| Name | Delia |
| Surname | Picone |
| Institution | University of Naples – Federico II |
| delia.picone@unina.it | |
| Address | Department of Chemical Sciences, University of Naples Federico II, Complesso Universitario di Monte Sant'Angelo, Via Cintia, I-80126 Napoli, Italy |
Cholecystokinin (CCK) is a peptide hormone endowed with several important biological activities, both in the central and peripheral nervous system. Previous conformational studies have dealt mainly with its C-terminal octapeptide fragment (CCK8), which represents the shortest fully circulating form of this hormone. We have undertaken a detailed NMR conformational study in a DMSOd6/H2O cryomixture at 278 K of the CCK analog H-Arg-Asp-Tyr(SO3H)-Thr-Gly-Trp-Nle-Asp-PheNH2 (CCK9) which retains all the bioactivities of CCK8, but was found to be remarkably more stable in acidic media and unaffected by air oxidation due to Met replacements. The predominant conformation contains a gamma-turn centered on Thr4, separated by Gly5 from a helical segment that comprises the C-terminal residues.
Many neuropeptides exert their action between the presynaptic vesicles and postsynaptic transmembrane receptors, crossing different layers of specialized cytoplasm. Biomimetic media usually employed to study bioactive peptides do not reproduce the physico chemical environment of cytoplasm--in particular, the high viscosity of this biological fluid. Here we describe a conformational study of a delta-selective opioid peptide, deltorphin I, at variable temperatures in several biocompatible media characterized by varying values of viscosity and dielectric constant. It was found that only viscosity, among these parameters, induces ordered conformations; that is, it acts as a conformational sieve. This finding suggests that the high viscosity of the intersynaptic fluid contributes, in addition to the membrane catalysis proposed by Schwyzer, in overcoming the so-called entropic barrier to the transition state of peptide-receptor interaction by selecting ordered conformations prior to receptor interaction. The folded conformer found in the 80:20 (v:v) DMSOd6/H2O cryoprotective mixture at 265 K has a shape consistent with those of rigid nonpeptidic opiates.
Deltorphin I, a delta-selective opioid peptide, has been studied in a DMSOd6/H2O cryoprotective mixture by two-dimensional (2D) NMR spectroscopy in the temperature range 260 K to 305 K. The high viscosity of the solvent at low temperature mimics a distinctive physico-chemical feature of cytoplasm and allows the measurement of a NOESY spectrum rich in intra- and inter-residue effects. Backbone NOEs at 265 K can be calculated with good accuracy in terms of only two limiting conformers: one folded, with a mole fraction of 0.30, and another extended with a mole fraction of 0.70. This calculation is still a rough approximation of the complex conformational equilibria existing in solution but, to the best of our knowledge, is the first one for a flexible peptide, and represents an encouraging starting point for a quantitative evaluation of NMR data of small, flexible peptides in solution. The folded conformer consistent with observed NOEs has a shape surprisingly similar to those of unrelated, rigid, delta-selective opiates.
The message domain of dermorphin (Tyr-D-Ala-Phe), a natural mu-opioid heptapeptide, has long been considered the main cause of the high mu selectivity of this peptide and of its analogues. The recent discovery, in the skin of Phyllomedusa sauvagei (i.e., the same natural source of dermorphin) and of Phyllomedusa bicolor of deltorphins, challenges this belief. Deltorphins, in fact, are three heptapeptides characterized by a message domain typical of mu-selective peptides, but endowed of an extremely high delta selectivity, the highest of all natural opioid peptides. A conformational analysis of dermorphin and deltorphins, based on nmr studies in DMSO and cryoprotective mixtures and internal energy calculations, showed that the enormous differences in receptor selectivity can be interpreted on the basis of receptor models for mu and delta opioids that recognize the same beta-turn in the N-terminal part, but discriminate for the conformation and polarity of the C-terminal part. Here we present the synthesis, biological activity, and conformational analysis in solution of three deltorphin analogues with very similar constitution, but with different net charge, different location of negative residues, or even without negative residues, which confirm these hypotheses and show that His4 can play a specific structural role.
The conformation of [Leu5]enkephalin has been studied by 1H-NMR spectroscopy in media more like the actual environment in which the agonist-receptor interaction takes place than water, i.e. in three cryoprotective mixtures (dimethylformamide/water, methanol/water and ethylene glycol/water), in aqueous SDS and in two neat solvents, dimethylformamide and acetonitrile, whose dielectric constants (36.7 and 37.5) are intermediate between that of water and that of the lipid phase. In all cases examined, contrary to the studies in water or dimethylsulfoxide, we were able to detect numerous nuclear Overhauser effects, indicating that the media employed favour well-defined structures and/or reduce the internal motions of the peptide. Data from both organic solvents and cryoprotective mixtures suggest a 4----1 beta turn as the most probable structure of [Leu5]enkephalin in solution, whereas in SDS/H2O micelles the structural picture appears completely different, suggesting the presence of a 5----2 beta turn. The existence of two different preferred conformations of enkephalins may possibly be related to their ability to be effective towards both mu and delta opioid receptors.
Deltorphin I is an opioid peptide of sequence H-Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2, recently isolated from the skin of Phyllomedusa bicolor. Its enormous selectivity towards the delta opioid receptor and the similarity of the conformation of the N-terminal part of the sequence with that of dermorphin (H-Tyr-D-Ala-he-Gly-Tyr-Pro-Ser-NH2), a mu selective peptide, prompted the synthesis, biological evaluation and comparative conformational study of four analogs. A 1H-NMR study showed that the conformational preferences of the N-terminal sequences of all peptides are similar. The different selectivities towards opioid receptors have been interpreted in terms of charge effects in the interaction with the membrane and at the receptor site and of hydrophobicity of the C-terminal part, when structured in a folded conformation.
The quaternary structure of bovine seminal ribonuclease, the only dimeric protein in the superfamily of ribonucleases, is maintained both by noncovalent forces and by two intersubunit disulfides. The available monomeric derivatives of the enzyme may not be reassembled into dimers. They are catalytically active, but do not retain certain properties of the dimeric enzyme, such as: (i) the ability to respond cooperatively to increasing substrate concentrations in the rate-limiting reaction step; and (ii) the antitumor and immunosuppressive actions. In this report we described the preparation of stable monomers of seminal ribonuclease which can be reassociated into covalent dimers indistinguishable from the native protein. With this procedure a hybrid dimer was constructed, made up of a native subunit associated to a subunit catalytically inactivated by selective alkylation of the active site His-119. This dimer was found to have enzymic properties typical of monomeric ribonucleases, such as a hyperbolic saturation curve in the hydrolytic rate-limiting step of the reaction. However, the hybrid dimer was one order-of-magnitude more active than the dimeric enzyme.
Deltorphin is an opioid peptide with the sequence H-Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2, recently isolated from the skin of Phyllomedusa sauvagei. Its enormous selectivity towards the delta-opioid receptor and the similarity of the N-terminal part of the sequence with that of dermorphin (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2), a mu selective peptide isolated from the same natural source, prompted a comparative conformational study. A 1H-NMR study in two different solvent systems showed that the conformational preferences of the N-terminal sequences of the two peptides are similar. The different selectivities towards opioid receptors have been interpreted in terms of charge effects. Besides a general trend consistent with the role of the membrane in the preselection of the peptides, the present study demonstrates the crucial role played by charged residues in the interaction inside the receptors.
The synthetic peptide of sequence H-Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr-OH, termed peptide T, a competitor of the Human Immunodeficiency Virus in the binding to human T cells, and its C-terminal pentapeptide fragment, were studied by 1H-nmr in DMSO solution to determine conformational preferences. The observation of nuclear Overhauser enhancements (NOEs) for both peptides, and unusual finding for small linear peptides, allowed complete sequence-specific resonance assignments. Long-range NOEs, ring-current shifts, and the very small temperature coefficient of the Thr8 NH chemical shift suggest, for the zwitterionic form of peptide T, the presence in solution of a beta-turn involving Thr5, Asn6, Tyr7 and Thr8. This conformational feature is consistent with previous structure-activity relationship studies indicating the invariance of the same residues in several potent pentapeptide analogues. The studied pentapeptide fragment, although less structured, shows some tendency to fold even in a polar solvent such as DMSO. Preliminary chemotaxis data on some pentapeptide analogues are consistent with our structural model.
Bioactive peptides of natural origin have, in general, short linear sequences, and are characterized by a large conformational flexibility. It is very difficult to study their conformation in solution since they exist, almost invariably, as a complex mixture of numerous conformers, most of which are extended. The so-called bioactive conformation may be one of them, although the solvents used in solution studies often have properties drastically different from those of the biological system in which the peptide acts. There is, however, no simple way of identifying the bioactive conformation amid the many existing conformers. It is possible to approach a solution to this problem using two distinct strategies: (a) Limiting the conformational freedom of the peptide, e.g., by increasing the viscosity of the solution and decreasing the temperature, in the assumption that the bioactive conformation is, even slightly, more stable than the others. (b) Trying to mimic in solution the physicochemical features of the more reliable receptor models. These two approaches will be illustrated with examples taken mainly from opioid peptides.