Conformational analysis of the tachykinins in solution: substance P and physalaemin.

A determination of the solution conformational behavior of two tachykinins, substance P and physalaemin, is described. Two-dimensional homonuclear Hartmann-Hahn (HOHAHA) and rotating-frame cross relaxation spectroscopy (ROESY) are used to obtain complete proton resonance assignments. Interproton distance restraints obtained from ROESY spectroscopy are used to characterize the conformational behavior. These data show that in solution both substance P and physalaemin exist in a mixture of conformational states, rather than as a single three-dimensional structure. In water both peptides prefer to be in an extended chain structure. In methanol, their behavior is described as a mixture of beta-turn conformations in dynamic equilibrium. Solvent titration data and chemical shift temperature coefficients complement the NMR estimate of interproton distances by locating hydrogen bonds and serving to identify predominant conformational states. The C-terminal tetrapeptide segment has the same conformational behavior for both substance P and physalaemin. In physalaemin, the midsegment of the peptide may also be constrained by formation of a salt bridge. The conformational behavior of substance P and physalaemin is discussed in relation to potency and receptor binding properties.     

Conformational Analysis of the Tachykinins in Solution: Substance P and Physalaemin

Abstract

A determination of the solution conformational behavior of two tachykinins, substance P and physalaemin, is described. Two-dimensional homonuclear Hartmann-Hahn (HOHAHA) and rotating-frame cross relaxation spectroscopy (ROESY) are used to obtain complete proton resonance assignments. Interproton distance restraints obtained from ROESY spectroscopy are used to characterize the conformational behavior. These data show that in solution both substance P and physalaemin exist in a mixture of conformational states, rather than as a single three-dimensional structure. In water both peptides prefer to be in an extended chain structure. In methanol, their behavior is described as a mixture of β-turn conformations in dynamic equilibrium. Solvent titration data and chemical shift temperature coefficients complement the NMR estimate of interproton distances by locating hydrogen bonds and serving to identify predominant conformational states. The C-terminal tetrapeptide segment has the same conformational behavior for both substance P and physalaemin. In physalaemin, the midsegment of the peptide may also be constrained by formation of a salt bridge. The conformational behavior of substance P and physalaemin is discussed in relation to potency and receptor binding properties.     

Preferential conformation of substance P in solution

The three-dimensional structure of substance P has been studied by 1H-NMR, (500 MHz), and by circular dichroism (CD) in different solvents. The analysis of the different NMR parameters suggest that substance P adopts a rather extended structure in dimethylsulfoxide and pyridine. In water, besides the aggregation phenomenon, the monomeric substance P presents a complex conformational equilibrium. The addition of sodium dodecylsulfate to the aqueous solution induces, as shown by CD spectroscopy, a preferential alpha-helical conformation. And in methanol three structural conclusions may be drawn: the flexibility of the N-terminal Arg-Pro-Lys, the alpha-helical structure of Pro4-Gln5-Gln6-Phe7-Phe8 and the interaction of the C-terminal carboxamide with the primary amides from both glutamines.     

Analysis of tachykinin-binding site interactions using NMR and energy calculation data of potent cyclic analogues of substance P

The three-dimensional structures of [Cys3,6,Tyr8]-, [Gly2,Cys3,6,Tyr8]- and [DCys3,Cys6]substance P, designed as conformational analogues of substance P, have been studied by 1H-NMR (500 MHz) in different solvents and by energy calculations. As previously observed for substance P and physalaemin, two tachykinins acting via the NK-1 receptor, [Cys3,6,Tyr8]substance P presents an alpha-helical structure of the 4----8 sequence in methanol. This structure is stabilized by a beta-turn III via the formation of three hydrogen bonds involving the Cys-6, Phe-7 and Tyr-8 NH groups. In contrast to substance P, two of these hydrogen bonds are still present in dimethyl sulfoxide and in water the Cys-6 NH hydrogen bond is the only one remaining, such that a beta-turn structure inside the ring can be envisaged. In close agreement with the NMR data, the energy calculations lead to three types of folding for the core of [Cys3,6,Tyr8]substance P: a beta-turn III, a less stable beta-turn I (delta E = 3 kcal), and a beta-turn II (delta E = 4.6 kcal). The structure of Gly-Leu-Met-NH2 is strongly affected by changing the hydrophobicity of the medium. The most stable calculated conformation is the helix; however, numerous unrelated structures are destabilized by about 2-3 kcal/mol. These data are analyzed and discussed in connection with the high potency of [Cys3,6,Tyr8]substance P for both the NK-1 and NK-3 binding sites; that is the internal region of tachykinins (non-homologous amino acids) might present a similar three-dimensional structure when bound to the receptors (which may be at the origin of some lack of selectivity), whereas paradoxically the selectivity may be due to the common C-terminal sequence.     

Chemical and immunochemical characterisation of substance P-like immunoreactivity and physalaemin-like immunoreactivity in a carcinoid tumour

Extracts of a carcinoid tumour, resected from the mid-portion of the ileum of a patient with no symptoms of endocrine disorder, were associated with a high concentration of substance P-like immunoreactivity. Using reverse-phase high performance liquid chromatography and antisera specifically directed against the C-terminal and N-terminal regions of substance P and against the N-terminal region of physalaemin, the following components were isolated and identified: substance P and its oxidised form, [pGlu⁵]substance P-(5–11) peptide and its oxidised form, and the oxidised form of physalaemin. The identity of tumour substance P with the undecapeptide was confirmed by amino acid analysis and high performance ion-exchange chromatography. In vitro incubation of tumour tissue from a lymph node metastasis from the same patient with [³H]leucine resulted in incorporation of radioactivity into newly synthesised substance P.     

Conformation of physalaemin

The conformational and spatial configuration of the biologically active undecapeptide physalaemin was studied using 350-MHz1H NMR. The NMR analyses suggested the existence of a strong hydrogen bond between the amide proton of the Phe7 and a carbonyl group in the N-terminal moiety, most likely the Pro4 one. Other bondings were postulated, involving the side-chain amine of Lys6 and the side-chain amide of Asn5 and respectively the side-chain carboxyl of Asp3 and the terminal amide carbonyl of Met-NH2. Thus unlike its shorter peptidic fragments, physalaemin exhibited a stable molecular structure in solution, giving some insight into the conformation required for interaction at the biological receptor of tachykinins.     

Circular-dichroism spectra of truncated and other analogs of angiotensin II.

Circular dichroism spectra on angiotensin II and analogs, and its truncated N-terminal and C-terminal peptides were determined in fluroinated alcohols under several conditions in the peptide or aromatic spectral regions. The following conclusions were suggested: (a) evidence for a beta structure for angiotensin II; (b) evidence for a folding at the N-terminal and C-terminal part of the molecule; (c) an interaction involving the C-terminal residue which decreases progressively when phenylalanine is replaced by isoleucine and then by alanine; (d) the N-terminal amino acid seems to play an important role in the overall conformation of the molecule possibly by interacting with the C-terminus, its absence in the 2 -- 8 heptapeptide giving rise to a more pronounced signal than angiotensin II; (e) in trifluoroethanol the conformation of these peptides is well defined and fits well with observed structure-activity relationships and observed binding data. There is a loss of this relationship when these solvents are diluted with water.     

Stability and unfolding studies on alkaline denatured state (Ip) of pepsin

Pepsin exists as alkaline denatured state (I_p) in pH range 8–10, where the N-terminal domain of the protein is mostly unfolded while the C-terminal domain is intact. The effects of fluorinated (TFE) and non-fluorinated (methanol) organic solvents on this partially unfolded state (I_p) of pepsin were investigated using various spectroscopic methods. Both, fluorinated (TFE) and non-fluorinated (methanol) organic solvents induce secondary structure (α-helix) after a critical concentration. The I_p state of pepsin unfolds in cooperative manner but the transition was found to be non-cooperative in the presence of 40% methanol or TFE. The differences in the unfolding of the protein in the presence and the absence of these organic solvents were interpreted. Our results indicate that unfolding transitions in I_p state are mostly dominated by unfolding of C-terminal domain because the N-terminal domain is largely unstructured in this state. The organic solvents (TFE and methanol) induce more secondary structure in N-terminal domain and make it another unfolding entity with different stability compare to C-terminal resulting into sequential unfolding of the domain.     

Conformation studies on oligoalanines, substance P, and the myoglobin sequence (66-73). Circular dichroism of polyethyleneglycol-bound peptides]

The conformation of polyethylene glycol-bound peptides, synthesized by the liquid-phase method, was investigated. This marcromolecular C-terminal protecting group is transparent in the visible and the ultraviolet range to 190 nm and solubilizes peptides in many different solvents. The CD spectra of the polymer-bound myoglobin sequence 66–73 and of the biologically active undecapeptide “substance P” were measured in each step of the synthesis. In both examples the formation of a secondary structure during the growth of the peptide chain was found. In the hydrophobic octapeptide containing the myoglobin sequence 66–73, the influence of either the blocked or the free N-terminal amino group on the conformation was observed. The blocked octapeptide in trifluoroethanol showed a higher degree of α-helix contribution than in its free state. The conformation of the polyethylene glycol-bound nona- and decaalanine in trifluoroethanol and water was determined. The peptide with a free amino end group has β-conformation in trifluoroethanol as well as in water. The corresponding N-Boc-protected derivatives show helical structure. The amino end group has a decisive influence on the formation of β-structure. The method of CD investigation of polymer-bound peptide sequences during the peptide synthesis in solution enables one to determine the influence of protecting groups and the chain end of a peptide on its conformation. It is also possible to study the relationship between the secondary structure, the chain length, and the kinetic of the coupling reaction in different solvents. Since the crystallization method for the liquid-phase peptide synthesis allows one to synthesize peptides in very short time, a new method of studying peptide conformations is opened.     

Substance K: a novel mammalian tachykinin that differs from substance P in its pharmacological profile

The primary structure of one of the bovine brain substance P precursors has shown the existence of a second mammalian tachykinin sequence, named substance K, that is remarkably homologous to that of the amphibian peptide kassinin. In this study, three substance K sequences were chemically synthesized and were submitted to parallel bioassays with kassinin, substance P and physalaemin. The results show that the three substance K peptides all possess biological activities characteristic of the tachykinin family and that their biological activities more closely resemble those of kassinin than those of substance P or physalaemin. This suggests that substance K may have a physiological role which is related to but different from that of substance P in mammalian organisms.     

Interaction of substance P and its N- and C-terminal fragments with Ca2+: implications for hormone action.

In an attempt to understand the role of Ca2+ on the bioactive conformation of peptide hormones, we have examined the interaction between Ca2+ and the neuropeptide substance P. Using CD spectroscopy to monitor conformational changes caused by Ca2+ binding, we found no significant binding of the cation by substance P in water. However, a substantial conformational change occurred in the hormone on Ca2+ addition in trifluoroethanol or an 80:20 (v/v) mixture of acetonitrile and trifluoroethanol. A biphasic binding of Ca2+ was observed in these solvents with saturation at 2 cations per hormone molecule. Mg2+ caused a relatively smaller conformational change in the hormone. A peptide corresponding to residues 1-7 at the N-terminal fragment of substance P showed a weak nonsaturating binding of Ca2+ in the nonpolar solvents whereas the 7-11 C-terminal fragment peptide displayed a binding indicative of an 1:1 Ca2+/peptide complex. Ca2+ binding by the hormone and the 7-11 fragment was also monitored by changes in fluorescence of the phenylalanyl residues. The results support the conclusion drawn from the CD data about a distinct Ca2+ binding site in the C-terminal part of substance P. The Kd values obtained from fluorescence data were 160 microM for Ca2+ and 1 mM for Mg2+ binding by substance P. The hormone and the two peptide fragments were also tested for their effect on the stability of dimyristoyl lecithin vesicles. Substance P and the N-terminal fragment caused no significant leakage of either fluorescent dyes or K+ trapped in the vesicles. Nor did they cause membrane fusion as monitored by the fluorescence quenching method.(ABSTRACT TRUNCATED AT 250 WORDS)     

CD and small-angle x-ray scattering of silk fibroin in solution

We investigated the structure of silk fibroin dissolved in water and in water-organic solvent mixtures by CD and small-angle x-ray scattering (SAXS). CD spectra indicated a disordered secondary structure in water and a beta-sheet conformation in aqueous organic solvents, such as methanol, dioxane, and trifluoroethanol (in trifluoroethanol a transient form evolving toward beta-sheet conformation was seen just after dissolution). The SAXS technique indicated the presence of fibroin particles of lamellar shape. The molecular weight was 188,000 daltons in water and 302,000 daltons in aqueous methanol.     

Circular dichroism studies on a synthetic peptide corresponding to the membrane-spanning region of vesicular stomatitis virus G protein and its fatty acyl derivative

The conformations of synthetic peptides Lys-Phe-Phe-Phe-Ile-Ile-Gly-Leu-Ile-Ile-Gly-Leu-Phe-OCH3 and Lys(epsilon-palmitoyl)-Phe-Phe-Phe-Ile-Ile-Gly-Leu-Ile-Ile-Gly-Leu-Phe-O CH3, which constitute a part of the membrane-spanning region of the vesicular stomatitis virus G protein, have been studied by circular dichroism (CD) spectroscopy. Secondary structural features are observed for both peptides in trifluoroethanol, methanol, aqueous mixtures of trifluoroethanol and methanol and in a micellar environment. In trifluoroethanol, the CD spectra indicate the presence of a helical conformation, whereas in aqueous mixtures of organic solvents, both helical and beta-conformations are observed. While fatty acid acylation does not directly modulate peptide conformation, it promotes self-association of the acylated peptide and association with micelles. In a micellar environment, the acylated peptide adopts an alpha-helical conformation.     

Conformation-based design of two cyclic physalaemin analogues

Two new cyclic analogues of physalaemin were designed on the basis of the conformation found in DMSO solution. Glp-Ala-cyclo(-Asp-Pro-Asn-Lys-)-Phe-Tyr-Gly-Leu-Met-NH2 (1) was synthesized by cyclization of physalaemin. In 2 the Asp residue was replaced by Glu. The linear analogue of 2 was synthesized by the solid phase method and subsequently cyclized. Two-dimensional nmr methods were employed to assign the proton and carbon resonances. Proton-proton distances were extracted from rotating frame nuclear Overhauser effect spectra and used as restraints in the molecular dynamics calculations. Analogue 1 was found to have a similar conformation as physalaemin, whereas 2 did not form intramolecular hydrogen bonds. The pharmacological evaluation revealed that both peptides have similar potencies as physalaemin in the dog carotid artery (NK-1 receptor). Therefore, the charged side chains of physalaemin appear not essential for NK-1 activation. However, the other tachykinin receptors show good sensitivity to the cyclic peptides. It is concluded that the replacement of a salt bridge by an amide bond connecting the side chains of natural residues might provide useful information about the biological significance of some charged side chains of neurokinins.     

Conformations of nicotinamide adenine dinucleotide (NAD(+)) in various environments

Enzymes bind NAD(+) in extended conformations and yet NAD(+) exists in aqueous solution as a compact, folded molecule. Thus, NAD(+) conformation is environment dependent. In an attempt to investigate the effects of environmental changes on the conformation of NAD(+), a series of molecular dynamics simulations in different solvents was performed. The solvents investigated (water, DMSO, methanol and chloroform) represented changes in relative permittivity and hydrophobic character. The simulations predicted folded conformations of NAD(+) to be more stable in water, DMSO and methanol. In contrast, extended conformations of NAD(+) were observed to be more stable in chloroform. Furthermore, the extended conformations observed in chloroform were similar to conformations of NAD(+) bound to enzymes. In particular, a large separation between the aromatic rings and a strong interaction between the pyrophosphate and nicotinamide groups were observed. The implications of these observations for the recognition of NAD(+) by enzymes is discussed. It is argued that a hydrophobic environment is important for stabilizing unfolded conformations of NAD(+).     

Structural analysis of substance P using molecular dynamics and NMR spectroscopy.

The present work is a combined structural study, using Nuclear Magnetic Resonance (NMR) and Molecular Dynamics(MD), of the amidated and the free acid forms of substance P in water and methanol. The results obtained using both approaches were compared in order to characterize the structural features of both peptides in solution. From the NMR experiments it was derived that the free acid form adopts an extended conformation at the N-terminus and a helical conformation at the C-terminal segment of the peptide in both water and methanol; these structural features are in qualitative agreement with the results of the MD simulations. No significant differences in behavior were observed between the amidated and the free acid forms of the peptide in the simulations and in the experiments carried out in water, suggesting that the different activities of these analogs are due to their different mode of interaction with the receptor rather than to their structural preferences. Finally, we propose that the structure of substance P can be partially inferred from its sequence due to the presence of a Pro-X-Pro motif on the N-terminus and a Gly-Leu sequence on the C-terminus.     

Lysis of chromaffin granules by phospholipase A2-treated plasma membranes. A cell-free model for exocytosis in adrenal medulla

The conformations of a synthetic peptide corresponding to the signal sequence of E. coli alkaline phosphatase, Lys-Gln-Ser-Thr-Ile-Ala-Leu-Ala-Leu-Leu-Pro-Leu-Leu-Phe-Thr-Pro-Val-Thr- Lys- Ala-OCH3, have been examined in different environments by circular dichroism spectroscopy. In trifluoroethanol, methanol and aqueous mixtures of these solvents, the signal peptide has largely random conformation (approximately 80%) with small amounts of alpha-helix and beta-structure. However, in micellar environment, there is a significant increase in ordered conformation with both alpha-helix and beta-structure being present, unlike in other signal sequences reported in the literature, where only the alpha-helical conformation has been observed. Hence, an alpha-helical conformation may not be as stringent a requirement as overall hydrophobicity for recognition of signal sequences by the cell's export machinery.     

Morphology of self‐assembled structures formed by short peptides from the amyloidogenic protein tau depends on the solvent in which the peptides are dissolved

The aggregation behavior of peptides Ac‐VQIVYK‐amide (AcPHF6) and Ac‐QIVYK‐amide (AcPHF5) from the amyloidogenic protein tau was examined by atomic force microscopy (AFM) and fluorescence microscopy. Although AcPHF5 did not show enhancement of thioflavin T (ThT) fluorescence in aqueous buffer, distinct aggregates were discernible when peptide was dissolved in organic solvents such as methanol (MeOH), trifluoroethanol (TFE), and hexafluoroisopropanol (HFIP) dried on mica and examined by AFM. Self‐association was evident even though the peptide did not have the propensity to form secondary structures in the organic solvents. In dried films, the peptide adopts predominantly β‐conformation which results in the formation of distinct aggregates. ThT fluorescence spectra and fluorescence images indicate the formation of fibrils when AcPHF6 solutions in organic solvents were diluted into buffer. AcPHF6 had the ability to organize into fibrillar structures when AFM samples were prepared from peptide dissolved in MeOH, TFE, HFIP, and also when diluted into buffer. AcPHF6 showed propensity for β‐structure in aqueous buffer. In MeOH and TFE, AcPHF6 showed helical and β‐structure. Morphology of the fibrils was dependent on peptide conformation in the organic solvents. The structures observed for AcPHF6 are formed rapidly and long incubation periods in the solvents are not necessary. The structures with varying morphologies observed for AcPHF5 and AcPHF6 appear to be mediated by surfaces such as mica and the organic solvents used for dissolution of the peptides. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis, conformational studies, and molecular dynamics calculations of two cyclic tetrapeptides with 17- and 18-membered rings

Two cyclic tetrapeptides [Boc-cyclo(-Xxx-Pro-Asn-Lys-)OMe (Xxx = Asp or Glu)] were synthesized and investigated by NMR spectroscopy. They were designed in order to mimic the salt bridge found in physalaemin. Isomers of the urethane bond were observed in DMSO solution. The ROESY spectrum allowed the assignment of many signals of the minor isomer of both compounds. Conformational studies based on the temperature gradients of the NH chemical shifts, coupling constants, and ROEs revealed a similar conformation for the Asp analogue as proposed for physalaemin. A beta I turn with Pro and Asn in the corner positions was found for the major isomer. No hydrogen bonds were detected for the major isomer of the cyclic Glu analogue. Molecular dynamics calculations, using the NMR based initial structures, yielded sets of conformations in agreement with the experimental data. It is concluded that the salt bridge in physalaemin is best approximated by a lactam formed from the original amino acids.     

Conformation of cyclosporin A in polar solvents.

A major conformation of cyclosporin A in methanol and in aqueous methanol was revealed by some simple NMR experiments. Thus, a stepwise transition of cyclosporin A conformation from 100% CDCl3 to 100% CD3OD was followed by 1H NMR, which showed that the chloroform conformation of cyclosporin A was still the major one in methanol. Employing the same technique, it was also shown that the chloroform conformation of cyclosporin A was one of the major conformations in 50% aqueous methanol. This may be the first experimental determination of a major conformation of cyclosporin A in polar solvents.