In a similar spirit, we describe here how NMR chemical shifts can be used to define the structures of the native states of proteins at high resolution without the requirement of any additional experimental measurements. In approaches of this type, the experimental information is used essentially to complement standard force fields and to guide the sampling of conformational space toward regions consistent with experimental observations related to the specific state under investigation ( 19). Such techniques have also been shown to be able to describe, simultaneously and with high accuracy, the structures and dynamics of native states of globular proteins by using both distance information (NOEs) and NMR order parameters ( 21) or RDC ( 22).
It has been demonstrated recently that strategies in which experimental data are used as restraints in molecular dynamics simulations can lead to a description of the structures of proteins, at least in outline, even in highly heterogeneous states such as those adopted by natively unfolded polypeptide molecules ( 19, 20).
If such effects could be interpreted in depth, therefore, they would enable the characterization of the detailed environment of virtually every atom in the structure and, in turn, the determination of a unique overall conformation compatible with all such environments. The chemical shift associated with a specific atom, by contrast, is a summation of many contributing factors ( 16– 18) so that the reliable identification of interaction partners is very difficult, even though they may be substantially influenced by contacts between residues, such as hydrogen bonding and proximity to aromatic rings, that are at very different locations in the protein sequence. The structural information contained in the chemical shifts is, however, very different in nature from that provided by NOEs, because the latter report on pairwise distances between specific protons and can thus provide unequivocal information about the relative spatial locations of different residues in a protein sequence ( 1). The unique fingerprints of proteins provided by their NMR spectra suggest that chemical shifts inherently carry sufficient information to determine their structures at high resolution, as indeed is often the case for molecules of low molecular weight ( 4). In many important cases, however, chemical shifts are the only NMR parameters that can be obtained on a given state of a protein with any degree of completeness ( 7, 8, 11– 15), prompting us to explore the extent to which these quantities alone can be used to determine high-resolution structures.
It has also been recognized that chemical shifts can aid in the determination of the tertiary structure of proteins when used in combination with other NMR probes that report on interproton distances (NOEs) and the relative orientations of the different nuclei in a protein structure ( 3, 6, 10). In structural biology, chemical shifts are most often used to predict regions of secondary structure in native and nonnative states of proteins ( 2, 5), to aid in the refinement of complex structures ( 6), and for the characterization of conformational changes associated with partial unfolding ( 7) or binding ( 8, 9). Indeed, it is this characteristic that is the origin of their unique value in probing in atomic detail the properties of systems ranging from simple organic and inorganic compounds to complex biological macromolecules, because it enables the resolution of distinct signals from even chemically identical groups when located in different local or global environments. Chemical shifts are exquisitely sensitive probes of molecular structure ( 1– 4).
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