Dennis R. Hare

Solution structures of the rabbit neutrophil defensin NP-5.

Solution structures of the rabbit neutrophil defensin NP-5 have been determined by 1H nuclear magnetic resonance (n.m.r.) spectroscopy and distance geometry techniques. This 33 amino acid peptide is part of the oxygen-independent mammalian defense system against microbial infection. The structures were generated from 107 n.m.r. derived inter-residue proton-proton distance constraints. A distance geometry algorithm was then used to determine the range of structures consistent with these distance constraints. These distance geometry calculations employed an improved algorithm that allowed the chirality constraints to be relaxed on prochiral centers when it was not possible to make stereo-specific assignments of protons on these centers. This procedure gave superior results compared with standard distance geometry methods and also produced structures that were more consistent with the original n.m.r. data. Analysis of the NP-5 structures shows that the overall folding of the peptide backbone is well defined by the n.m.r. distance information but that the side-chain group conformations are generally less well defined.

Determining stereo-specific 1H nuclear magnetic resonance assignments from distance geometry calculations

Stereo-specific 1H nuclear magnetic resonance assignments can be obtained following distance geometry structure calculations. The key to this method is to allow stereo-related atoms or methyls to float between pro-R and pro-S configurations, the final configuration being determined by the experimental constraints. Resonances from stereo-related pairs are given initial random assignments (either pro-R or pro-S) for identifying nuclear Overhauser effects (NOEs). A list of distance constraints using these assignments is compiled and a series of structures calculated where the chirality of non-C alpha chiral centers is not constrained; no pseudoatom corrections are required. Calculated structures are both locally and globally well-determined since the assignments rely upon the structure determination rather than the structure quality relying upon stereo-specific assignments. The method represents a global approach to determining stereo-specific assignments versus previously reported methods where only intraresidue NOEs and J-coupling information are used.