Combined effect of the DeltaPhe or DeltaAla residue and the p-nitroanilide group on a didehydropeptides conformation

Two series of dehydropeptides of the general formulae Boc-Gly-X-Phe-p-NA, Boc-Gly-Gly-X-Phe-p-NA, Gly-X-Gly-Phe-p-NA.TFA, and Boc-Gly-X-Gly-Phe-p-NA, with X = Delta(Z)Phe and DeltaAla, were studied with NMR in DMSO and CDCl(3)-DMSO, and with CD in MeOH, MeCN, and TFE. The NMR spectra measured in DMSO suggest that peptides with the DeltaPhe residue next to Phe are folded whereas peptides with Gly between DeltaPhe and Phe are less ordered. NMR spectra of DeltaAla-containing peptides indicate that these peptides are flexible and their conformational equilibria are populated by many different conformations. The CD spectra show that conformational properties of the peptides studied are distinctly influenced by a mutual position of the dehydroamino acid residue and the p-NA group. They indicate that all dehydropeptides with the DeltaPhe residue, Boc-Gly-DeltaAla-Phe-p-NA, and Boc-Gly-Gly-DeltaAla-Phe-p-NA adopt ordered conformations in all solvents studied, presumably of the beta-turn type. The last two peptides exhibit surprising chiroptical properties. Their spectra show exciton coupling-like couplets in the region of the p-NA group absorption. This shape of CD spectra suggests a rigid, chiral conformation with a fixed disposition of the p-NA group. The CD spectra indicate that Boc-Gly-DeltaAla-Gly-Phe-p-NA and Gly-DeltaAla-Gly-Phe-p-NA.TFA are unordered, independently of the solvent.     

Interaction of antimicrobial peptides from Australian amphibians with lipid membranes

Solid-state NMR and CD spectroscopy were used to study the effect of antimicrobial peptides (aurein 1.2, citropin 1.1, maculatin 1.1 and caerin 1.1) from Australian tree frogs on phospholipid membranes. 31P NMR results revealed some effect on the phospholipid headgroups when the peptides interact with DMPC/DHPC (dimyristoylphosphatidylcholine/dihexanoylphosphatidylcholine) bicelles and aligned DMPC multilayers. 2H NMR showed a small effect of the peptides on the acyl chains of DMPC in bicelles or aligned multilayers, suggesting interaction with the membrane surface for the shorter peptides and partial insertion for the longer peptides. 15N NMR of selectively labelled peptides in aligned membranes and oriented CD spectra indicated an alpha-helical conformation with helix long axis approximately 50 degrees to the bilayer surface at high peptide concentrations. The peptides did not appear to insert deeply into PC membranes, which may explain why these positively charged peptides preferentially lyse bacterial rather than eucaryotic cells.     

The use of p-fluorobenzenesulfonyl chloride as a reagent for studies of proteins by fluorine nuclear magnetic resonance

The reagent p-fluorobenzenesulfonyl chloride modifies the protein side chains of tyrosine, lysine, and histidine and the alpha-NH2 group. The p-fluorobenzenesulfonyl (Fbs-) group, identified by the 19F nuclear magnetic resonance signal, exhibits a different 19F chemical shift for each functional group modified. The Fourier-transformed spectra of the Fbs- group displayed the expected nine-line multiplet in Fbs- amino acids and simple Fbs- peptides but not in the Fbs- proteins, where the resolution was less. Lysozyme, RNase, DNase, and chymotrypsin react with this reagent and each Fbs- protein exhibits a distinctive pattern of 19F NMR signals due to the label, suggesting that the reaction of the reagent varies with the reactivity of the side chains in a protein. The three major 19F signals of the unfolded Fbs-RNase in 8 M urea are due to the Fbs- label on the imidazolium, alpha-NH2, and epsilon-NH2 groups. Based upon results from amino acid and 19F NMR analyses of the tryptic-chymotryptic peptides of Fbs-RNase, portions of the imidazolium and epsilon-NH2 resonances were assigned to the Fbs- label on His-105 and Lys-41, respectively, while the alpha-NH2 resonance was entirely due to the Fbs- label on the alpha-NH2 of Lys-1. Because Fbs-RNase has an unchanged, near-ultraviolet circular dichroism spectrum and because it retains approximately 80% of the RNase activity, the conformation of Fbs-RNase is probably not altered from the folded conformation of the native enzyme. Upon unfolding in 8 M urea or heating at 70 degrees C, Fbs-RNase gave a 19F NMR spectrum differing from that of the folded Fbs-RNase. In the presence of uridylic acid, Lys-41 was the only residue protected from modification by the reagent with a concomitant reduction of the epsilon-NH2 resonance, and the RNase thus modified was fully active. Hence, 19F NMR analysis of protein, via the reaction with p-fluorobenzenesulfonyl chloride, provided not only information about the protein conformation but also direct measurements of the modification status.     

The application of DOSY NMR and molecular dynamics simulations to explore the mechanism(s) of micelle binding of antimicrobial peptides containing unnatural amino acids

Anionic and zwitterionic micelles are often used as simple models for the lipids found in bacterial and mammalian cell membranes to investigate antimicrobial peptide‐lipid interactions. In our laboratory we have employed a variety of 1D, 2D, and diffusion ordered (DOSY) NMR experiments to investigate the interactions of antimicrobial peptides containing unnatural amino acids with SDS and DPC micelles. Complete assignment of the proton spectra of these peptides is prohibited by the incorporation of a high percentage of unnatural amino acids which don't contain amide protons into the backbone. However preliminary assignment of the TOCSY spectra of compound 23 in the presence of both micelles indicated multiple conformers are present as a result of binding to these micelles. Chemical Shift Indexing agreed with previously collected CD spectra that indicated on binding to SDS micelles compound 23 adopts a mixture of α‐helical structures and on binding to DPC micelles this peptide adopts a mixture of helical and β‐turn/sheet like structures. DOSY NMR experiments also indicated that the total positive charge and the relative placement of that charge at the N‐terminus or C‐terminus are important in determining the mole fraction of the peptide that will bind to the different micelles. DOSY and ¹H‐NMR experiments indicated that the length of Spacer #1 plays a major role in defining the binding conformation of these analogs with SDS micelles. Results obtained from molecular simulations studies of the binding of compounds 23 and 36 with SDS micelles were consistent with the observed NMR results. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 548–561, 2013.     

Peptides and proteins in a confined environment: NMR spectra at natural isotopic abundance.

Confinement of proteins and peptides in a small inert space mimics the natural environment of the cell, allowing structural studies in conditions that stabilize folded conformations. We have previously shown that confinement in polyacrylamide gels (PAGs) is sufficient to induce a change in the viscosity of the aqueous solution without changing the composition and temperature of the solvent. The main limitation of a PAG to run NMR experiments in a confined environment is the need for labelling the peptides. Here we report the use of the agarose gel to run the NMR spectra of proteins and peptides. We show that agarose gels are completely transparent in NMR experiments, relieving the need for labelling. Although it is necessary to expose biomolecules to fairly high temperatures during sample preparation, we believe that this is not generally an obstacle to the study of peptides, and found that the method is also compatible with temperature-resistant proteins. The mesh of agarose gels is too wide for direct effects of confinement on the stability of proteins but confinement can be easily exploited to interact the proteins with other reagents, including crowding macromolecules that can eventually lead to fold stabilization. The use of these gels is ideally suited for low-temperature studies; we show that a very flexible peptide at subzero temperatures is stabilized into a well-folded conformation.     

Solution conformational preferences of immunogenic peptides derived from the principal neutralizing determinant of the HIV-1 envelope glycoprotein gp120

With standard one- and two-dimensional proton NMR techniques, a common structural motif has been identified in water solutions of short peptide sequences derived from the envelope glycoprotein gp120 of HIV-1. Three peptides of lengths 12, 24, and 40 residues (termed RP342, RP142, and RP70, respectively) were synthesized, each containing a central amino acid sequence common to many HIV-1 isolates. In addition, RP70 contained a disulfide bond between cysteine residues close to the ends of the molecule, forming a loop that is thought to constitute an important structural and immunological component of the intact glycoprotein. Peptides RP70 and RP142 showed evidence for the presence of a significant population of conformations containing a beta-turn in the conserved sequence Gly-Pro-Gly-Arg. Strong nuclear Overhauser effect (NOE) connectivities were observed between the amide protons of the arginine and the adjacent glycine. A weak NOE connectivity was observed between the C alpha H of the proline residue and the NH of the Arg [a d alpha N(i,i + 2) NOE connectivity], confirming the presence of a conformational preference for a turn conformation in this sequence. The remainder of the peptide showed evidence of conformational averaging: no NMR evidence for a uniquely folded structure was obtained for any of the peptides in water solution. Circular dichroism (CD) spectra indicated that no ordered helix was present in water solutions of RP70, although a CD spectrum that indicated the presence of approximately 30% helix could be induced by the addition of trifluoroethanol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spectroscopic studies of structural changes in two beta-sheet-forming peptides show an ensemble of structures that unfold noncooperatively

A characterization of the conformation and stability of model peptide systems that form beta-sheets in aqueous solutions is considerably important in gaining insights into the mechanism of beta-sheet formation in proteins. We have characterized the conformation and equilibrium folding and unfolding of two 20-residue peptides whose NMR spectra suggest a three-stranded beta-sheet topology in aqueous solution: Betanova [Kortemme, T., Ramirez-Alvarado, M., and Serrano, L. (1998) Science 281, 253-256] and (D)P(D)P with d-Pro-Gly segments at the turns [Schenck, H. L., and Gellman, S. H. (1998) J. Am. Chem. Soc. 120, 4869-4870]. Both circular dichroism (CD) and infrared measurements indicate only 20-26% beta-sheet-like structure at 5 degrees C for Betanova and 42-59% beta-sheet for (D)P(D)P. For both peptides, the CD and infrared spectra change nearly linearly with increasing temperatures (or urea concentrations) and lack a sigmoidal signature characteristic of cooperative unfolding. Fluorescence resonance energy transfer (FRET) measurements between donor and acceptor molecules attached to the two ends confirm that Betanova is largely unstructured even at 10 degrees C; the average end-to-end distance estimated from FRET is closer to that of a random coil than a structured beta-sheet. In (D)P(D)P, the FRET results indicate a more compact structure that remains compact even at high temperatures (approximately 80 degrees C) or high urea concentrations (approximately 8 M). These results indicate that both these peptides access an ensemble of conformations at all temperatures or denaturant concentrations, with no significant free energy barrier separating the "folded" and "unfolded" conformations.     

Configurationally driven folding of model tetrapeptides containing L- or D-morpholine-3-carboxylic acids as beta-turn nucleators

The conformational analysis by NMR, IR, and molecular modeling of tetrapeptides containing morpholine-3-carboxylic acid (Mor) as a proline surrogate is presented. The relationship between the chirality of the cyclic amino acid at position i+1 and the turn propensity is maintained with respect to the reference proline-containing peptides, although marked differences in the type of folded structures were observed. The conformational profile of morpholine-containing turn peptides as a function of the chirality of the cyclic amino acid indicated that the heterochiral tetrapeptide containing the D-isomer of the cyclic amino acid is more prone to nucleate compact folded structures, although with no resemblance to the beta-turn structures of D-proline-containing peptides. Also, the solvation system proved to influence the organization of folded structures, as in the more interactive CD(3)CN the model peptides showed more compact conformations. The L-Mor-containing peptide displayed two rotamers at the Val-Mor amide bond. The trans isomer did not experience any turn structures, nor any intramolecular hydrogen-bonds, whereas the cis isomer showed a strong preference for a type VI beta-turn structure, thus providing a different conformational asset with respect to the beta-turn structure as reported for the reference L-proline model peptide.     

Autonomous folding of a peptide corresponding to the N-terminal beta-hairpin from ubiquitin.

The N-terminal 17 residues of ubiquitin have been shown by 1H NMR to fold autonomously into a beta-hairpin structure in aqueous solution. This structure has a specific, native-like register, though side-chain contacts differ in detail from those observed in the intact protein. An autonomously folding hairpin has previously been identified in the case of streptococcal protein G, which is structurally homologous with ubiquitin, but remarkably, the two are not in topologically equivalent positions in the fold. This suggests that the organization of folding may be quite different for proteins sharing similar tertiary structures. Two smaller peptides have also been studied, corresponding to the isolated arms of the N-terminal hairpin of ubiquitin, and significant differences from simple random coil predictions observed in the spectra of these subfragments, suggestive of significant limitation of the backbone conformational space sampled, presumably as a consequence of the strongly beta-structure favoring composition of the sequences. This illustrates the ability of local sequence elements to express a propensity for beta-structure even in the absence of actual sheet formation. Attempts were made to estimate the population of the folded state of the hairpin, in terms of a simple two-state folding model. Using published "random coil" values to model the unfolded state, and values derived from native ubiquitin for the putative unique, folded state, it was found that the apparent population varied widely for different residues and with different NMR parameters. Use of the spectra of the subfragment peptides to provide a more realistic model of the unfolded state led to better agreement in the estimates that could be obtained from chemical shift and coupling constant measurements, while making it clear that some other approaches to population estimation could not give meaningful results, because of the tendency to populate the beta-region of conformational space even in the absence of the hairpin structure.     

Solution structure of a peptide derived from the oncogenic protein beta-Catenin in its phosphorylated and nonphosphorylated states

Beta-Catenin plays an essential role in the Wingless/Wnt signaling cascade. Phosphorylation of beta-Catenin in its N-terminal region by the kinase GSK-3beta is required for the interaction with the SCF-beta-TrCP protein complex that targets beta-Catenin for proteasome degradation. In the present work, we used two peptides of 32 amino acids referred to beta-Cat17-48 and P-beta-Cat17-48 for the phosphorylated peptide at the two sites Ser33 and Ser37. Circular dichroism and NMR techniques were used to assess the influence of the phosphorylation. The spectra of the peptides at pH 7.2 were completely assigned. Analysis of the medium-range NOE connectivities indicated that beta-Cat17-48 seems to be only poorly folded. These data are in agreement with the result of structure calculations. P-beta-Cat17-48 possesses two helical segments around the DpSGXXpS motif, which forms a large bent with the phosphate groups pointing out of the structure. On the contrary, beta-Cat17-48 shows less well-defined secondary structures and appears as a more flexible peptide, but adopts in the motif DSGXXS a more compact conformation than P-beta-Cat17-48. Differences in this molecular region suggest that conformational changes of phosphorylated beta-Catenin play an important role for the interaction with the SCF-beta-TrCP protein complex.     

Preferential conformation of the endogenous opiate-like pentapeptide met-enkephalin in DMSO-d6 solution determined by high field 1H NMR

A preferential conformation of the endogenous opiate-like pentapeptide Met-Enkephalin in DMSO-d₆ solution was proposed from high field ¹H NMR experiments at variable temperature and complete analysis of the coupling constants in relation with conformational energy steric maps.This conformation is characterized by a highly folded secondary structure with a β_I turn involving a head-to-tail interaction and a quasi-axial position of the methionine side chain. The N-terminal Tyr-Gly moiety which exhibits a relative degree of freedom shows all the steric requirements found in opiates for a stereospecific interaction with the receptor. All these results are discussed in relation with the physicochemical and biological properties of opiate-like peptides.     

Role of peptide backbone conformation on biological activity of chemotactic peptides

To investigate the role of peptide backbone conformation on the biological activity of chemotactic peptides, we synthesized a unique analog of N-formyl-Met-Leu-Phe-OH incorporating the C alpha,alpha disubstituted residue, dipropylglycine (Dpg) in place of Leu. The conformation of the stereochemically constrained Dpg analog was examined in the crystalline state by x-ray diffraction and in solution using NMR, IR, and CD methods. The secretagogue activity of the peptide on human neutrophils was determined and compared with that of a stereochemically constrained, folded type II beta-turn analog incorporating 1-aminocyclohexanecarboxylic acid (Ac6c) at position 2 (f-Met-Ac6c-Phe-OMe), the parent peptide (f-Met-Leu-Phe-OH) and its methyl ester derivative (f-Met-Leu-Phe-OMe). In the solid state, the Dpg analog adopts an extended beta-sheet-like structure with an intramolecular hydrogen bond between the NH and CO groups of the Dpg residue, thereby forming a fully extended (C5) conformation at position 2. The phi and psi values for Met and Phe residues are significantly lower than the values expected for an ideal antiparallel beta conformation causing a twist in the extended backbone both at the N and C termini. Nuclear magnetic resonance studies suggest the presence of a significant population of the peptide molecules in an extended antiparallel beta conformation and the involvement of Dpg NH in a C5 intramolecular hydrogen bond in solutions of deuterated chloroform and deuterated dimethyl sulfoxide. IR studies provide evidence for the presence of an intramolecular hydrogen bond in the molecule and the antiparallel extended conformation in chloroform solution. CD spectra in methanol, trifluoroethanol, and trimethyl phosphate indicate that the Dpg peptide shows slight conformational flexibility, whereas the folded Ac6c analog is quite rigid. The extended Dpg peptide consistently shows the highest activity in human peripheral blood neutrophils, being approximately 8 and 16 times more active than the parent peptide and the folded Ac6c analog, respectively. However, the finding that all four peptides have ED50 (the molar concentration of peptide to induce half-maximal enzyme release) values in the 10(-8)-10(-9) M range suggests that an induced fit mechanism may indeed be important in this ligand-receptor interaction. Moreover, it is also possible that alterations in the backbone conformation at the tripeptide level may not significantly alter the side chain topography and/or the accessibility of key functional groups important for interaction with the receptor.     

Solution structure of monomeric peptide YY supports the functional significance of the PP-fold

Peptide YY (PYY) belongs to a family of peptides including neuropeptide Y (NPY) and pancreatic peptide (PP) that regulate numerous functions through both central and peripheral receptors. The solution structure of these peptides is hypothesized to be critically important in receptor selectivity and activation, based on prior demonstration of a stable tertiary conformation of PP called the "PP-fold". Circular dichroism (CD) spectra show a pH-dependent structural transition in the pH range 3-4. Thus we describe the tertiary structure of porcine PYY in water at pH 5.5, 25 degrees C, and 150 mM NaCl, as determined from 2D (1)H NMR data recorded at 500 MHz. A constraint set consisting of 396 interproton distances from NOE data was used as input for distance geometry, simulated annealing, and restrained energy minimization calculations in X-PLOR. The RMSDs of the 20 X-PLOR-generated structures were 0.71 +/- 0.14 and 1.16 +/- 0.17 A, respectively, for backbone and heavy atom overlays of residues 1-34. The resulting structure consists of two C-terminal helical segments from residues 17 to 22 and 25 to 33 separated by a kink at residues 23, 24, and 25, a turn centered around residues 12-14, and the N-terminus folded near residues 30 and 31. The well-defined portions of the PYY structure reported here bear a marked similarity to the structure of PP. Our findings strongly support the importance of the stable folded structure of this family of peptides for binding and activation of Y receptor subtypes.     

Conformational features of bradykinin. A circular dichroism study of some peptide fragments and structural analogues of bradykinin.

The vasoactive hormone bradykinin, its N-and C-terminal fragments and some structural analogues were studied by Circular Dichroism. Conformational features of the peptide can be detected by comparative analysis of the various CD spectra recorded as a function of aqueous pH, solvent and temperature. It is shown that the two biologically essential arginine residues (Arg1 and Arg9) are important for the specific folded bradykinin conformation. Differences between bradykinin, its fragments and analogues become clearly established in conformational terms, and are discussed in relation to the biological activity of these peptides.     

The crystal structure of a Dcp-containing peptide

We have investigated the conformational preferences of a newly synthesized C(alpha,alpha) symmetrically disubstituted glycine, namely alpha,alpha-dicyclopropylglycine (Dcp). We report here the crystal structure of a fully protected dipeptide containing Dcp, namely Z-Dcp(1)-Dcp(2)-OCH(3). Both Dcp residues are in a folded conformation. The overall peptide structural organization corresponds to an alpha-pleated sheet conformation, similar to that observed in linear peptides made up of alternating D- and L-residues and in Z-Aib-Aib-OCH(3) (Aib: alpha,alpha-dimethylglycine). These preliminary data suggest that the Dcp could represent an alternative as molecular tool to stabilize folded conformations.     

Effect of substitution of D-alanine for L-alanine on activity and conformation of an encephalitogenic peptide.

Synthetic peptides Phe-Ser-Trp-Gly-Ala-Glu-Gly-Gln-Lys and its D-Ala analog were tested for induction of experimental allergic encephalomyelitis (EAE) in guinea pig. The L-Ala peptide was highly active at 0.5 μg dosage and the D-Ala peptide was inactive even at 10 μg dosage. NMR spectra indicated backbone conformational differences between the two isomers. Energy calculations delineate conformations that are high in energy for the D-form and low for the L-form. A conformation for the physiologically active peptide is suggested that is in accord with both clinical and physical data.     

Mapping molecular flexibility of proteins with site-directed spin labeling: a case study of myoglobin

Site-directed spin labeling (SDSL) has potential for mapping protein flexibility under physiological conditions. The purpose of the present study was to explore this potential using 38 singly spin-labeled mutants of myoglobin distributed throughout the sequence. Correlation of the EPR spectra with protein structure provides new evidence that the site-dependent variation in line shape, and hence motion of the spin label, is due largely to differences in mobility of the helical backbone in the ns time range. Fluctuations between conformational substates, typically in the μs-ms time range, are slow on the EPR time scale, and the spectra provide a snapshot of conformational equilibria frozen in time as revealed by multiple components in the spectra. A recent study showed that osmolyte perturbation can positively identify conformational exchange as the origin of multicomponent spectra ( López et al. ( 2009 ) , Protein Sci. 18 , 1637 ). In the present study, this new strategy is employed in combination with line shape analysis and pulsed-EPR interspin distance measurements to investigate the conformation and flexibility of myoglobin in three folded and partially folded states. The regions identified to be in conformational exchange in the three forms agree remarkably well with those assigned by NMR, but the faster time scale of EPR allows characterization of localized states not detected in NMR. Collectively, the results suggest that SDSL-EPR and osmolyte perturbation provide a facile means for mapping the amplitude of fast backbone fluctuations and for detecting sequences in slow conformational exchange in folded and partially folded protein sequences.     

Solid-state nuclear magnetic resonance studies of HIV and influenza fusion peptide orientations in membrane bilayers using stacked glass plate samples

The human immunodeficiency virus (HIV) and influenza virus fusion peptides are approximately 20-residue sequences which catalyze the fusion of viral and host cell membranes. The orientations of these peptides in lipid bilayers have been probed with 15N solid-state nuclear magnetic resonance (NMR) spectroscopy of samples containing membranes oriented between stacked glass plates. Each of the peptides adopts at least two distinct conformations in membranes (predominantly helical or beta strand) and the conformational distribution is determined in part by the membrane headgroup and cholesterol composition. In the helical conformation, the 15N spectra suggest that the influenza peptide adopts an orientation approximately parallel to the membrane surface while the HIV peptide adopts an orientation closer to the membrane bilayer normal. For the beta strand conformation, there appears to be a broader peptide orientational distribution. Overall, the data suggest that the solid-state NMR experiments can test models which correlate peptide orientation with their fusogenic function.     

Context-dependent conformation of diethylglycine residues in peptides.

Diethylglycine (Deg) residues incorporated into peptides can stabilize fully extended (C5) or helical conformations. The conformations of three tetrapeptides Boc-Xxx-Deg-Xxx-Deg-OMe (Xxx=Gly, GD4; Leu, LD4 and Pro, PD4) have been investigated by NMR. In the Gly and Leu peptides, NOE data suggest that the local conformations at the Deg residues are fully extended. Low temperature coefficients for the Deg(2) and Deg(4) NH groups are consistent with their inaccessibility to solvent, in a C5 conformation. NMR evidence supports a folded beta-turn conformation involving Deg(2)-Gly(3), stabilized by a 4-->1 intramolecular hydrogen bond between Pro(1) CO and Deg(4) NH in the proline containing peptide (PD4). The crystal structure of GD4 reveals a hydrated multiple turn conformation with Gly(1)-Deg(2) adopting a distorted type II/II' conformation, while the Deg(2)-Pro(3) segment adopts a type III/III' structure. A lone water molecule is inserted into the potential 4-->1 hydrogen bond of the Gly(1)-Deg(2) beta-turn.