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The backbone dihedral angle φ in polypeptides is characterized by four different J couplings:  3 J H N H α , 3 J H N C ‘, 3 J H N C β , and 3 J H α C ‘. E.COSY and quantitative J correlation techniques have been used to measure these couplings in the protein human ubiquitin, uniformly enriched in 13C and 15N. Assuming that the dihedral backbone angles in solution are identical to those in the X-ray structure of this protein and that HN is located in the C‘−N−Cα plane, Karplus relations for 3 J H N H α , 3 J H α C ‘, and 3 J H N C β , have been reparametrized. The root-mean-square (rms) difference between measured values of 3 J H N H α , 3 J H α C ‘, 3 J H N C β , and 3 J H N C ‘ and their corresponding Karplus curves are 0.53, 0.25, 0.24, and 0.36 Hz, respectively, whereas the precision of these measurements is considerably better. For any given residue, the differences between the four measured J couplings and values predicted by their Karplus curves on the basis of the X-ray structure-derived φ angle are highly correlated with one another. On average, a root-mean-square change of 5.7° in the X-ray derived φ angles is needed to obtain optimal agreement with all four measured J couplings. There is no clear correlation between the φ angle correction needed and the out-of-plane position of the amide proton predicted by ab initio calculations. The small differences in φ angles therefore presumably result from small uncertainties in the atomic positions of the 1.8 Å X-ray structure. However, they may also be caused by genuine differences between the structure of the protein in solution and in the crystalline state or contain a contribution resulting from deviations from the assumption that the HN−N−Cα−Hα dihedral angle equals φ − 60°.

The toxin 3-nitropropionic acid 5 is produced by certain plants and fungi. It is a specific inhibitor of mitochondrial respiratory complex II. Fatalities after eating moldy sugarcane have been linked to 3-NP toxicity (1, 2). Ruminants grazing in regions with 3-NP-producing plants acquire resistance because of reduction of the nitro group to an amine by ruminal bacteria (3).The effectiveness of 3-NP in vivo after injection or oral administration has made it useful in studies involving tissues or whole animals. Ingestion of 3-NP results in neurodegeneration with symptoms resembling those of Huntington disease (4), and conversely Huntington disease results in a loss of complex II activity (5); thus 3-NP has been used to produce an animal model for the study of Huntington disease (6, 7). Symptoms also include convulsions, and 3-NP is being looked at for inducing a model of epilepsy (8). Prior subacute 3-NP poisoning seems to provide resistance to ischemic damage to nervous tissue by a preconditioning effect (9) similar to that resulting from mild ischemia.The target of 3-NP is Complex II, which is both a member of the Krebs tricarboxylic acid cycle (oxidizing succinate to fumarate) and an entry point for electrons into the respiratory chain at the level of ubiquinol. It consists of a large flavoprotein subunit containing covalently bound FAD, an iron-sulfur protein (IP) with three different iron-sulfur clusters, and two small membrane anchor subunits (chains C and D) ligating a single low spin heme of type B. Human genetic defects in the IP subunits or chains C or D lead to development of paragangliomas (10, 11). A mutation in chain C leads to premature aging in nematodes, presumably through excessive production of free radicals (12). Bacterial homologs succinate:quinone oxidoreductase (SQR) and menaquinol: fumarate oxidoreductase (FRD) in Escherichia coli have been studied as genetically manipulable models for the mitochondrial protein. Recent reviews cover this family of enzymes (13-18). X-ray crystallographic structures are available for a number of members of the family. The available mitochondrial structures and representative bacterial examples are listed in Table 1.The toxin 3-NP, structurally similar to and isoelectronic with the substrate succinate, is believed to be a suicide inactivator of Complex II. Alston et al. (19) proposed, based on previous observations and on their own experience with another flavoprotein, that the normal reaction pathway involves a temporary adduct with the N-5 nitrogen of flavin, which in the case of 3-NP collapses to a stable adduct resulting in permanent inactivation. Irreversible covalent modification of the flavin was ruled out by later work (20) examining the spectral change induced and showing that unmodified flavin peptide could be isolated from the inactivated complex by mild proteolysis. It was proposed that 3-NP is oxidized to 3-nitroacrylate, an unstable molecule that then reacts with some residue in the active site. A cysteine that was believed to be in the active ...

The utility of three-bond Jcouplings for determining backbone and side-chain conformation in peptides and proteins has long been established.' Historically, the focus has been primarily on three-bond 1HJH couplings to obtain information on the dihedral backbone angle 4 and the side-chain angle XI, but measurement of heteronuclear 3 J c~ and 3 J~~ couplings is becoming increasingly popular.2-I'J Recently, measurement of 3 J~~ to methyl carbons in proteins was also demonstrated to be feasible," facilitating the stereospecific assignment of the methyl groups of valines and leucines and providing information about the side-chain x1 (Val, Ile, Thr) and x2 (Leu) angles.'* Here, an experiment is described that provides quantitative information on the Jcoupling between methyl carbons and backbone amide 15N nuclei. Together with the measurement of ~J c c , this allows one to determine the x1 angle in valine, isoleucine, and threonine residues.Most methods available to date for measurement of unresolved Jcouplings in proteins rely on the E.COSY principle,13 in which the Jcoupling is measured from the frequency difference between two resonances in a 2D or 3D spectrum. More recently, a different approach for measurement of unresolved Jcouplings was proposed which relies on quantitating coherence transfer" or measuring the magnetization loss due to dephasing caused by the unresolved Jc0up1ing.l~ As will be demonstrated below, this approach is also very well suited for measurement of small unresolvable 3 J~~ couplings in proteins.The method described here is essentially a 2D difference experiment, and the pulse scheme is sketched in Figure 1. When the 15N 180° pulse is applied in position a, the scheme is identical to the constant-time 1HJ3C correlation experiment (CT-HSQC) described elsewhere.15-1' The effect of one-bond 13C-13C J couplings during the constant-time evolution period is suppressed by adjusting the duration (277 of the constant-time evolution (1) Bystrov, V. F. Prog. Nucl. Magn. Reson. Spectrosc. 1976,10,41-81. (2) Montelione, G. T.; Winkler, M. E.; Rauenbiihler, P.; Wagner, G. J. (3) Wider, G.; Neri, D.; Otting, G.; Wiithrich, K. J. Magn. Reson. 1989, Magn. Reson. 1989, 82, 198-204. 85. 426-43 I . --. (4) Kurz, M.; Schmieder, P.; Kessler, H . Angew. Chem., Int. Ed. Engl. ( 5 ) Edison, A. S.; Westler, W. M.; Markley, J. L. (10) Eggenberger, U.; Karimi-Nejad, Y.; Thiiring, H.; Riiterjans, H.; (11) Bax,A.; Max, D.; Zax, D. J. Am. Chem. SOC. 1992,114,6924-6925. (12) Powers, R.; Garrett, D. S.; March, C. J.; Frieden, E. A.; Gronenborn, (13) Griesinger, C.; Sarensen, 0. W.; Ernst, R. R. A. M.; Clore, G. M. Biochemistry, in press. 85,6837-6852. Z. H.; Bax, A. J. Biomol. NMR 1992, 2, 527-533. 637-644.

It is demonstrated that sequential resonance assignment of the backbone 1H alpha and 15N resonances of proteins can be obtained without recourse to the backbone amide protons, an approach which should be useful for assignment of regions with rapidly exchanging backbone amide protons and for proteins rich in proline residues. The method relies on the combined use of two 2D experiments, HA(CA)N and HA(CACO)N or their 3D analogs, which correlate 1H alpha with the intraresidue 15N and with the 15N resonance of the next residue. The experiments are preferably conducted in D2O, where very high resolution in the 15N dimension can be achieved by using 2H decoupling. The approach is demonstrated for a sample of human ubiquitin, uniformly enriched in 13C and 15N. Complete backbone and 13C beta/1H beta resonance assignments are presented.

Application of radio-frequency power in multidimensional NMR experiments can significantly increase the sample temperature compared to that of the surrounding gas flow. Radio-frequency heating effects become more severe at higher magnetic field strengths and ionic strengths. The effects are particularly noticeable for experiments that utilize 1H and/or 13C isotropic mixing and broadband decoupling. If radio-frequency power is applied during the systematically increasing evolution period t1, the sample temperature can change with t1 and thereby cause line-shape distortions. Such distortions are easily avoided by ensuring that the average radio-frequency power remains constant during the entire experiment.

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Mitochondrial Complex II (succinate:ubiquinone oxidoreductase) is purified in a partially inactivated state, which can be activated by removal of tightly bound oxaloacetate (E.B. Kearney, et al., Biochem. Biophys. Res. Commun. 49 1115-1121). We crystallized Complex II in the presence of oxaloacetate or with the endogenous inhibitor bound. The structure showed a ligand essentially identical to the "malate-like intermediate" found in Shewanella Flavocytochrome c crystallized with fumarate (P. Taylor, et al., Nat. Struct. Biol. 6 1108-1112) Crystallization of Complex II in the presence of excess fumarate also gave the malate-like intermediate or a mixture of that and fumarate at the active site. In order to more conveniently monitor the occupation state of the dicarboxylate site, we are developing a library of UV/Vis spectral effects induced by binding different ligands to the site. Treatment with fumarate results in rapid development of the fumarate difference spectrum and then a very slow conversion into a species spectrally similar to the OAA-liganded complex. Complex II is known to be capable of oxidizing malate to the enol form of oxaloacetate (Y.O. Belikova, et al., Biochim. Biophys. Acta 936 1-9). The observations above suggest it may also be capable of interconverting fumarate and malate. It may be useful for understanding the mechanism and regulation of the enzyme to identify the malate-like intermediate and its pathway of formation from oxaloacetate or fumarate.