No abstract

Cyclosporin A bound to the presumed receptor protein cyclophilin was studied in aqueous solution at pH 6.0 by nuclear magnetic resonance spectroscopy using uniform 15N- or 13C-labeling of cyclosporin A and heteronuclear spectral editing techniques. Sequence-specific assignments were obtained for all but one of the cyclosporin A proton resonances. With an input of 108 intramolecular NOEs and four vicinal 3JHN alpha coupling constants, the three-dimensional structure of cyclosporin A bound to cyclophilin was calculated with the distance geometry program DISMAN, and the structures resulting from 181 converged calculations were energy refined with the program FANTOM. A group of 120 conformers was selected on the basis of the residual constraint violations and energy criteria to represent the solution structure. The average of the pairwise root-mean-square distances calculated for the backbone atoms of the 120 structures was 0.58 A. The structure represents a novel conformation of cyclosporin A, for which the backbone conformation is significantly different from the previously reported structures in single crystals and in chloroform solution. The structure has all peptide bonds in the trans form, contains no elements of regular secondary structure and no intramolecular hydrogen bonds, and exposes nearly all polar groups to its environment. The root-mean-square distance between the backbone atoms of the crystal structure of cyclosporin A and the mean of the 120 conformers representing the NMR structure of cyclosporin A bound to cyclophilin is 2.5 A.

An alternative method for the stereospecific assignment of the methyl groups of valine and leucine in the nuclear magnetic resonance (NMR) spectra of peptides and proteins is proposed, and its practical application is demonstrated with the assignment of all valine and leucine methyl groups in cyclosporin A. The method is based on the use of a mixture of fully i3C-labelled and unlabelled glucose as the sole carbon source for the biosynthetic production of the polypeptide studied, knowledge of the independently established stereoselectivity of the reaction pathways of valine and leucine biosynthesis, and analysis of the distribution of W labels in the valyl and leucyl residues of the product by two-dimensional heteronuclear NMR correlation experiments.

The immunologically dominant central portion of the circumsporozoite (CS) surface protein on the malaria parasite Plasmodium falciparum contains a large number of tandemly repeated NPNA tetrapeptide motifs. The preferred secondary structure of this repeat unit in aqueous solution has been investigated with the aid of the secondary structure-inducing amino acid (8-a-methylproline (PMe). \u27H-Nuclear magnetic resonance (Nh4R) and circular dichroism (CD) spectroscopy have been used to probe the structures of synthetic peptides containing one to three tetrapeptide NPMeNA units. The far-UV CD spectra of these peptides show more intense negative bands at 215 nm than do similar peptides based on the NPNA motif. This and the temperature dependence of the peptide amide chemical shifts, the pattern of NOE connectivities, and the magnitude of 3J coupling constants, derived from oneand two-dimensional NMR spectra of Ac(NPMeNA)3-OH, provide strong evidence for stable tumlike structures. From NOE distance and dihedral angle restraints, structures consistent with the NMR parameters were calculated. These reveal a stable hydrogen-bonded type-I p-tum conformation (most likely present at 70-80% population) within each NPMeNA motif, stabilized by the backbone C, methylation. Side chain to backbone hydrogen bonds involving the side chain amide groups of both asparagine residues also appear to impart stabilization to the turn conformation. No regular repeating conformations were detected in the linker regions connecting each NPMeNA unit. Polyclonal antisera raised in rabbits against (NPMeNA)3 recognized intact P. falciparum sporozoites in an immunofluorescence assay as efficiently as antisera raised against (NPNA)3. This indicates that the type-I ,\u26turn detected in the PMecontaining peptide is closely related to the immunologically dominant portion of the folded CS protein. An improved knowledge of the three-dimensional structure of this protein may be of value for the design of second-generation synthetic malaria vaccines

The initial velocities of hydrolysis of nineteen glycopeptides by peptide: N-glycosidase F and A were determined. Substrates were prepared from bovine fetuin, hen ovalbumin, pineapple stem bromelain, bovine fibrin and taka-amylase. From these glycopeptides, several variants with regard to peptide and carbohydrate structure were prepared and derivatized with dabsyl chloride, dansyl chloride or activated resorufin. Tyrosine containing glycopeptides were also used without an additional chromophore. Enzymatic hydrolysis of glycopeptides was quantified by narrow bore, reversed phase HPLC with turnaround cycle times of down to 6 min, but usually 15 min. KM values ranging from 30 to 64 microM and from 4 to 36 microM were found for N-glycosidase F and A, respectively. Relative velocities of hydrolysis of the different substrates by each enzyme varied considerably. Little, if any, similarity of the performance of N-glycosidase F and A with the different substrates was observed. The minimal carbohydrate structure released by peptide: N-glycosidase F was a di-N-acetylchitobiose. N-glycosidase A could release even a single N-acetylglucosamine, albeit 3000 times slower than a di-N-acetylchitobiose or larger glycans. In general the structure of the intact glycan had little effect on activity, and with both enzymes the rate of hydrolysis appeared to be primarily governed by peptide structure and length. However, N-glycosidase F did not release glycans alpha 1,3-fucosylated at the asparagine linked N-acetylglucosamine irrespective of the presence of xylose in the substrate.