Serge Fermandjian

Proton NMR studies on thyrotropin releasing factor

The synthesis of the tripeptide pyroglutamylhistidyl-prolineamide (TRF), the thyreotropin releasing factor [ 1,2], and its labelling [3] have promoted studies of its mode of action at the molecular level [4]. One important step towards this aim is to define the shape of the molecule and the relationships between biological activity and conformations. For this purpose ‘H-NMR investigations of TRF and related peptides have been carried out. The conformation of the histidyl residue was deduced from the relationships between the vicinal coupling constant J (NH-CctH) and the dihedral angle (C&H-NH). @ was derived from the equation 8 = (O-60) [5-S]. The behaviour of the C&H-CPH, histidyl protons and the non equivalence of the two prolyl C6 protons, on one hand, the differences between the cis and the trans-protons of the terminal amide group on the other hand suggested additional ~onformational restrictions. On this basis a model of TRF conformation is presented.

13C-nuclear magnetic resonance studies of 85% 13C-enriched amino acids and small peptides: pH effects on the chemical shifts, coupling constants, kinetics of cis-trans isomerisation and conformation aspects

The 13C chemical shifts of several 85% 13C-enriched amino acids and small peptides were studied as a function of pH. The results show that the chemical shifts of carbon atoms of ionizable groups vary significantly within the zone of their pK. Generally with the pH GOING FROM 7 to 1 all the deltaC are shifted more or less upfield with the exception of the carbonyl group carbon of the second last residue which is shifted slightly downfield. This suggests the formation of an hydrogen bond at acid pH involving in a seven-membered ring the C=O in question and the COOH terminal. The percentage of cis and trans conformers of glycyl-L-proline and glycyl-L-prolylglycine were studied as a function of pH. The trans form is always preponderant whatever the pH. The accessibility of the carbonyl group to protonation of the proline residue strongly influences the cis-trans equilibrium. Thus, with the pH varying from 7 to 1, the trans isomer changes from 61 to 85% for glycyl-L-proline and only from 77 to 80% for glycyl-L-prolylglycine. The proton NMR studies underline the important differences existing between the two molecular forms of glycyl-L-proline. The cis conformation is characterized with regard to the trans form by the non-equivalence of the alpha-protons of the glycine residue, by a lower pK(1) and by a larger deltadeltaHalpha of the proline residue as a function of pH. These results could suggest an end-to-end interaction in the cis form of the glycyl-L-proline molecule. The 13C-13C coupling constants were also studied as a function of pH. The results show that J(Co-Calpha) of a C-terminal residue, varying from 5 to 6 Hz and reflecting thhe pK of the carboxylate group, is a linear function of delta(Co) and delta(Calpha) as in the case of the amino acids. The total variation of the electron density of those two carbons in an amino acid is approximately 40% weaker than in a C-terminal residue. The charge distribution along the Calpha-C(o) bond, however, is practically the same in both cases. Finally the ratios of the conversion rate constants of the two isomers cis-trans of glycyl-proline were calculated at different pH values; the relations between the isomer percentages and delta(Co), delta(Calpha) on the one hand and the J(Co-Calpha) on the other were established.

A comparative 1H-N.M.R. study of MurNAc-l-Ala-d-iGln (MDP) and its analogue murabutide: Evidence for a structure involving two successive β-turns in MDP

The active principle, MurNAc-L-Ala-D-iGln (MDP), of complete Freunds adjuvant and its analogue, MurNAc-L-Ala-D-Gln-OnBu (murabutide), which express immunomodulatory as well as other biological properties, have been studied by 2D-1H-n.m.r. spectroscopy at 500 MHz. The results suggest the presence in MDP of two successive turns involving the MurNAc-L-Ala and L-Ala-D-iGln moieties, respectively, whereas only the former turn persists in murabutide. This turn mimics the type II beta-turn found in L-D depsipeptides, whereas the other is a typical type II beta-turn for L-D peptides.

Pre-organized structure of viral DNA at the binding-processing site of HIV-1 integrase.

The integration of the human immunodeficiency virus type 1 DNA into the host cell genome is catalysed by the viral integrase (IN). The reaction consists of a 3′-processing [dinucleotide released from each 3′ end of the viral long terminal repeat (LTR)] followed by a strand transfer (insertion of the viral genome into the human chromosome). A 17 base pair oligonucleotide d(GGAAAATCTCTAGCAGT), d(ACTGCTAGAGATTTTCC) reproducing the U5-LTR extremity of viral DNA that contains the IN attachment site was analysed by NMR using the classical NOEs and scalar coupling constants in conjunction with a small set of residual dipolar coupling constants (RDCs) measured at the 13C/15N natural abundance. The combination of these two types of parameters in calculations significantly improved the DNA structure determination. The well-known features of A-tracts were clearly identified by RDCs in the first part of the molecule. The binding/cleavage site at the viral DNA end is distinguishable by a loss of regular base stacking and a distorted minor groove that can aid its specific recognition by IN.

Effects of the binding of a dextran derivative on fibroblast growth factor 2: secondary structure and receptor-binding studies

CMDB (carboxymethyldextran-benzylamide) are dextrans statistically substituted with carboxymethyl and benzylamide groups which can mimick some of the biological properties of heparin. It has previously been shown that CMDB inhibit autocrine growth of breast tumor cells (Bagheri-Yarmand et al., Biochem. Biophys. Res. Commun. 239: 424-428, 1997) and selectively displace fibroblast growth factor 2 (FGF-2) from its receptor. Here, we used circular dichroism and fluorescence anisotropy measurements to show that the conformation of FGF-2 was significantly altered upon its binding to CMDB and to short CMDB fragments prepared within this study. CMDB and fragments formed a stable 1:1 complex with FGF-2, with affinities being estimated as 20+/-10 nM from fluorescence anisotropy analysis. No such a complex was formed with insulin-like growth factor (IGF-1) or epidermal growth factor (EGF). CMDB competed with the FGF-2 receptor for binding to FGF-2 but did not disturb the binding of IGF-1 and EGF to their receptors. Thus, our results highlight the selectivity of CMDB and their fragments towards FGF-2. Heparin, however, competes with CMDB and their fragments for binding to FGF-2. The carboxymethyl and benzylamide groups of these molecules likely interact directly with a heparin-binding region of FGF-2. The resulting change in conformation disturbs the binding of FGF-2 to its receptor and consecutively its mitogenic activity.

Self-association and Domains of Interactions of an Amphipathic Helix Peptide Inhibitor of HIV-1 Integrase Assessed by Analytical Ultracentrifugation and NMR Experiments in Trifluoroethanol/H2O Mixtures

EAA26 (VESMNEELKKIIAQVRAQAEHLKTAY) is a better inhibitor of human immunodeficiency virus, type 1, integrase than its parent Lys-159, reproducing the enzyme segment 147–175 with a nonpolar-polar/charged residue periodicity defined by four helical heptads (abcdefg) prone to collapse into a coiled-coil. Circular dichroism, nuclear magnetic resonance, sedimentation equilibrium, and chemical cross-linking were used to analyze EAA26 in various trifluoroethanol/H2O mixtures. In pure water the helix content is weak but increases regularly up to 50–60% trifluoroethanol. In contrast the multimerization follows a bell-shaped curve with monomers in pure water, tetramers at 10% trifluoroethanol, and dimers at 40% trifluoroethanol. All suggest that interhelical interactions between apolar side chains are required for the coiled-coil formation of EAA26 and subsist at medium trifluoroethanol concentration. The NH temperature coefficients measured by nuclear magnetic resonance show that at low trifluoroethanol concentration the amide groups buried in the hydrophobic interior of four α-helix bundles are weakly accessible to trifluoroethanol and are only weakly subject to its hydrogen bond strengthening effect. The increased accessibility of trifluoroethanol to buried amide groups at higher trifluoroethanol concentration entails the reduction of the hydrophobic interactions and the conversion of helix tetramers into helix dimers, the latter displaying a smaller hydrophobic interface. The better inhibitory activity of EAA26 compared with Lys-159 could arise from its better propensity to form a helix bundle structure with the biologically important helical part of the 147–175 segment in integrase.

A proton NMR investigation of proline-24 cis-trans isomerism in corticotropin1–32 and related peptides

250 MHz 1H-NMR studies performed on aqueous solutions of corticotropin1-32, corticotropin1-24, corticotropin15-32, corticotropin20-32 and corticotropin15-24 have allowed the location and the subsequent assignment of the signals of Tyrosine-23 aromatic protons and valine-22 methyl protons. These signals are sensitive to the geometry of proline-24, clearly transcribe its isomerism and yield the ratio of the cis-trans conformers. It is concluded that for large peptides in specific cases, the proton signals of side chains can be used to probe the backbone conformation.

Proton NMR study of the conformation of bradykinin: pH titration

Abstract pH titration by 1H-NMR spectroscopy of the peptide hormone bradykinin was carried out in 2H2O. Assignment of all α-proton signals and of most of the other resonances permitted the extraction of vicinal coupling constants 3Jαβ,β′ from which side chain conformation of all residues could be followed and analyzed as a function of pH. It is shown that the ionization of the terminal COOH group affects simultaneously the Arg-9 and Phe-8 chemical shifts and side chain orientation, and the non-equivalence of the Gly-4 methylene protons. Cooperative effects along the peptide backbone or a folded structure of the C-terminal part of bradykinin could explain this effect.

Circular dichroism comparative studies of two bacterial collagenases and thermolysin

The recently isolated and purified collagenase produced by Achromobacter iophagus, the collagenase from Clostridium histolyticum, and thermolysin, three enzymes having common properties, were studied by circular dichroism. From the spectra of the aqueous solutions obtained in the peptide region, the fraction of alpha helix, beta sheet and aperiodic segments in the three proteins could be estimated. Good similarity was found between Achromobacter collagenase and thermolysin, which both contain a high fraction of alpha helix. Side-chain contributions were analyzed in the aromatic region of thespectra: effects of pH and of organic solvents were observed, showing the strong influence of surroundings on the stabilization of the proteins.

Circular dichroism signatures of features simultaneously present in structured guanine-rich oligonucleotides: A combined spectroscopic and electrophoretic approach

In order to identify possible signatures of the most typical structures adopted by guanine‐rich oligonucleotides, we submitted them to the crossed fire of circular dichroism (CD) and electrophoresis. These signatures show up in the circular dichroism spectra even when simultaneously present within the same molecule. Guanine‐rich oligonucleotides, when structured, manifest themselves by CD contributions around 260 or 295 nm. For instance, positive bands at 264 nm and 295 nm, respectively, signal the parallel and antiparallel guanine quartets, while a positive band around 261 nm may indicate the presence of a (parallel?) Hoogsteen duplex. A positive band at 264 nm may also reflect the presence of rigidly and unusually oriented GpT and TpG steps within loops. The signatures are additive with those of other structural features of the same molecule, such as hairpins or Watson‐Crick duplexes, whose bands are observed at 280 nm.