Conformational analysis of a glycosylated human myelin oligodendrocyte glycoprotein peptide epitope able to detect antibody response in multiple sclerosis.

Myelin oligodendrocyte glycoprotein (MOG), a minor myelin component, is an important central nervous system specific target autoantigen for primary demyelination in autoimmune diseases such as multiple sclerosis (MS). The native structure of MOG presents a glycosylation site at position 31 (Asn(31)). It has been recently described that glycosylation of a MOG peptide epitope improved the detection of specific autoantibodies in sera of MS patients. The solution conformational behavior of two MOG derived peptides-hMOG(30-50) (1) and the glycosylated analogue [Asn(31)(N-beta-Glc)]hMOG(30-50) (2)-were investigated through NMR analysis in a water/HFA solution. Conformational studies revealed that peptides 1 and 2 adopted similar conformations in this environment. In particular, they showed strong propensity to assume a well-defined amphipatic structure encompassing residues 41-48. The N-terminal region resulted to be almost completely unstructured for both peptides. The presence in 1 of a low populated Asx-turn conformation characteristic of the Asn-Xaa-Thr glycosylation sites was the only conformational difference between peptides 1 and 2. Thus, the specific antibody recognition of peptide 2 is most likely driven by direct interactions of the antibody binding site with the Asn-linked sugar moiety.

A galpha(s) carboxyl-terminal peptide prevents G(s) activation by the A(2A) adenosine receptor.

The molecular mechanisms of interaction between G(s) and the A(2A) adenosine receptor were investigated using synthetic peptides corresponding to various segments of the Galpha(s) carboxyl terminus. Synthetic peptides were tested for their ability to modulate binding of a selective radiolabeled agonist, [(3)H]2-[4-(2-carboxyethyl)phenylethylamino]-5'-N-ethylcarboxam idoade nosine ([(3)H]CGS21680), to A(2A) adenosine receptors in rat striatal membranes. The Galpha(s) peptides stimulated specific binding both in the presence and absence of 100 microM guanosine-5'-O-(3-thiotriphosphate) (GTPgammaS). Three peptides, Galpha(s)(378-394)C(379)A, Galpha(s)(376-394)C(379)A, and Galpha(s)(374-394)C(379)A, were the most effective. In the presence of GTPgammaS, peptide Galpha(s)(374-394)C(379)A increased specific binding in a dose-dependent fashion. However, the peptide did not stabilize the high-affinity state of the A(2A) adenosine receptor for [(3)H]CGS21680. Binding assays with a radiolabeled selective antagonist, [(3)H]5-amino-7-(2-phenylethyl)-2-(2-furyl)pyrazolo[4, 3-e]-1,2,4-triazolo[1,5-c]pyrimidine ([(3)H]SCH58261), showed that the addition of the Galpha(s) peptide modified the slope of the 5'-N-ethylcarboxamidoadenosine (NECA) competition curve, suggesting modulation of receptor affinity states. In the presence of GTPgammaS, the displacement curve was right-shifted, whereas the addition of Galpha(s)(374-394)C(379)A caused a partial left-shift. Both curves were fitted by one-site models. This same Galpha(s) peptide was also able to disrupt G(s)-coupled signal transduction as indicated by inhibition of the A(2A) receptor-stimulated adenylyl cyclase activity without affecting either basal or forskolin-stimulated enzymatic activity in the same membrane preparations. Shorter peptides from Galpha(s) and Galpha(i1/2) carboxyl termini were not effective. NMR spectroscopy showed the strong propensity of peptide Galpha(s)(374-394)C(379)A to assume a compact carboxyl-terminal alpha-helical conformation in solution. Overall, our results point out the conformation requirement of Galpha(s) carboxyl-terminal peptides to modulate agonist binding to rat A(2A) adenosine receptors and disrupt signal transduction.

Conformational studies on a synthetic C-terminal fragment of the alpha subunit of G(S) proteins.

It has recently been reported that synthetic peptides corresponding to the C-terminal sequence of G alpha, can be used to study the molecular mechanisms of interaction between this protein and G protein coupled receptors (Hamm et al., Science, 1988, Vol. 241, pp. 832-835). A conformational analysis on a 11 amino acids peptide from the G alpha(S) C-terminus, G alpha(S)(384-394) (H-QRMHLRQYELL-OH), was performed by nmr spectroscopy and molecular modeling methods. Two-dimensional nmr spectra, recorded in hexafluoroacetone/water, a mixture with structure stabilizing properties, showed an unusually high number of nuclear Overhauser effects, forming significative pattern to the drawing of a secondary structure. Conformations consistent with experimental NOE distances were obtained through molecular dynamics and energy minimization methods. These calculations yielded two stable conformers corresponding to an alpha-turn and a type III beta-turn involving the last five C-terminal residues. Interestingly, the alpha-turn conformation was found to overlap with good agreement the crystallographic structure of the same fragment in the G alpha(S) protein.

Solution conformation of a potent cyclic analogue of tuftsin: low- temperature nuclear magnetic resonance study in a cryoprotective mixture.

Tuftsin, a linear tetrapeptide (Thr-Lys-Pro-Arg), corresponding to the sequence 289-292 of the heavy chain of leukokinin, has been the object of intensive SAR studies during the past 30 years, owing to its numerous biological activities and to the possibility of generating a novel anticancer drug. A cyclic tuftsin analogue, c-[T-K-P-R-G], has biological activity 50 times higher than that of the parent linear peptide. Here we present a conformational study of c-[T-K-P-R-G] based on NMR data in a cryoprotective DMSO/water mixture. The preferred conformation is a type VIa turn centered on the K-P residues. The orientation of the side chains of the two basic residues (K and R) may represent the essential feature of the bioactive conformation of tuftsin. A possible role of tuftsin as a DNA binding motif is suggested by the similarity of the bioactive conformation of c-[T-K-P-R-G] and of the beta-turn conformation proposed by Suzuki for the [T,S]-P-K-R motif.

Assignment and secondary-structure determination of monomeric bovine seminal ribonuclease employing computer-assisted evaluation of homonuclear three-dimensional 1H-NMR spectra.

Monomeric bovine seminal ribonuclease (mBS-RNase), the subunit of dimeric bovine seminal ribonuclease (BS-RNase), is an unusual monomer: for its structural stability, its catalytic activity, which is even higher than that of the parent dimeric enzyme, and for its role as an intermediate in the refolding of dimeric BS-RNase. Here we present the proton NMR assignment and secondary-structure determination of mBS-RNase, with a comparison of its structure to the structure of its parent protein, and to the structure of RNase A, a homologue with more than 80% identity in amino acid sequence. Proton NMR assignment was performed using a computer-assisted procedure, through a partially automated analysis of homonuclear three-dimensional spectra [Oschkinat, H., Holak, T. A. & Cieslar, C. (1991) Biopolymers 31, 699-712]. The secondary structures of mBS-RNase, of the A chain of dimeric BS-RNase, and of RNase A, are found to be similar. Significant differences are found instead, between mBS-RNase and RNase A in the more flexible stretches of the molecule, where a higher number of substitutions is present. Furthermore, a preliminary tertiary-structure model is reported, showing that the overall folding of mBS-RNase is closer to that of RNase A rather than that of (dimeric) BS-RNase.

Solution conformation of CCK9, a cholecystokinin analog.

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.

Solution conformation of tuftsin.

Tuftsin, a natural linear tetrapeptide (Thr-Lys-Pro-Arg) of potential antitumor activity, has been studied in DMSO-d6 solution by 2D NMR spectroscopy. 1H and 13C spectra show the presence of two families of conformations characterized by a trans or cis Lys-Pro bond, respectively. The family of conformers containing the cis peptide bond is a mixture of extended structures as expected for a short linear peptide. On the contrary, the trans isomer appears to be a rigid, folded conformer, as indicated by crucial NOEs and by the exceptionally low temperature coefficient of Arg NH. Analysis of the solution data by means of energy calculations leads to a unique structure, characterized by a Lys-Pro inverse gamma-turn.

Conformational preferences of [Leu5]enkephalin in biomimetic media. Investigation by 1H NMR.

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.