. High resolution NMR has become an important tool for determination of solution structures of proteins and nucleic acids, but there are still important questions to be resolved about how precise and accurate these structures are. The research proposed here focuses on careful comparisons of calculated and observed 2D-correlated nuclear Overhauser effect spectroscopy (NOESY) as a means of analyzing and improving structures. The key elements are: (1) the use of gradient-based refinement techniques to improve agreement between calculated and observed spectra; (2) the development and calibration of models for molecular tumbling and internal motion that will lead to more reliable estimates of NOESY intensities; (3) molecular dynamics simulations of proteins in water to help design internal motion models and to develop simulated data against which refinement methods can be tested; and (4) the incorporation of direct matrix inversion techniques in the refinement scheme, providing for smoothing of the data and improved estimation of weak peaks. Two additional projects will be pursued that should also help to make NMR refinement a more robust and accurate process. First, systematic search procedures at the di- and tri-peptide level will be explored as a means of generating better distance bounds, of making reliable stereospecific assignments, and of detecting inconsistencies in experimental data. Second, empirical models for conformation-dependent chemical shifts will be developed and tested against a large database of experimental information, and the extent to which refinement against such data can be used to improve structures will be tested. The techniques developed here will be used to refine solution structures of plastocyanin, a """"""""zinc finger"""""""" peptide, and two oligonucleotides, d(ATATATAUAT)2 and d(CAUAUAUAUG)2 using experimental data provided by Peter Wright and Tom James. The computer codes incorporating these refinement techniques will be assembled into a portable and documented package and made available to the NMR community.
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