Showing papers on "Structural biology published in 1993"

Heteronuclear three-dimensional NMR spectroscopy of a partially denatured protein: the A-state of human ubiquitin.

Abstract: Human ubiquitin is a 76-residue protein that serves as a protein degradation signal when conjugated to another protein. Ubiquitin has been shown to exist in at least three states: native (N-state), unfolded (U-state), and, when dissolved in 60% methanol:40% water at pH 2.0, partially folded (A-state). If the A-state represents an intermediate in the folding pathway of ubiquitin, comparison of the known structure of the N-state with that of the A-state may lead to an understanding of the folding pathway. Insights into the structural basis for ubiquitin's role in protein degradation may also be obtained. To this end we determined the secondary structure of the A-state using heteronuclear three-dimensional NMR spectroscopy of uniformly 15N-enriched ubiquitin. Sequence-specific 1H and 15N resonance assignments were made for more than 90% of the residues in the A-state. The assignments were made by concerted analysis of three-dimensional 1H-15N NOESY-HMQC and TOCSY-HMQC data sets. Because of 1H chemical shift degeneracies, the increased resolution provided by the 15N dimension was critical. Analysis of short- and long-range NOEs indicated that only the first two strands of β-sheet, comprising residues 2–17, remain in the A-state, compared to five strands in the N-state. NOEs indicative of an α-helix, comprising residues 25–33, were also identified. These residues were also helical in the N-state. In the N-state, residues in this helix were in contact with residues from the first two strands of β-sheet. It is likely, therefore, that residues 1–33 comprise a folded domain in the A-state of ubiquitin. On the basis of 1Hα chemical shifts and weak short-range NOEs, residues 34–76 do not adopt a rigid secondary structure but favor a helical conformation. This observation may be related to the helix-inducing effects of the methanol present. The secondary structure presented here differs from and is more thorough than that determined previously by two-dimensional 1H methods [Harding et al. (1991) Biochemistry, 30, 3120–3128].

The use of osmolytes to facilitate protein NMR spectroscopy

Abstract: A method of stabilizing folded proteins is described, which allows NMR studies under conditions where a protein would normally be unfolded. This enables stable proteins to be examined at elevated temperatures, or spectra recorded on samples that are insufficiently stable under normal conditions. Up to two molar perdeuterated glycine, a potent osmolyte, can be added to aqueous protein NMR samples without altering the folded three-dimensional structure or function of the protein. However, the stability of the folded form is dramatically increased. This is illustrated for the protein lysozyme at high temperature (348 K) where the structural integrity is destroyed in standard aqueous solution, but is retained in the osmolyte solution. We hope that the technique will be of value to those studying by NMR the structural biology of protein fragments and mutants, which are often of reduced stability compared with the original proteins.

Hydration of Biological Macromolecules in Solution: Surface Structure and Molecular Recognition

Abstract: The presently available three-dimensional protein and nucleic acid structures at atomic resolution have, with few exceptions, been determined either by X-ray diffraction in protein crystals (Blundell and Johnson 1976) or by nuclear magnetic resonance (NMR) spectroscopy in protein solutions (Wuthrich 1986). These two techniques produce a rapidly growing pool of data (Hendrickson and Wuthrich 1991, 1992, 1993), which represent today's structural basis for detailed investigations on the functionality of biological macromolecules. NMR spectroscopy and X-ray diffraction can provide complementary information on the same molecule, which results primarily from the facts that the time scales of the two types of measurements are widely different, and that the two techniques use, respectively, proteins in solution and protein single crystals (see, e.g., Wuthrich 1990, 1991). Since protein structure determinations by NMR or by X-ray diffraction can be performed independently (Blundell and Johnson 1976; Wuthrich 1986), meaningful comparisons of corresponding structures in single crystals and in noncrystalline states can be obtained. This is highly relevant, since the solution conditions for NMR studies may coincide closely with the physiological fluids, and comparative studies of corresponding crystal and solution structures promise to result in more relevant ways of analyzing crystal data with regard to protein functions in physiological milieus. Providing this kind of fundamental information may well turn out to be the major impact of NMR in structural biology, considering that X-ray crystallography continues to provide a dominant fraction of the new macromolecular structures (Hendrickson and Wuthrich 1993). Although NMR structure determination in solution will foreseeably be limited to proteins with molecular weights below about 30,000-40,000 (see, e.g., Wuthrich 1990), many conclusions from comparative studies with relatively small proteins should also be applicable to crystal structures of bigger molecules. In comparisons of corresponding crystal and solution structures of proteins, both global conformational rearrangements and extensive conservation between the two states have been observed. Major rearrangements are usually seen in nonglobular polypeptides (see, e.g., Braun et al. 1983) and on the surface of globular proteins, whereas close coincidence is commonly encountered for the core of globular proteins (Billeter 1992). However, even for proteins with virtually identical molecular architecture in crystals and in solution, the two techniques provide different information on the internal mobility of the molecular core. NMR can provide direct, quantitative measurements of the frequencies of certain high-activation-energy motional processes in the interior of globular proteins, and at least semiquantitative information on additional, higher-frequency processes. The corresponding information from X-ray structure determinations commonly consists of an outline of the conformation space covered by the combination of static disorder and high-frequency structure fluctuations. The protein surface is most likely to be influenced by crystal packing, or by solvent interactions, respectively, and it is thus of special interest to investigate these effects by combined use of NMR and crystallography. On the one hand, a detailed description of the protein surface in near-physiological solution represents a proper reference for investigating mechanistic aspects of intermolecular interactions with proteins, and novel NMR techniques enabling the observation of hydration water on the molecular surface (Otting et al. 1991a) have greatly added to the characterization of protein surfaces in solution. On the other hand, the structural basis of intermolecular recognition between different macromolecules can be investigated in a large sample of X-ray crystal structures of proteins in binary complexes or multimolecular assemblies, and even the influence of crystal-packing effects on the protein surface may be indicative of the types of protein-protein interactions that are important in physiological recognition processes. In this paper, I survey novel insights into protein structures in solution that result from NMR investigations of protein hydration. Special emphasis is on protein surface hydration in aqueous solution and comparison with corresponding data from diffraction experiments with protein crystals. As an illustration, hydration data on a homeodomain-DNA complex (Billeter et al. 1993; Qian et al. 1993a) are discussed.