W2A-025 Invertebrate peptides-0.txt
Conformational Studies on Analogues of the Invertebrate Peptide pyroGlu-Asp-Pro-Phe-Leu-Arg-Phe-NH 2 using H NMR

David J. S. Guthrie, Robert F. Geraghty, G. Brent Irvine and Carvell H. Williams Division of Biochemistry, School of Biology Biochemistry, Medical Biology Centre, Queen's University, Belfast, UK BT9 7BL

The peptide pQDPFLRFamide is one of several closely related heptapetides found in invertebrates, including molluscs. Electrophysiological findings and ligand binding studies have both suggested that these heptapeptides probably interact with receptors distinct from those that are activated by the related tetrapeptide FMRFamide (also found in the same group of invertebrates), depite the fact that some syntetic N -terminally extended analogues of the latter show marked tetrapeptidelike activity. We have carried out structural studies, using 1- and 2-dimensional H NMR, on pQDPFLRFamide and some synthetic analogues in which Asp-2 was replaced by Asn and Pro-3 by either Aib (α -amino isobutyric acid) of by Gly. The results are consistent with the suggestion that pQDPFLRFamide can adopt a bent conformation, which might form the basis of the selectivity of this and related heptapeptides.

Two types of FMRFamide-related peptides (Farps) containing either four or seven amino acids have been found in several groups of invertebrates, including molluscs. These peptides are thought to participate in a range of neuroregulatory functions. In the land snail Helix aspersa, the peptides include FXRFamide (X = M or L) and XDPFLRFamide (X = pQ, S or N). Cloning of the gene that codes for the heptapeptides in this mollusc has revealed the presence of other, closely related heptapeptide sequences. Electrophysiological studies in this species have indicated that these two groups of peptides probably act through different receptors. Although ligandbinding studies have shown the presence of high and low affinity binding sites on membrane preparations from Helix tissues for the tetrapeptides and their analogues, so far no specific high affinity binding site for the heptapeptides has been unequivocally demonstrated in such preparations.

High affinity binding at the tetrapeptide-binding site is not restricted to peptides containing four amino acids. For example, some N -terminal extensions of FMRFamide or its analogues also interact efficiently at this site, in some cases with an affinity better than that of the natural ligand. These include N -3 (p -hydroxyphenyl) propanoyl-FMRFamide, YFMRFamide and YGGFMRFamide.

This is not the case for the naturally occurring heptapeptide Farps, which compete only weakly with tetrapeptides such as FMRFamide. It is clear therefore that the particular N -terminal tripeptide sequence XDP in the heptapeptides must be crucial for discriminating between the tetrapeptide and putative heptapeptide receptors. One possible way in which this might happen is if the heptapeptides contained a turn in the region of the Asp-Pro sequence, since it has been shown that the sequence Asx-Pro is frequently found to occur at positions 1 and 2 of β -turns in proteins and peptides. Such a conformation might also be stabilised by electrostatic interactions between the side chains of the Asp and Arg residues. We have previously shown that the effects on agonist activity and ligand binding of substitutions of Asp or Pro in PQDPFLRFamide are broadly in keeping with the presence of a bent conformation, though the electrostatic interaction may not be obligatory for biological activity. Evidence that might support this viewpoint has been sought by an NMR study of the pQDPFLRamide and some analogues. A preliminary report of part of this work has already been published.

It is unlikely that peptides such as those considered here will adopt a single conformation. All studies to date point to small linear peptides as possessing considerable conformational freedom and existing as an equilibrium over several conformations of similar energy. The object of a study such as this must be to see if any conformation(s) or families of conformations exist with sufficient population to permit detection. It is also relevant to consider the most appropriate solvent in which to study biomolecules. Water is often seen as the obvious choice, but, as well as introducing some technical problems, a solution in water cannot really be considered typical of conditions prevailing in vivo, when many other ions and molecules will be present. Furthermore, we are more interested in predicting what may be the conformation of a peptide in a binding site, and such regions are usually assumed to be a lot less polar than bulk water. DMSO has often been used as a solvent not only because many peptides are readily soluble in it, but also with the hope that its polarity may be similar to that of the binding pocket in a receptor or enzyme. However, it has also been argued that, in vivo, layers of water near to a protein surface are more viscous than bulk water and that this increased viscosity, by reducing conformational mobility, may 'filter' an otherwise flexible peptide into a predominant conformation. Mixtures of water and DMSO have higher viscosity than either solvent alone and have been suggested as an appropriate solvent for conformational studies.

Experimental
Synthesis. Ñ The following peptides used in this study were prepared by solid-phase synthesis, as described previously;. As reported, all peptides were purified using reversed-phase C 18 HPLC μ -Bondapak, Waters), gave satisfactory amino acid analyses (Picotag Analyser, Waters) and the expected molecular ions were observed in FAB mass spectrometry (VG AutoSpec. instrument at the SERC Mass Spectrometry Service Centre). In addition, the Gly analogue was subjected to additional analysis to eliminate the possibility that complexities observed in the NMR spectrum were due to impurities. Capillary electrophoresis of this analogue (Thermo Separation Products) at pH 3.0, 4.5 and 9.1 and in the presence of SDS (sodium dodecyl sulfate) at pH 9.0 showed only one major component with an integrated value of 93%. Similarly, electrospray mass spectrometry (VG Quattro instrument fitted with electrospray ionisation), which because it analyses the total sample is more likely than FABMS to detect all components present, identified one main component m/z = 864.9; M, 863.4) with other species present at levels less than 1%.
NMR Studies. Ñ Experiments were carried out on samples prepared as follows. Approximately 5 mg of peptide was dissolved in deionised water and the pH adjusted to near 6.0. The solvent was removed under vacuum in a Speed VAC concentrator (Savant). The samples were further dried in vacuo over phosphorous pentoxide for 24 h. The peptide was then dissolved in and in some cases this was further diluted with H 2 O. In one case the solvent used was. Spectra were recorded on a Varian VXR-600S spectrometer at the Edinburgh University Ultra High Field NMR Service.

H NMR spectra were acquired with 32 K data points and Fourier transformed with zero filling into 64K points. For some spectra, an exponential line broadening factor of 0.5 was applied. 2D spectra were obtained by the Hypercomplex method of States, Heberkorn and Reuben as 512 FIDs of 2 K data points and transformed with zero filling into 1K • 2K points using either a sine bell or Gaussian window. Assignments of proton resonances in the peptides were made using several types of 2D spectra, i.e. DQF-COSY, TOCSY and ROESY. ROCSY spectra were acquired with a mixing time of 65 ms (for DMSO solution) or 80 ms (for DMSO/H 2 O solution) and ROESY spectra with mixing times of 150 (DMSO and H 2 O) or 200 ms (DMSO/H 2 O). For ROESY spectra, the frequency offset was varied to ensure that the observed cross peaks did not arise from Hartmann-Hahn transfer coupled with incoherent magnetisation transfer.

The temperature dependence of amide protons was determined from 1D spectra recorded at 5 ̊ C intervals from 25 to 55 ̊ C. In all cases the variations of the chemical shift of the amide proton with temperature was linear and the temperature coefficient was determined by linear regression analysis (correlation coefficients ≤ 0.95).

Results and Discussion

H Resonance Assignments. Ñ The amide regions of the 1D spectra of the peptides are shown in Fig. 1. Detailed analyses of the NMR spectra of the peptides, under a variety of conditions were made using DQF-COSY, TOCSY and ROESY experiments and are summarised for pQDPFLRFamide and the Asn and Aib analogues in Tables 1 to 3, respectively. The temperature coefficients of the amide protons of the peptides are listed in Table 4. The effects of pH on the amide protons of pQDPFLRFamide are shown in Fig. 2. The nuclear Overhauser connectivities observed in ROESY spectra are summarised in Table 5 and two examples of the spectra obtained are shown in Figs. 3 and 4. Moving the frequency offset in selected ROESY spectra by up to 300 Hz yielded essentially identical spectra.

Conformational Studies. Ñ As mentioned above, the available evidence suggests that most small peptides exist as an ensemble of interconverting conformations. Inspection of the assignments reported in Tables 1 to 3 shows that considerable differences exist between the spectrum of pQDPFLRFamide and those of the Asn, Aib and Gly analogues and between the spectra of pQDPFLRFamide recorded under different conditions. At the very least, this points to the various conformations available to the peptides being populated to different extents for each peptide or set of conditions. Examination of all the observed NMR spectra (Tables 1 to 3 and Fig. 1), the amide proton temperature coefficient (Table 4) and the nuclear Overhauser connectivities (Table 5), allows certain conclusions to be drawn. These will now be presented for pQDPFLRFamide and the Asn and Aib analogues. The Gly analogue, which displays rather different behaviour, will be discussed later.

Configuration of the Asx-Pro peptide bond. Under all the conditions used, the ROESY spectra obtained for pQDPFLRFamide and the Asn analogue display a strong d (i,i + 1) connectivity between Asx-2 and Pro-3, indicating that the peptide exists predominantly as the trans isomer of the Asx-Pro peptide bond. In several of the 1D spectra (e.g. PQDPFLRFamide in H 2 O at pH 3.0 and the Asn analogue in DMSO) a set of minor peaks is visible which could be due to a small fraction of molecules existing as the cis isomer, but no d (i,i + 1) or of d N α (i,i + 1) connectivities between Asx-2 and Pro-3, characteristics of a cis peptide bond, were ever observed.

Existence of a salt bridge between Asp-2 and Arg-6. Several types of evidence suggest that such a salt bridge exists and has an influence on the conformation.

(a) Appearance of amide proton resonances. The amide proton resonances of the Asn analogue in both DMSO and DMSO/H 2 O are sharp, consistent with the averaging over the available conformations being essentially complete. However, for pQDPFLRFamide and the Aib analogue in DMSO the amide resonances are broad. Again, this could be interpreted as a case of equilibria over an ensemble of conformers but now, because of the additional constraint of an internal salt bridge between the sidechains of Asp-2 and Arg-6, the rates of interconversion are slower ant the averaging of the NMR signals is less complete for the Asn analogue.

(b) Differences in chemical shift values. Compared with the Asn analogue in DMSO, the amide proton resonance of Phe-4 in pQDPFLRFamide in DMSO has moved from 7.97 to 7.59 and the amide and α proton resonances of Leu-5 have moved from 7.30 and 4.29, respectively, to 7.77 and 3.95 (Tables 1 and 2). The corresponding residues in the Aib analogue, where a salt bridge is also possible, have chemical shifts close to those of pQDPFLRFamide (Table 3). It is difficult to account for differences between pQDPFLRFamide and the Asn analogue other than by postulating a major conformational difference due to a salt bridge.

(c) Variation with pH of the amide protons in pQDPFLRFamide. Not only does the chemical shift of the Asp-2 amide proton vary as the pH is altered over a range covering the pK a of the Asp sidechain carboxyl group, but the Phe-4 and Arg-6 amide resonances also change dramatically (Fig. 2). Such changes imply that a major alteration in conformation, affecting most of the peptide, occurs on ionisation of the Asp sidechain. It seems most likely that this involves an increase in number of those conformers which allow a salt bridge between the sidechains af Asp-2 and Arg-6. If such a salt bridge exists in aqueous solution, it is even more likely to exist in solvents of lower dielectric constant.
