Network-editing experiments are variants of the basic NOESY experiment that allow more accurate direct measurement of interproton distances in macromolecules by defeating specific spin-diffusion pathways. Two network-editing approaches, block-decoupled NOESY and complementary-block-decoupled-NOESY, were applied as three-dimensional, heteronuclear-edited experiments to distance measurement in a small protein, turkey ovomucoid third domain (OMTKY3). Two-hundred and twelve of the original 655 distance constraints observed in this molecule (Krezel AM et al., 1994, JMol Biol242:203-214) were improved by their replacement by distances derived from networkedited spectra, and distance geometryhimulated annealing solution structure calculations were performed from both the unimproved and improved distance sets. The resulting two families of structures were found to differ significantly, the most important differences being the hinge angle of a 0-turn and an expansion of the sampled conformation space in the region of the reactive-site loop. The structures calculated from network-editing data are interpreted as a more accurate model of the solution conformation of OMTKY3.Keywords: BD-NOESY-HSQC; CBD-NOESY-HSQC; cross relaxation; network editing; "N NMR assignments; NOESY; nuclear magnetic resonance; protein structure; spin diffusionThe accuracy and reliability of structural models derived by NMR is a key issue in the methodology of structure determination. In the context of a drug-development study, for example, a relatively minor structural distortion can lead to a tremendous amount of wasted effort on the part of synthetic chemists. NMR data directly determine only short-range constraints, and those with relatively low precision, but yield those constraints in great abundance such that converged and apparently highly precise structures are obtained. Therefore, the extent to which the accuracy and precision of distance measurements will affect the quality of derived structural models has been a source of controversy for some time.Two different approaches can be taken in improving the accuracy of distance measurements derived from NMR data. One approach makes use of a model structure in the analysis of the full relaxation matrix (Boelens et al
Genetic engineering studies of ovomucoid domains have been hindered by the lack of an efficient procedure for overproducing this protein. The novel scheme presented here has led to the isolation of chicken ovomucoid third domain (OMCHI3) at a level of 22 mg pure protein/l Escherichia coli culture medium. The gene coding for OMCHI3 was fused to the 3' end of the gene encoding staphylococcal nuclease (SNase). Expression of the chimeric gene was placed under control of the strong transcription and translation signals of the phage T7 promoter. Upon isopropyl-beta-D-galactopyranoside induction, the cells harboring the target plasmid efficiently overproduced the protein (30% of the total soluble protein). The 56-residue fragment corresponding to OMCHI3 was then liberated by cyanogen bromide (CNBr) cleavage at a genetically engineered methionine residue located at the nuclease--OMCHI3 junction (OMCHI3 lacks an internal methionine). SDS--PAGE, enzyme inhibition studies and NMR spectroscopy all indicated that the recombinant OMCHI3 has properties identical to those of OMCHI3 isolated from its natural source. The expression system was easily adapted for the production of [98% U 15N] OMCHI3. The expression vector was mutated for overexpression of turkey ovomucoid third domain (OMTKY3), which differs from OMCHI3 by three amino acid substitutions. Since many other avian ovomucoid domains also lack methionine residues, this approach should be suitable for large-scale production and isotope labeling of homologous proteinase inhibitors with a variety of inhibitory specificities.
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