We have been working on obtaining the solution structures for w-conotoxins GVIA, MVIIA, MVIIC and SVIB. These small peptides contain 25 to 27 amino acid residues, respectively, and three disulfide bridges. They come from the venom of the fish hunting sea snails Conus Geographus and Conus Magus, respectively. They bind to certain subtypes of the calcium channel proteins, and have been used to identify different types of calcium channels. We have determined the NMR solution structure of the w-conotoxin GVIA, which we have published, with a backbone atom average pairwise RMS deviation of 0.7 for the 22 structures obtained. The methodology for the structure determination involved using the program MARDIGRAS based on a complete relaxation matrix analysis of the crosspeak intensities in NOESY spectra to obtain accurate distance constraints. These constraints were used in conjunction with angle constraints based on vicinal coupling constants for the input to the Distance Geometry and Molecular Dynamics calculations to obtain structures consistent with the experimental data. The correlation time necessary in order to use MARDIGRAS was obtained by determining the T1 and T2 relaxation times for the alpha-carbons at natural abundance on the same 10mM sample (5mm diameter sample tube) used for all the other spectra. These measurements were obtained using 1H detection. The structures for the w-conotoxin MVIIA, MVIIC and SVIB were also determined with the same procedure, with the average pairwise RMSDs for the backbone atoms of 0.8A, 1.0, and 0.9 respectively. We have published the results for w-conotoxin MVIIA and MVIIC. Use of the Computer Graphics Laboratory is critical in the structure determination process, since many structures are generated, and then compared via an rms fit of the atomic coordinates using MIDAS. Comparison of the distances in the models with the experimentally determined distances is done using noeshow, which is part of the MIDAS package of programs. This allows us to identify which distances may be erroneous, directing our efforts towards re-investigating the assignment of NOESY crosspeaks and the measurement of their intensities. Future planned developments by CGL are extremely important to us, especially the fast link between structure and crosspeaks in two- and three-dimensional NMR spectra. This would greatly speed the structure determination process.
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