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The structure of a recently reported neurotrophic ligand, 3-(3-pyridyl)-1-propyl(2S)-1-(3,3-dimethyl-1, 2-dioxopentyl)-2-pyrrolidinecarboxylate, in complex with FKBP12 was determined using heteronuclear NMR spectroscopy. The inhibitor exhibits a binding mode analogous to that observed for the macrocycle FK506, used widely as an immunosuppressant, with the prolyl ring replacing the pipecolyl moiety and the amide bond in a trans conformation. However, fewer favourable protein-ligand interactions are detected in the structure of the complex, suggesting weaker binding compared with the immunosuppressant drug. Indeed, a micromolar dissociation constant was estimated from the NMR ligand titration profile, in contrast to the previously published nanomolar inhibition activity. Although the inhibitor possesses a remarkable structural simplicity with respect to FK506, 15N relaxation studies show that it induces similar effects on the protein dynamics, stabilizing the conformation of solvent-exposed residues which are important for mediating the interaction of immunophilin/ligand complexes with molecular targets and potentially for the transmission of the neurotrophic action of FKBP12 inhibitors.

The interaction of Li+ with myo-inositol monophosphatase was studied by 7Li-NMR spectroscopy. Li+ binding to the enzyme induces a downfield shift and broadening of the 7Li-NMR signal. Changes of the chemical shift were used to follow the titration of the enzyme with lithium and to determine a dissociation constant, Kd = (1.0 +/- 0.1) mM. Only one major binding site/enzyme subunit was inferred. The complex forms independently of the presence of inorganic phosphate. Metals from the group IIa of the periodic table compete with Li+ binding with the affinity increasing in the order Mg2+ < Ca2+ < Be2+. In contrast to lithium, their binding is enhanced by phosphate.

The structure of cGMP-dependent protein kinase I alpha-(546-576)-peptide amide (peptide-546) and its effects on cGMP-dependent protein kinase I alpha (G-kinase) have been studied. By primary sequence analysis and analogy to a peptide that stimulates protein kinase C, peptide-546 was predicted to form part of the protein/peptide binding site of G-kinase, and it was proposed that it would stimulate the enzyme by interaction with an autoinhibitory site. The portion of cAMP-dependent protein kinase analogous to peptide-546 forms part of the peptide substrate binding site, interacting with the peptide inhibitor residues Argp-2 and Phep-11 (where p is the pseudophosphorylation site), through residues at positions corresponding to Glu4, Pro10 and Ser13 in peptide-546. Peptide-546 is a reasonably potent G-kinase activator, increasing the turnover number with the peptide substrate Arg-Lys-Arg-Ser-Arg-Lys-Glu by about threefold with an activation constant that is about fivefold lower than the Km value of this peptide substrate. Peptide-546 does not appear to change the affinity of the enzyme for the above substrate, ATP or cGMP and does not affect the binding of [3H]cGMP to G-kinase. The activation does not seem to result from an interaction between peptide-546 and peptide substrates, and a kinetic scheme is proposed which is compatible with an action of peptide-546 on G-kinase independent of substrates. The activation is additive with that given by cGMP and causes the enzyme to enter a hitherto unrecognised superactive state. Peptide conformation has been monitored in mixed 2,2,2-trifluoroethanol/H2O solvents by circular dichroism: helical structure is observed in these mixtures when the 2,2,2-trifluoroethanol content is above 25%. The structure is lost only gradually on raising the temperature to 80 degrees C with no clear melting transition. Assignment of the resonances in the 1H-NMR spectrum has allowed the identification of elements of secondary structure from detected nuclear Overhauser effects. In particular, a helical segment from Met18 to Arg26 is observed. The four proline residues (Pro10, Pro11, Pro15 and Pro17) are all seen to be in the trans conformation, although additional, weaker peaks in the spectra may correspond to a minor conformer in which one or more of the prolines is in a cis conformation. The N-terminal residues are less structured but show some helical character.(ABSTRACT TRUNCATED AT 400 WORDS)

The structure of porcine neuropeptide Y in 0.05 M CD3COOD/D2O was determined by nuclear magnetic resonance spectroscopy. Nuclear Overhauser spectra yielded 377 distances which define a helical segment formed by residues 11-36. An additional set of 24 distances were interpreted as intermolecular distances within a dimer. A combination of distance geometry calculations, energy minimization and molecular dynamics yielded a model of the dimer having antiparallel packing of two curved helical units whose hydrophobic sides form a well defined core. The N-terminus (residues 1-9) appears as an unstructured mobile segment. Large changes in the intrinsic fluorescence intensity of neuropeptide Y tyrosine residues allowed the determination of the dimer dissociation constant as 1.6 +/- 0.6 microM at pH 2-8 in aqueous buffers and also indicated the enclosure of several tyrosine residues in the hydrophobic environment of the interface region in the dimeric species. Fluorescence anisotropy data reveals the slow rotation of such shielded residues.

Detailed biophysical studies have been carried out on echistatin, a member of the disintegrin family of small, cysteine-rich, RGD-containing proteins, isolated from the venom of the saw-scaled viper Echis carinatus. Analysis of circular-dichroism spectra indicates that, at 20 degrees C, echistatin contains no alpha-helix but contains mostly beta-turns and beta-sheet. Two isobestic points are observed as the temperature is raised, the conformational changes associated with that observed between 40 degrees C and 72 degrees C being irreversible. Raman spectra also indicate considerable beta-turn and beta-sheet (20%) structure and an absence of alpha-helical structure. Three of the four disulphide bridges are shown to be in an all-gauche conformation, while the fourth adopts a trans-gauche-gauche conformation. The 1H-NMR spectrum of echistatin has been almost fully assigned. A single conformation was observed at 27 degrees C with the four proline residues adopting only the trans conformation. A large number of backbone amide protons were found to exchange slowly, but no segments of the backbone were found to be in either alpha-helical or beta-sheet conformation. A number of turns could be characterised. An irregular beta-hairpin contains the RGD sequence in a mobile loop at its tip. Two of the four disulphide cross-links have been identified from the NMR spectra. The data presented in this paper will serve to define the structure of echistatin more closely in subsequent studies.

The antigenic properties of acylphosphatase are compared with its various sequential characteristics (hydrophobicity, chemical shift of the main-chain 1H-NMR resonances, numbers and intensities of the nuclear Overhauser enhancements, hydrogen-deuterium exchange and sequential arrangement of the secondary structure units). The discussion is based on the complete sequential assignment of the 1H-NMR spectrum and the knowledge of the three-dimensional fold of the protein obtained by NMR spectroscopy from distance geometry calculations. Regions with very different degrees of mobility can be distinguished. It is found that all major antigenic sites are located in the most mobile surface loops.

A complete range of two-dimensional NMR experiments was used for the assignment of the 1H-NMR spectrum of horse muscle acylphosphatase. Firstly the spin systems of some easily identifiable amino acid side chains were assigned. These side chains involved all the aromatic residues and all the leucine, valine, isoleucine, threonine, alanine, proline as well as some of the glycine residues. Analysis of nuclear Overhauser enhancement spectra in our previous work had identified the sequential and long-range patterns characteristics for secondary structure elements. This result had also provided the identification of the main-chain alpha and amide proton resonances. Several of the completely assigned spin systems were then identified as being part of the secondary structure units which led, after analysis of the primary amino acid sequence, to unambiguous sequence-specific assignments. The identification and assignment of the remaining side-chain resonances was then completed and are reported here. These results provide a complete data base for the three-dimensional structure determination of this enzyme in solution.