Substance P (SP) interacts with the neurokinin-1 (NK-1) G-protein-coupled receptor, which has been cloned in several species. In the present study, the domains of the NK-1 receptor involved in the binding of SP and SP-(7-11) C-terminal fragment have been analyzed using two peptide analogs containing the photoreactive amino acid para-benzoylphenylalanine

Two binding sites NK-1M (major, more abundant) and are associated with the neurokinin-1 receptor. For the first time with a bioactive peptide, the C a methylation constraint, shown to be a helix stabiliser in model peptides, was systematically used to probe the molecular requirements of NK-1M and NK-1m binding sites and the previously postulated bioactive helical conformation of substance P (SP). Seven C a methylated analogues of the undecapeptide SP (from position 5±11) have been assayed for their affinities and their potencies to stimulate second messenger production. The consequences of C a methylation on the structure of SP have been analysed by circular dichroism and nuclear magnetic resonance combined with restrained molecular dynamics. The decreased potencies of six out of these seven C a methylated SP analogues do not allow the identification of any clear-cut differences in the structural requirements between the two binding sites. Strikingly, the most active analogue, [aMeMet5]SP, leads to variable subnanomolar affinity and potency when interacting with the NK-1m binding site. The conformational analyses show that the structural consequences associated with C a methylation of SP are sequence dependent. Moreover, a single C a methylation is not sufficient by itself to drastically stabilize a helical structure even pre-existing in solution, except when Gly9 is substituted by an a-aminoisobutyric acid. Furthermore, C a methylation of residues 5 and 6 of SP in the middle of the postulated helix does not stabilize, but decreases (to different extents) the stability of the helical structure previously observed in the 4±8 domain of other potent SP analogues.Keywords: binding sites; C a methylation; conformational analysis; molecular recognition; tachykinins.The structure determination of peptide/receptor complexes is still indirect and mainly based on the comparison of binding affinities and biological potencies of modified peptides and/or mutated receptors. These structure±activity relationships are nowadays enriched by the evolution of peptide chemistry, with new strategies for the design of constrained peptides, by spectroscopic analyses combined with calculations and modelization [1±4] and also by molecular biology leading to mutated receptors [5]. Over the past years, the tools to restrict the conformational space of a peptide have been clearly identified and their conformational behaviours have been analysed by spectroscopy, mainly NMR and CD, but also by X-ray diffraction associated with potential energy calculations. However, all these chemical modifications designed to stabilize a`b ioactive' conformation can also create repulsive interactions in the binding site, and/or induce conformational change of the peptide and (or) the receptor. Therefore, a combination of various nonredundant constraints must be introduced in the endogenous peptide to ascertain its postulated bioactive conformation.In this study, the C a methylation constraint [6] was used to probe the bioactive conformation(s) of substance ...

Protochlorophyllide reductases are key enzymes in the process of chlorophyll biosynthesis. In this review, current knowledge on the molecular organization, substrate specificity and assembly of the light-dependent NADPH : protochlorophyllide oxidoreductases are dis¬ cussed. Characteristics of light-independent enzymes are then briefly described and the possible reasons for the selection of light-dependent enzymes in the course of evolution are discussed.