The 1H resonances of human ubiquitin were studied by two-dimensional nuclear magnetic resonance techniques. A recently introduced assignment algorithm termed the main chain directed (MCD) assignment [Englander, S. W., & Wand, A. J. (1987) Biochemistry 26, 5953-5958] was applied. This approach relies on an ordered series of searches for prescribed patterns of connectivities in two-dimensional J-correlated and nuclear Overhauser effect spectra and centers on the dipolar interactions involving main-chain amide NH, alpha-CH, and beta-CH. Unlike the sequential assignment procedure, the MCD approach does not rest upon definition of side-chain J-coupled networks and is generally not sequential with the primary sequence of the protein. The various MCD patterns and the general algorithm are reiterated and applied to the analysis of human ubiquitin. With this algorithm, the vast majority of amino acid residue amide NH-C alpha H-C beta H J-coupled subspin systems could be associated with and aligned within units of secondary structure without any knowledge of the identity of the side chains. This greatly simplified recognition of side-chain spin systems by restricting their identity. Essentially complete resonance assignments are presented. The MCD method is compared with the sequential assignment method in some detail. The MCD method is highly amenable to automation. Human ubiquitin is found, at pH 5.8 and 30 degrees C, to be composed of an extensive beta-sheet structure involving five strands. Three of these strands form an antiparallel set sharing a common strand and have a parallel orientation to two antiparallel strands. Two helical segments were also observed. The largest, spanning 13 residues, shows dipolar interactions consistent with an alpha-helix while the smaller 4-residue helical segment appears, on the basis of observed nuclear Overhauser effects, to be a 3(10) helix. Five classical tight turns could be demonstrated.

A model for the solution structure of horse heart ferrocytochrome c has been determined by nuclear magnetic resonance spectroscopy combined with hybrid distance geometry-simulated annealing calculations. Forty-four highly refined structures were obtained using a total of 1940 distance constraints based on the observed magnitude of nuclear Overhauser effects and 85 torsional angle restraints based on the magnitude of determined J-coupling constants. The all-residue root mean square deviation about the average structure is 0.47 +/- 0.09 A for the backbone N, C alpha, and C' atoms and 0.91 +/- 0.07 A for all heavy atoms. The overall topology of the model for solution structure is very similar to that seen in previously reported models for crystal structures of homologous c-type cytochromes. However, a detailed comparison between the model for the solution structure and the available model for the crystal structure of tuna ferrocytochrome c indicates significant differences in a number of secondary and tertiary structural features. For example, two of the three main helices display 3(10) to alpha-helical transitions resulting in bifurcation of main-chain hydrogen bond acceptor carbonyls. The N- and C-terminal helices are tightly packed and display several interhelical interactions not seen in previously reported models. The geometry of heme ligation is well-defined and completely consistent with the crystal structures of homologous cytochromes c as are the locations of four of six structural water molecules. Though the total solvent-accessible surface area of the protoporphyrin ring is similar to that seen in crystal studies of tuna ferrocytochrome c, the distribution is somewhat different. This is mainly due to a difference in packing of residues Phe-82 and Ile-81 such that Ile-81 crosses the edge of the heme in the solution structure. These and other observations help to explain a range of physical and biological data spanning the redox properties, folding, molecular recognition, and stability of the protein.

Two-dimensional nuclear magnetic resonance spectroscopy (2D NMR) was used to obtain extensive resonance assignments in the 1H NMR spectrum of horse ferricytochrome c. Assignments were made for the main-chain and C beta protons of 102 residues (all except Pro-44 and Gly-84) and the majority of side-chain protons. As starting points for the assignment of the oxidized protein, a limited set of protons was initially assigned by use of 2D NMR magnetization transfer methods to correlate resonances in the oxidized form with assigned resonances in the reduced form [Wand, A. J., Di Stefano, D. L., Feng, Y., Roder, H., & Englander, S. W. (1989) Biochemistry (preceding paper in this issue)]. Given the complexity of the spectrum due to the size of this protein (104 residues) and its paramagnetic center, the initial search for side-chain spin systems in J-correlated spectra was successful only for the simplest side chains, but the majority of NH-C alpha H-C beta H subspin systems (NAB sets) could be identified at this stage. The subsequent search for sequential NOE connectivities focused on NAB sets, with use of previously assigned residues to place NOE-connected segments within the amino acid sequence. Selective proton labeling of either the slowly or the rapidly exchanging amide sites was used to simplify the spectra, and systematic work at two temperatures was used to resolve ambiguities in the 2D NMR spectra. These approaches, together with the use of magnetization transfer methods to correlate reduced and oxidized cytochrome c spectra, provide multiple cross-checks to verify assignments.

Reduced (GSH), oxidized (GSSG), and protein-bound (PSSG) glutsthione were-determined in dry and hydrated wheat embryos. Dry embryos contained-about 0.6 tkmoles per gramdry weight each of GSSG and PSSG, and thege levels declned 5-to lO4fold within minutes after the onset of imbibition. GSH declined from about 8 to 2pmoles per gram over a peiod of 90 minutes. Similar changes occurred when embryos were hydrated by storage at 100% relative humidity. The decline in glutathione levels was not reversed-upon redrying hydrated enbryos. About 40% of the cysteine residues of embryo protein was found to be in the disulfide form in both dry and inbibd embryos. he -ability of wheat embryos to withstandheat shock was shown to correlate with water content but not GSSG content.Incorporation of 135SImethlonine-into protein was studied using a systen based upon wheat embryo extract (S23). Incorportion rate was found to be snsitive-to the-nature of thiol added to the system and to be decreased. by GSSG. S23 exhibited a substantial capacity to reduce GSSG and preparation of S23 having a GSSG content comparable to dry embryos requlredladltlon of large amounts of GSSG to-the extraction buffer .823 prepared in this fashion exhibited a marked decrease in ability to suppo protein syn-thesis. These results suggest that the early decrease in GSSG. during germination is necessary for optimal protein synthesis in wheat embryo. This is one ofa series ofstudies undertaken to identify biological systems in which changes in the glutathione thiol-disulfide status (9) might be of potential importance in regulating biological activity. -In an earlier study (6) we demonstrated that GSSG3 levels are higher in the asexual spores (conidia) than in the vegetative cells of the fungus Neurospora crassa and that this difference disappears during the first minutes of conidial germination. P.arallel changes occur in the level of PSSG, suggesting that reversible conversion of protein thiol groups to disulfide forms might be involved as part of a control process. The initial objective of the present studies was to ascertain whether similar changes in glutat" thione thiol-disulfide status occur during germination of seed embryos. The results obtained with wheat and-barley embryos, proved generally similar to those obtained with N. crassa conidia.What role do such thiol-disulfide changes play in seed embryos?Since disulfide proteins generally exhibit greater thermal stability than proteins lacking disulfide bonds (12) confer thermal stability upon proteins. The second objective in the present study was to differentiate the effects of dehydration and thiol-disulfide changes upon thermal stability in seed embryos.Another possibility is that thiol-disulfide reactions are involved in the control of protein synthesis. It has been shown that GSSG inhibits the initiation of protein synthesis in cell-free extracts from rabbit reticulocytes (4, 10). Wheat embryos lack polysomes in the dry, quiescent state but polysomes appear shortly after initiation of germinat...

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