glycoproteins. According to this model, the peptide chain traverses the interior of a globular protein, reversing its direction at the protein surface, a likely point for the attachment of hydrophilic carbohydrate residues. In order to search for plausible conformations of glycosylated β-turns in asparagine-linked glycoproteins, we have adapted the conformational energy calculation method of Scheraga and coworkers for use in carbohydrates. The parameters for nonbonded and hydrogen-bonded interactions have been published, and electrostatic parameters are derived from a CNDO calculation on a model glycopeptide. Our results indicate that the orientation of the glycosyl amide bond having the amide proton nearly trans to the anomeric proton of the sugar has the lowest energy. Although CD and nmr experiments in our laboratory have consistently found this conformation, our calculations show the conformation having these two protons in a cis relationship to lie very close in energy. Calculations on the glycopeptide linkage model, α-N-acetyl, δ-N(2-acetamido-1,2-dideoxy-β-D -glucopyranosyl)-N′-methyl-L -asparaginyl amide show that several distinct geometries are allowed for glycosylated β-turns. For a type I β-turn, three conformations of the glycosylated side chain are found within 4 kcal of the minimum, while two conformations of the glycosylated side chain are allowed for a type II turn. The hydrogen-bonded C7 conformation is also allowed. Stereoviews of the low-energy conformations reveal no major hydrogen-bonding interaction between the peptide and sugar.     

Proton nuclear magnetic resonance study of an active pentapeptide fragment of ubiquitin

The aqueous solution conformation of Tyr-Asn-Ile-Gln-Lys (UB5) corresponding to positions 59-63 of the polypeptide, ubiquitin, has been investigated by proton NMR. Like the parent protein, UB5 induces nonspecifically both T and B lymphocyte differentiation. The various NH and CH resonances of this pentapeptide have been assigned, and its solution conformation has been probed through a study of chemical shift variations with pH, temperature dependence of amide hydrogen chemical shifts, vicinal NH--C alpha H and C alpha H--C beta H2 coupling constant data, and amide hydrogen-exchange rates. The latter were measured in H2O by using a combination of transfer of solvent saturation and saturation recovery NMR experiments. The data are compatible with the assumption of a highly motile dynamic equilibrium among different conformations for this peptide. The various secondary amide hydrogens remain essentially exposed to the solvent. The temperature-dependence study of the amide hydrogen chemical shifts also did not reveal any strong internal hydrogen bonds. A rotamer population analysis of tyrosine and asparagine side chains suggests that two of the rotomers are predominantly populated for each of these residues. From these results, a picture emerges of the dynamic conformation of UB5 in aqueous solution.     

The use of N-methylated peptides and depsipeptides to probe the binding of heptapeptide substrates to cAMP-dependent protein kinase

Peptide 1, Leu-Arg-Arg-Ala-Ser-Leu-Gly, is an excellent substrate for cAMP-dependent protein kinase. While the importance of both arginines for effective enzyme-substrate interactions has been shown, it has not been known whether the kinase will catalyze phosphorylation of substrates which contain other than peptide bonds. We report that analogs of peptide 1 which contain depsi linkages replacing selected amide bonds are good protein kinase substrates. Therefore, with the possible exception of the serine amide proton, no peptide 1 amide hydrogens are involved in peptide-peptide or peptide-enzyme hydrogen bonding crucial to defining the high substrate activity of this peptide. It is thus unlikely that peptide 1 is bound by the protein kinase while in an alpha-helical or a beta-turn structure. Three peptides were found to be very poor substrates for protein kinase, those containing N-methyl amino acids in place of Ser5 or Leu6 and a peptide containing Pro in place of Leu6. These peptides are poor substrates for the enzyme possibly because they are unable to adopt a conformation necessary for catalysis of phosphoryl group transfer to occur or due to steric effects in the enzymatic active site.     

Nmr analyses of conformations of linear peptides in solution

Important aspects in detailed nmr analyses of the conformations of linear peptides are discussed using enkephalin and the α-mating factor of Saccharomyces cerevisiae as examples. The cationic, dipolar, and anionic forms in dimethyl sulfoxide solution may be identified by ir analyses. Because of the electrostatic interaction between the N- and C-terminal groups, the dipolar form of enkephalin takes the folded conformation, as well as extended conformation(s), in dimethyl sulfoxide solution. Such conformational equilibrium is responsible for anomalous temperature dependences and solvent-composition dependences of the amide and C^α proton chemical shifts. Active analogs, enkephalinamide and enkephalinol, take extended conformation(s) in solution. These opioid peptides probably take a specific active conformation upon binding with a receptor. For the α-mating factor and active peptide analogs in aqueous solution, a folded conformation with two βturn structures is responsible for the biological activity.     

Evidence for a folded structure of Met-enkephalin in membrane mimetic systems: 1H-nmr studies in sodiumdodecylsulfate, lyso-phosphatidylcholine, and mixed lyso-phosphatidylcholine/sulfatide micelles

¹H-nmr spectra of Met-enkephalin dissolved in aqueous solution of sodiumdodecylsulfate (SDS) micelles are reported as a function of pH and temperature. The temperature behavior of the amide protons is compared with that observed for the same peptide dissolved in aqueous solution of lyso-phosphatidylcholine (LPC) and lyso-phosphatidylcholine-sulfatide (LPC-SH) micelles. The temperature coefficients are affected by the micelle polarity, which suggests that the peptide backbone is not remote from the micelle surface. pH titration performed in the presence of SDS micelles gives a number of intrinsic and extrinsic pK_a values, indicative of a folded structure of the opioid molecule. This conformation is characterized by the existence of an intramolecular hydrogen bond involving the Met-5 amide proton and an interaction of the N-terminal residue with the aliphatic side chains of both Phe-4 and Met-5.     

Influence of N-terminal residue stereochemistry on the prolyl amide geometry and the conformation of 5-tert-butylproline type VI beta-turn mimics.

The effects of N-terminal amino acid stereochemistry on prolyl amide geometry and peptide turn conformation were investigated by coupling both L- and D-amino acids to (2S, 5R)-5-tert-butylproline and L-proline to generate, respectively, N-(acetyl)dipeptide N'-methylamides 1 and 2. Prolyl amide cis- and trans-isomers were, respectively, favored for peptides 1 and 2 as observed by proton NMR spectroscopy in water, DMSO and chloroform. The influence of solvent composition on amide proton chemical shift indicated an intramolecular hydrogen bond between the N'-methylamide proton and the acetamide carbonyl for the major conformer of dipeptides (S)-1, that became less favorable in (R)-1 and 2. The coupling constant (3J(NH,alpha)) values for the cis-isomer of (R)-1 indicated a phi2 dihedral angle value characteristic of a type VIb beta-turn conformation in solution. X-ray crystallographic analysis of N-acetyl-D-leucyl-5-tert-butylproline N'-methylamide (R)-lb showed the prolyl residue in a type VIb beta-turn geometry possessing an amide cis-isomer and psi3-dihedral angle having a value of 157 degrees, which precluded an intramolecular hydrogen bond. Intermolecular hydrogen bonding between the leucyl residues of two turn structures within the unit cell positioned the N-terminal residue in a geometry where their phi2 and psi2 dihedral angle values were not characteristic of an ideal type VIb turn. The circular dichroism spectra of tert-butylprolyl peptides (S)- and (R)-1b were found not to be influenced by changes in solvent composition from water to acetonitrile. The type B spectrum exhibited by (S)-1b has been previously assigned to a type VIa beta-turn conformation [Halab L, Lubell WD. J. Org. Chem. 1999; 64: 3312-3321]. The type C spectrum exhibited by the (R)-lb has previously been associated with type II' beta-turn and alpha-helical conformations in solution and appears now to be also characteristic for a type VIb geometry.     

FTIR spectroscopic studies of the conformation and amide hydrogen exchange of a peptide model of the hydrophobic transmembrane alpha-helices of membrane proteins.

The conformation and amide hydrogen exchangeability of the hydrophobic peptide Lys2-Gly-Leu24-Lys2-Ala-amide were studied by Fourier transform infrared spectroscopy. In these studies information on the secondary structure of the peptide was obtained from an examination of the contours of both the amide I and amide II absorption bands. The conformationally sensitive amide I and amide II regions of the infrared spectra suggest that the peptide is predominantly alpha-helical and that it contains some non-alpha-helical structures which are probably in an extended conformation. Studies of the exchangeability of the amide protons of the peptide indicate that there are two populations of amide protons which differ markedly with respect to their exchangeability with the bulk solvent phase, whether the peptide is dissolved in methanol or dispersed in hydrated lipid bilayers. One population of amide protons is very readily exchangeable, and our data suggest that it arises primarily but not exclusively from the extended regions of the peptide. The other population exchanges very slowly with the bulk solvent and appears to originate entirely from the alpha-helical domain of the peptide. This latter population is virtually unexchangeable when the peptide is dispersed in hydrated phosphatidylcholine bilayers but can be largely exchanged when the peptide is solubilized with methanol. We suggest that this slowly exchanging population of amide protons arises from the central part of the hydrophobic polyleucine core which forms a very stable alpha-helix that would be deeply buried in the hydrophobic domain of hydrated lipid bilayers.(ABSTRACT TRUNCATED AT 250 WORDS)     

The conformational characteristics in solution of the synthetic cyclic hexapeptide 5L-ala-D-ala

An attempt to elucidate the solution conformation(s) of the synthetic cyclic hexapeptide 5L -ala·D-ala is described. Nuclear magnetic resonance (nmr) spectra are recorded for the purpose of measuring the vicinal coupling constant between the amide and α-protons in each residue and to observe the deuterium exchange rate and temperature dependence of the chemical shift of each amide proton. Low-energy cyclic conformations, whose individual residues are in conformations consistent with the observed amide to α-proton coupling constant, are searched for in an approximate theoretical treatment. The two lowest energy, all trans peptide bond conformations generated are distinguishable by the presence or absence of a single intramolecular hydrogen bond. The observed temperature independence of the chemical shift of one of the amide protons is consistent with the presence of a single intramolecular hydrogen bond, while the observation of similar deuterium exchange rates for each of the amide protons indicates their comparable availability to solvent. Consequently, it is concluded that 5L -ala·D-ala is in rapid equilibrium between conformations with and without a single internal hydrogen bond and possesses considerable conformational flexibility in solution.     

Conformation and interaction with the membrane models of the amino-terminal peptide of influenza virus hemagglutinin HA2 at fusion pH.

Conformations of a 48-mer peptide corresponding to the amino-terminal region of influenza HA2 in aqueous and membranous environments were studied. In aqueous solution the peptide was found to be oligomeric and its helicity was enhanced at higher concentrations. The conformation in phospholipid bilayer and insertion depth into the sodium dodecyl sulfate (SDS) micelle for the fusion peptide were in line with those determined for the amino-terminal 25-mer analog. The turn of residues 28-31 found in the crystal structure of hemagglutinin at neutral pH persisted in the presence of SDS at pH 5.0. Except for the turn, conformational lability of the amino portion of HA2 is suggested by comparison of the secondary structure determined herein with that obtained with the influenza fusion protein crystallized in the aqueous phase at neutral pH. The backbone amide proton exchange experiment suggested an interaction with the micellar surface for the segment carboxy-terminal to the fusion peptide domain.     

1H-NMR studies of receptor-selective substance P analogues reveal distinct predominant conformations in DMSO-d6

Proton nmr parameters are reported for DMSO-d6 solutions of two receptor-selective substance P analogues: Ac[Arg6,Pro9]SP6-11, which is selective for the NK-1 (SP-P) receptor and [pGlu6,N-MePhe8]SP6-11, which selectively activates the NK-3 (SP-N) receptor. Full peak assignments of both analogues were obtained by COSY experiments. The chemical shifts, coupling constants, and temperature coefficients of amide proton chemical shifts as well as NOESY effects and calculated side-chain rotamer populations of Phe side chains are reported for both peptides. Analysis of coupling constants and temperature coefficients together with the nuclear Overhauser enhancement spectroscopy effects suggest that Ac[Arg6,Pro9]SP6-11 has a trans configuration about the Phe8-Pro9 amide bond and the preferred conformation of this analogue has a type I beta-turn. The nmr data for [pGlu6,N-MePhe8]SP6-11 suggest that this peptide exists as a mixture of cis-trans isomers in which the cis isomer can preferably adopt a type VI beta-turn conformation, and the trans isomer can adopt a gamma-turn conformation. There are indications that the two last turns are stabilized by a hydrogen bond between the syn carboxamide proton and the pGlu ring carbonyl.     

Experimental and calculated conformational characteristics of the bicyclic heptapeptide phalloidin

Several conformations generated from approximate potential energy calculations are presented for the bicyclic heptapeptide phalloidin which are consistent with the conformation-dependent information obtained from proton nuclear magnetic resonance measurements performed on phalloidin in dimethylsulfoxide solution. In each conformation, the cysteine amide proton is intramolecularly hydrogen bonded, the tryptophan amide is internally buried and the methyl group of the alanine residue preceding tryptophan is shielded by the tryptophan ring. Thus, phalloidin appears to be a relatively rigid molecule in solution.     

The orientation and dynamics of substance P in lipid environments.

The membrane-associated conformation of substance P (RPKPQQFFGLM-NH2) has been previously proposed to be the NK1-receptor-active conformation. In this work, NMR methods are applied to explore the orientation and dynamics of substance P at lipid surfaces for which the peptide's three-dimensional structure had been previously determined. Here the presence of dodecylphosphocholine (DPC) or sodium dodecylsulfate (SDS) micelles has been found to cause sequence specific changes in the acid- and base-catalyzed amide proton exchange rates relative to the solution state values. On binding of substance P to SDS micelles, the FFG portion showed the largest decreases in the base-catalyzed amide exchange rates. Similar sequence-specific changes in substance P are observed in the presence of DPC micelles, albeit at much weaker levels due to fast exchange between free and bound forms of the peptide. These differences are attributed to the location of the amide protons either in the surface double layer (via electrostatic effect) or inserted into the polar head group region of the micelles (via low dielectric). The sequence-specific effects of micelle association were also observed in the homonuclear nonselective spin-lattice relaxation time; these, in combination with spin-spin relaxation times, were used to calculate correlation times for the backbone amide protons. These data combined with paramagnetic broadening observations on peptide protons in the presence of spin-labeled lipids yield a detailed model of the interaction of substance P with lipid surfaces.