Solution structure determination by NMR spectroscopy of a synthetic peptide corresponding to a putative amphipathic alpha-helix of spiralin: resonance assignment, distance geometry and simulated annealing.

Spiralin is the major protein of the plasma membrane of several spiroplasmas. Neither the function of this protein nor the crystallographic structure is known. Analysis of the primary structure of spiralin from Spiroplasma melliferum BC3 suggests the presence of an amphipathic peptide in the 143-162 region (Chevalier, C., Saillard, C. and Bové, J.M. (1990) J. Bacteriol. 172, 6090-6097). The structure of a synthetic peptide, H2N-L-N-A-V-N-T-Y-A-T-L-A-K-A-V-L-D-A-I-Q-N-amide, corresponding to this fragment has been examined by 1H-NMR spectroscopy. This 20 amino acid peptide adopts a random coil structure in solution, but the addition of trifluoroethanol stabilizes a structure exhibiting alpha-helical character. The 1H-NMR spectrum has been fully assigned in CF3CD2OD/H2O (30:70, v/v) and the three-dimensional structure has been elucidated using NMR-derived distance information. The calculated structures have been obtained by dynamical simulated annealing or distance geometry followed by simulated annealing. Both sets of structures have been energy-minimized using CHARMm potential. The resulting structures are very similar in terms of constraint violations and energies. It is demonstrated that whereas the first three residues exhibit a large flexibility, the remaining sequence is helical.

A particularly labile Asp-Pro bond in the green mamba muscarinic toxin MTX2. Effect of protein conformation on the rate of cleavage.

The single Asp53-Pro54 bond of the MTX2 toxin from the mamba snake Dendroaspis angusticeps is rapidly and efficiently cleaved in acidic solution (pH 1.5-2.5) at 45 degrees C. Unfolding of the toxin slows down the cleavage reaction by several times. Modelling studies indicate that the native toxin conformation can catalyse the Asp53-Pro54 bond cleavage. The implications of this study are: (i) cleavage of Asp-Pro bond for sequence determination may occur better in absence than in presence of denaturant, (ii) mild acid conditions, commonly used in NMR structure determinations, may irreversibly affect the structural integrity of Asp-Pro containing peptides and proteins.

Three-dimensional structure of the ion-channel forming peptide trichorzianin TA VII bound to sodium dodecyl sulfate micelles.

Trichorzianin TA VII, Ac0 U1 A2 A3 U4 J5 Q6 U7 U8 U9 S10 L11 U12 P13 V14 U15 I16 Q17 Q18 Fol19, is a nonadecapeptide member of the peptaibol antibiotics biosynthesized by Trichoderma soil fungi, which is characterized by a high proportion of the alpha, alpha-dialkylated amino acids, alpha-aminoisobutyric acid (Aib, U) and isovaline (Iva, J), an acetylated N-terminus and a C-terminal phenylalaninol (Pheol, Fol). The main interest in such peptides stems from their ability to interact with phospholipid bilayers and form voltage-dependent transmembrane channels in planar lipid bilayers. In order to provide insights into the lipid-peptide interaction promoting the voltage gating, the conformational study of TA VII in the presence of perdeuterated sodium dodecyl sulfate (SDS-d25) micelles has been carried out. 1H sequential assignment have been performed with the use of two-dimensional homo- and -heteronuclear nmr techniques including double quantum filtered correlated spectroscopy, homonuclear Hartmann-Hahn, nuclear Overhauser effect spectroscopy, 1H-13C heteronuclear single quantum correlation, and heteronuclear multiple bond correlation. Conformational parameters, such as 3JNHC alpha H coupling constants, temperature coefficients of amide protons (delta gamma/delta TNH) and quantitative nuclear Overhauser enhancement data, lead to detailed structural information. Ninety-eight three-dimensional structures consistent with the nmr data were generated from 231 interproton distances six phi dihedral angle restraints, using restrained molecular dynamics and energy minimization calculations. The average rms deviation between the 98 refined structures and the energy-minimized average structure is 0.59 A for the backbone atoms. The structure of trichorzianin TA VII associated with SDS micelles, as determined by these methods, is characterized by two right-handed helical segments involving residues 1-8 and 11-19, linked by a beta-turn that leads to an angle about 90 degrees-100 degrees between the two helix axes; residues 18 and 19 at the end of the C-terminal helix exhibit multiple conformations.

Solution structure of a green mamba toxin that activates muscarinic acetylcholine receptors, as studied by nuclear magnetic resonance and molecular modeling.

The three-dimensional solution structure of the MTX2 toxin (65 amino acids and 4 disulfides) from the green mamba venom (Dendroaspis angusticeps), a toxin that activates the pharmacological M1 muscarinic acetylcholine receptors, has been determined by nuclear magnetic resonance and molecular modeling. Seventeen structures were calculated from 810 distance and 68 dihedral angle restraints using DIANA and X-PLOR. The average rms deviation between the 17 refined structures and the energy-minimized average structure is 0.95 A for the backbone atoms. The overall folding of MTX2 consists of three loops stabilized by the four disulfides and forming a two- and a three-stranded beta-sheet. This structure appears to be very similar to that of other snake toxins, such as neurotoxins, fasciculins, and cardiotoxins, that also possess the same three-finger fold. For instance, the RMSd for the backbone atoms between MTX2 and the curaremimetic toxin alpha (from Naja nigricollis), the acetylcholinesterase inhibitor fasciculin 1 (from Dendroaspis angusticeps), and the cardiotoxic toxin gamma (from Naja nigricollis) are 1.86, 1.87, and 2.04 A, respectively. Local differences are observed between this toxin and the other structurally related toxins. Some of these differences could be relevant for the functional specificity of MTX2. In particular, this toxin presents a large twist at the tip of loop II due to a bulge (V31, T32; N35) that accommodates an inserted amino acid in the loop. This spatial arrangement brings the side chain of K34 in the beta-turn of the loop to be aligned with the beta-sheet. Hypotheses about a possible functional role of this lysine are described. Other characteristics in the side-chain distribution that could be related to the MTX2 function are presented.