Multinuclear magnetic resonance studies of the 2Fe.2S* ferredoxin from Anabaena species strain PCC 7120. 1. Sequence-specific hydrogen-1 resonance assignments and secondary structure in solution of the oxidized form

Complete sequence-specific assignments were determined for the diamagnetic 1H resonances from Anabaena 7120 ferredoxin (Mr = 11,000). A novel assignment procedure was followed whose first step was the identification of the 13C spin systems of the amino acids by a 13C(13C) double quantum correlation experiment [Oh, B.-H., Westler, M. W., Darba, P., & Markley, J. L. (1988) Science 240, 908-911]. Then, the 1H spin systems of the amino acids were identified from the 13C spin systems by means of direct and relayed 1H(13C) single-bond correlations [Oh, B.-H., Westler, W. M., & Markley, J. L. (1989) J. Am. Chem. Soc. 111, 3083-3085]. The sequential resonance assignments were based mainly on conventional interresidue 1H alpha i-1HNi + 1 NOE connectivities. Resonances from 18 residues were not resolved in two-dimensional 1H NMR spectra. When these residues were mapped onto the X-ray crystal structure of the homologous ferredoxin from Spirulina platensis [Fukuyama, K., Hase, T., Matsumoto, S., Tsukihara, T., Katsube, Y., Tanaka, N., Kakudo, M., Wada, K., & Matsubara, H. (1980) Nature 286, 522-524], it was found that they correspond to amino acids close to the paramagnetic 2Fe.2S* cluster. Cross peaks in two-dimensional homonuclear 1H NMR spectra were not observed for any protons closer than about 7.8 A to both iron atoms. Secondary structural features identified in solution include two antiparallel beta-sheets, one parallel beta-sheet, and one alpha-helix.     

Refined X-ray structures of the oxidized, at 1.3 A, and reduced, at 1.17 A, [2Fe-2S] ferredoxin from the cyanobacterium Anabaena PCC7119 show redox-linked conformational changes

The chemical sequence of the [2Fe-2S] ferredoxin from the cyanobacterium AnabaenaPCC7119 (Fd7119) and its high-resolution X-ray structures in the oxidized and reduced states have been determined. The Fd7119 sequence is identical to that of the ferredoxin from the PCC7120 strain (Fd7120). X-ray diffraction data were collected at 100 K with an oxidized trigonal Fd7119 crystal, at 1.3 A resolution, and with an orthorhombic crystal, previously reduced with dithionite and flash frozen under anaerobic conditions, at 1.17 A resolution. The two molecular models were determined by molecular replacement with the [2Fe-2S] ferredoxin from the strain PCC7120 (Rypniewski, W. R., Breiter, D. R., Benning, M. M., Wesenberg, G., Oh, B.-H., Markley, J. L., Rayment, I., and Holden, H. M. (1991) Biochemistry 30, 4126-4131.) The final R-factors are 0. 140 (for the reduced crystal) and 0.138 (for the oxidized crystal). The [2Fe-2S] cluster appears as a significantly distorted lozenge in the reduced and oxidized redox states. The major conformational difference between the two redox forms concerns the peptide bond linking Cys46 and Ser47 which points its carbonyl oxygen away from the [2Fe-2S] cluster ("CO out") in the reduced molecule and toward it ("CO in") in the oxidized one. The "CO out" conformation could be the signature of the reduction of the iron atom Fe1, which is close to the molecular surface. Superposition of the three crystallographically independent molecules shows that the putative recognition site with the physiological partner (FNR) involves charged, hydrophobic residues and invariant water molecules.     

Multinuclear magnetic resonance studies of the 2Fe.2S* ferredoxin from Anabaena species strain PCC 7120. 2. Sequence-specific carbon-13 and nitrogen-15 resonance assignments of the oxidized form

Multinuclear two-dimensional NMR techniques were used to assign nearly all diamagnetic 13C and 15N resonances of the plant-type 2Fe.2S* ferredoxin from Anabaena sp. strain PCC 7120. Since a 13C spin system directed strategy had been used to identify the 1H spin systems [Oh, B.-H., Westler, W. M., & Markley, J. L. (1989) J. Am. Chem. Soc. 111, 3083-3085], the sequence-specific 1H assignments [Oh, B.-H., & Markley, J. L. (1990) Biochemistry (first paper of three in this issue)] also provided sequence-specific 13C assignments. Several resonances from 1H-13C groups were assigned independently of the 1H assignments by considering the distances between these nuclei and the paramagnetic 2Fe.2S* center. A 13C-15N correlation data set was used to assign additional carbonyl carbons and to analyze overlapping regions of the 13C-13C correlation spectrum. Sequence-specific assignments of backbone and side-chain nitrogens were based on 1H-15N and 13C-15N correlations obtained from various two-dimensional NMR experiments.     

Multinuclear magnetic resonance studies of the 2Fe.2S* ferredoxin from Anabaena species strain PCC 7120. 3. Detection and characterization of hyperfine-shifted nitrogen-15 and hydrogen-1 resonances of the oxidized form

All the nitrogen signals from the amino acid side chains and 80 of the total of 98 backbone nitrogen signals of the oxidized form of the 2Fe.2S* ferredoxin from Anabaena sp. strain PCC 7120 were assigned by means of a series of heteronuclear two-dimensional experiments [Oh, B.-H. Mooberry, E. S., & Markley, J. L. (1990) Biochemistry (second paper of three in this issue )]. Two additional nitrogen signals were observed in the one-dimensional 15N NMR spectrum and classified as backbone amide resonances from residues whose proton resonances experience paramagnetic broadening. The one-dimensional 15N NMR spectrum shows nine resonances that are hyperfine shifted and broadened. From this inventory of diamagnetic nitrogen signals and the available X-ray coordinates of a related ferredoxin [Tsukihara, T., Fukuyama, K., Nakamura, M., Katsube, Y., Tanaka, N., Kakudo, M., Wada, K., Hase, T., & Matsubara, H. (1981) J. Biochem. 90, 1763-1773], the resolved hyperfine-shifted 15N peaks were attributed to backbone amide nitrogens of the nine amino acids that share electrons with the 2Fe.2S* center or to backbone amide nitrogens of two other amino acids that are close to the 2Fe.2S* center. The seven 15N signals that are missing and unaccounted for probably are buried under the envelope of amide signals. 1H NMR signals from all the amide protons directly bonded to the seven missing and nine hyperfine-shifted nitrogens were too broad to be resolved in conventional 2D NMR spectra.(ABSTRACT TRUNCATED AT 250 WORDS)     

1H NMR spectra of vertebrate [2Fe-2S] ferredoxins. Hyperfine resonances suggest different electron delocalization patterns from plant ferredoxins

We report the observation of paramagnetically shifted (hyperfine) proton resonances from vertebrate mitochondrial [2Fe-2S] ferredoxins. The hyperfine signals of human, bovine, and chick [2Fe-2S] ferredoxins are described and compared with those of Anabaena 7120 vegetative ferredoxin, a plant-type [2Fe-2S] ferredoxin studied previously [Skjeldal, L., Westler, W. M., & Markley, J. L. (1990) Arch. Biochem. Biophys. 278, 482-485]. The hyperfine resonances of the three vertebrate ferredoxins were very similar to one another both in the oxidized state and in the reduced state, and slow (on the NMR scale) electron self-exchange was observed in partially reduced samples. For the oxidized vertebrate ferredoxins, hyperfine signals were observed downfield of the diamagnetic envelope from +13 to +50 ppm, and the general pattern of peaks and their anti-Curie temperature dependence are similar to those observed for the oxidized plant-type ferredoxins. For the reduced vertebrate ferredoxins, hyperfine signals were observed both upfield (-2 to -18 ppm) and downfield (+15 to +45 ppm), and all were found to exhibit Curie-type temperature dependence. This pattern and temperature dependence are distinctly different from those found with reduced plant-type ferredoxins which have signal centered around +120 ppm with Curie-type temperature dependence, assigned to cysteines which interact with Fe(III), and signals centered around +20 ppm with anti-Curie temperature dependence, assigned to cysteines which interact with Fe(II) [Dugad, L. B., La Mar, G. N., Banci, L., & Bertini, I. (1990) Biochemistry 29, 2263-2271].(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystallization and preliminary analysis of oxidized, recombinant, heterocyst [2Fe-2S] ferredoxin from Anabaena 7120.

The [2Fe-2S] ferredoxin produced in the heterocyst cells of Anabaena 7120 plays a key role in nitrogen fixation, where it serves as an electron acceptor from various sources and an electron donor to nitrogenase. Crystals of recombinant heterocyst ferredoxin, coded for by the fdx H gene from Anabaena 7120 and overproduced in Escherichia coli, have been grown from ammonium sulfate solutions and are suitable for high resolution X-ray crystallographic analysis. They belong to the hexagonal space group P6(1) or P6(5) with unit cell dimensions of a = b = 44.2 A and c = 80.6 A. The crystals contain one molecule per asymmetric unit and diffract to a nominal resolution of 1.6 A. The molecular structure of this heterocyst ferredoxin is of special interest in that 4 of the 22 amino acid positions thought to be absolutely conserved in nonhalophilic ferredoxins are different and, based on amino acid sequence alignments, three of these positions are located in the metal-cluster binding loop. Consequently, a high-resolution X-ray analysis of this [2Fe-2S] ferredoxin, and subsequent three-dimensional comparisons with other known ferredoxin models, will provide new insight into structure/function relationships for this class of redox proteins.     

Detection and characterization of hyperfine-shifted resonances in the proton nuclear magnetic resonance spectrum of Anabaena 7120 ferredoxin at high magnetic fields

This paper presents previously unobserved signals in the 1H NMR spectra of oxidized and reduced [2Fe-2S]-ferredoxin from Anabaena 7120 detected at 400, 500, and 600 MHz. The signals shifted to low field exhibited longitudinal relaxation (T1) values in the range of 100-400 microseconds and line widths in the range of 1-10 kHz (at 400 MHz), and the chemical shifts of all signals showed strong temperature dependence. Although the line widths were smaller at lower magnetic fields, the resolution was better at higher magnetic fields. In the oxidized state, a broad signal was detected at 37 ppm, which corresponds to at least 6 protons, and whose chemical shift exhibits positive temperature dependence. This signal also was found in oxidized ferredoxin reconstituted in 2H2O, which excludes the signal as arising from solvent-exchangeable amide protons. In the reduced state, four signals detected between 90 and 140 ppm exhibited negative temperature dependence. These consisted of two pairs of signals, each pair having one component with half the linewidth of the other. On the basis of their chemical shifts, linewidths, longitudinal relaxation properties, and temperature dependence we assigned these resonances to four of the beta hydrogens of the ligated cysteines. Two solvent-exchangeable hyperfine-shifted signals were found in the reduced state; these are located upfield of the diamagnetic region. The low-field hyperfine resonances of half-reduced ferredoxin in the presence of sodium dithionite showed a self electron transfer exchange rate that was slow on the NMR scale as observed earlier (Chan, T., and Markley, J. L. (1983) Biochemistry 22, 5982-5987), but the exchange rate was accelerated in the presence of methyl viologen.     

Oxygen disruption of the 2[4Fe-4S] clusters in Clostridium pasteurianum ferredoxin shown by 1H-NMR.

The ferredoxin from Clostridium pasteurianum, which contains two [4Fe-4S] clusters, was investigated in its oxidized and reduced states by two-dimensional (2D) (1)H-(1)H nuclear Overhauser enhancement spectroscopy (NOESY). Comparison of the data from the oxidized ferredoxin with those published previously revealed the same NOE connectivities. No previous (1)H-(1)H NOESY study of the fully reduced ferredoxin has previously been published. However, it was possible to compare our results with those of a 2D exchange spectroscopy investigation of half-reduced C. pasteurianum ferredoxin. The present results with reduced C. pasteurianum ferredoxin confirm many of the (1)H peaks and NOE interactions reported earlier, revise others, and locate resonances previously undetected. When the ferredoxin was slightly exposed to oxygen, several of the hyperfine shifted resonances were irreversibly influenced. A resonance at 34 ppm in the (1)H NMR spectra of both redox states is indicative of oxygen exposure. These results indicate the importance of keeping the ferredoxin strictly anaerobic during purification and solvent exchange.     

Resonance Raman and magnetic circular dichroism studies of reduced [2Fe-2S] proteins.

The structural and electronic properties of the [2Fe-2S] clusters in reduced putidaredoxin, Spinacea oleracea ferredoxin, and Clostridium pasteurianum [2Fe-2S] ferredoxin have been investigated by resonance Raman and variable temperature magnetic circular dichroism spectroscopies. Both techniques are shown to provide diagnostic fingerprints for identifying [2Fe-2S]+ clusters in more complex multicomponent metalloenzymes. The Fe-S stretching modes of oxidized and reduced putidaredoxin are assigned via 34S and D2O isotope shifts and previous normal mode calculations for adrenodoxin (Han, S., Czernuszewicz, R. S., Kimura, T., Adams, M. W. W., and Spiro, T. G. (1989) J. Am. Chem. Soc. 111, 3505-3511). The close similarity in the resonance Raman spectra of reduced [2Fe-2S] centers, in terms of both the vibrational frequencies and enhancement profiles of the Fe-S stretching modes, permits these assignments to be generalized to all clusters of this type. Modes primarily involving Fe(III)-S(Cys) stretching are identified in all three reduced [2Fe-2S] proteins, and the frequencies are rationalized in terms of the conformation of the cysteine residues ligating the Fe(III) site of the localized valence reduced cluster. D2O isotope shifts indicate few, if any, amide NH-S hydrogen bond interactions involving the cysteines ligating the Fe(III) site. Preliminary resonance Raman excitation profiles suggest assignments for the complex pattern of electronic bands that comprise the low temperature magnetic circular dichroism spectra of the reduced proteins. S----Fe(III) and Fe(II)----S charge transfer, Fe d-d, and Fe(II)----Fe(III) intervalence bands are identified.     

Engineering a three-cysteine, one-histidine ligand environment into a new hyperthermophilic archaeal Rieske-type [2Fe-2S] ferredoxin from Sulfolobus solfataricus

We heterologously overproduced a hyperthermostable archaeal low potential (E(m) = -62 mV) Rieske-type ferredoxin (ARF) from Sulfolobus solfataricus strain P-1 and its variants in Escherichia coli to examine the influence of ligand substitutions on the properties of the [2Fe-2S] cluster. While two cysteine ligand residues (Cys(42) and Cys(61)) are essential for the cluster assembly and/or stability, the contributions of the two histidine ligands to the cluster assembly in the archaeal Rieske-type ferredoxin appear to be inequivalent as indicated by much higher stability of the His(64) --> Cys variant (H64C) than the His(44) --> Cys variant (H44C). The x-ray absorption and resonance Raman spectra of the H64C variant firmly established the formation of a novel, oxidized [2Fe-2S] cluster with one histidine and three cysteine ligands in the archaeal Rieske-type protein moiety. Comparative resonance Raman features of the wild-type, natural abundance and uniformly (15)N-labeled ARF and its H64C variant showed significant mixing of the Fe-S and Fe-N stretching characters for an oxidized biological [2Fe-2S] cluster with partial histidine ligation.     

Potential ligands to the [2Fe-2S] Rieske cluster of the cytochrome bc1 complex of Rhodobacter capsulatus probed by site-directed mutagenesis.

The Rieske protein of the ubiquinol-cytochrome c oxidoreductase (bc1 complex or b6f complex) contains a [2Fe-2S] cluster which is thought to be bound to the protein via two nitrogen and two sulfur ligands [Britt, R. D., Sauer, K., Klein, M. P., Knaff, D. B., Kriauciunas, A., Yu, C.-A., Yu, L., & Malkin, R. (1991) Biochemistry 30, 1892-1901; Gurbiel, R. J., Ohnishi, T., Robertson, D. E., Daldal, F., & Hoffman, B. M. (1991) Biochemistry 30, 11579-11584]. All available Rieske amino acid sequences have carboxyl termini featuring two conserved regions containing four cysteine (Cys) and two or three histidine (His) residues. Site-directed mutagenesis was applied to the Rieske protein of the photosynthetic bacterium Rhodobacter capsulatus, and the mutants obtained were studied biochemically in order to identify which of these conserved residues are the ligands of the [2Fe-2S] cluster. It was found that His159 (in the R. capsulatus numbering) is not a ligand and that the presence of the Rieske protein in the intracytoplasmic membrane is greatly decreased by alteration of any of the remaining six His or Cys residues. Among these mutations, only the substitution Cys155 to Ser resulted in the synthesis of Rieske protein (in a small amount) which contained a [2Fe-2S] cluster with altered biophysical properties. This finding suggested that Cys155 is not a ligand to the cluster. A comparison of the conserved regions of the Rieske proteins with bacterial aromatic dioxygenases (which contain a spectrally and electrochemically similar [2Fe-2S] cluster) indicated that Cys133, His135, Cys153, and His156 are conserved in both groups of enzymes, possibly as ligands to their [2Fe-2S] clusters. These findings led to the proposal that Cys138 and Cys155, which are not conserved in bacterial dioxygenases, may form an internal disulfide bond which is important for the structure of the Rieske protein and the conformation of the quinol oxidation (Qo) site of the bc1 complex.     

Electron-nuclear interactions in two prototypical [2Fe-2S] proteins: selective (chiral) deuteration and analysis of (1)H and (2)H NMR signals from the alpha and beta hydrogens of cysteinyl residues that ligate the iron in the active sites of human ferredoxin and Anabaena 7120 vegetative ferredoxin

A vertebrate ferredoxin (human ferredoxin) and a plant-type ferredoxin (the ferredoxin from the vegetative form of Anabaena 7120) were labeled selectively with deuterium at their active site cysteines. The recombinant proteins were produced in Escherichia coli and labeled by replacing natural abundance cysteine in the defined culture medium with (2)H(alpha)-cysteine, (2)H(beta2), (2)H(beta3)-cysteine, or (2)H(beta2)-cystine. The chiral labeled cystine ((2)H(beta2)-cystine) was prepared by selective hydrogen exchange catalyzed by cystathionine gamma-synthase. NMR spectra of these samples in their oxidized and reduced states support unambiguous identifications by atom type of (1)H and (2)H NMR signals from the cysteine alpha and beta hydrogens. These signals lie outside the normal diamagnetic spectral region as a result of interaction of the hydrogens with unpaired electron density from the iron-sulfur cluster, and their chemical shifts are highly dependent on local conformation at the active site. The very different chemical properties of the iron centers of plant-type and vertebrate ferredoxins reflect relatively small differences in the conformation of the iron-sulfur cluster ligands.     

Electrostatic effects in electron transfer reactions of [2Fe-2S] ferredoxins with inorganic reagents.

The kinetics of electron transfer from the reduced [2Fe-2S] ferredoxins from the cyanobacterium Anabaena 7120 and the protozoan Trichomonas vaginalis to select cobalt coordination compounds have been studied in order to gain insight into the mechanism of electron transfer and intrinsic reactivity of [2Fe-2S] active sites. With tripositive cobalt complexes, reactions of both proteins displayed saturation kinetics; values of association constants of 12,900 and 1,400 M-1 and limiting rate constants of 7.6 and 3.5 s-1 were found for oxidation of T. vaginalis and Anabaena ferredoxins, respectively, by Co(NH3)6(3+) at room temperature and I = 0.1 M. An activation enthalpy of 12.1 kcal/mol and activation entropy of -14.3 cal/mol K for oxidation of T. vaginalis ferredoxin by Co(NH3)6(3+) contrasted with corresponding values of 13.4 kcal/mol and -10.5 cal/mol K for the Spirulina platensis protein, which is homologous to Anabaena ferredoxin. The dependence of the reaction rates on ionic strength were measured to probe the importance of electrostatics on the reactivity of the proteins. Analysis of the ionic strength dependence of the oxidation of the proteins by Co(NH3)6(3+) by the "parallel plate" model of Watkins et al. (1994, Protein Sci 3:2104-2114) afforded values for active site charges of -0.7 and -1.1 and limiting rate constants at infinite ionic strength of 25,800 and 76 M-1 S-1 for T. vaginalis and Anabaena ferredoxins, respectively. These results suggest that the [2Fe-2S] center of the protozoal ferredoxin is more accessible and adjacent to a less highly charged, more compact patch of negative charges than the photosynthetic protein.     

Flavodoxin from Anabaena 7120: uniform nitrogen-15 enrichment and hydrogen-1, nitrogen-15, and phosphorus-31 NMR investigations of the flavin mononucleotide binding site in the reduced and oxidized states

Interactions between flavin mononucleotide (FMN) and apoprotein have been investigated in the reduced and oxidized states of the flavodoxin isolated from Anabaena 7120 (Mr approximately 21,000). 1H, 15N, and 31P NMR have been used to characterize the FMN-protein interactions in both redox states. These are compared with those seen in other flavodoxins. Uniformly enriched [15N]flavodoxin (greater than 95% isotopic purity) was isolated from Anabaena 7120 grown on K15NO3 as the sole nitrogen source. 15N insensitive nucleus enhanced by polarization transfer (INEPT) and nuclear Overhauser effect (NOE) studies of this sample provided information regarding protein structure and dynamics. A 1H-detected 15N experiment allowed the correlation of nitrogen resonances to those of their attached protons. Over 90% of the expected N-H cross peaks could be resolved in this experiment.     

Crystallization and structure determination to 2.5-A resolution of the oxidized [2Fe-2S] ferredoxin isolated from Anabaena 7120

The molecular structure of the oxidized form of the [2Fe-2S] ferredoxin isolated from the cyanobacterium Anabaena species strain PCC 7120 has been determined by X-ray diffraction analysis to a nominal resolution of 2.5 A and refined to a crystallographic R factor of 18.7%. Crystals used in this investigation belong to the space group P2(1)2(1)2(1) with unit cell dimensions of a = 37.42 A, b = 38.12 A, and c = 147.12 A and two molecules in the asymmetric unit. The three-dimensional structure of this ferredoxin was solved by a method that combined X-ray data from one isomorphous heavy-atom derivative with noncrystallographic symmetry averaging and solvent flattening. As in other plant-type [2Fe-2S] ferredoxins, the iron-sulfur cluster is located toward the outer edge of the molecule, and the irons are tetrahedrally coordinated by both inorganic sulfurs and sulfurs provided by protein cysteine residues. The main secondary structural elements include four strands of beta-pleated sheet and three alpha-helical regions.     

Two-dimensional NMR studies of staphylococcal nuclease. 2. Sequence-specific assignments of carbon-13 and nitrogen-15 signals from the nuclease H124L-thymidine 3',5'-bisphosphate-Ca2+ ternary complex

Samples of staphylococcal nuclease H124L (cloned protein overproduced in Escherichia coli whose sequence is identical with that of the nuclease isolated from the V8 strain of Staphylococcus aureus) were labeled uniformly with carbon-13 (26% ul 13C), uniformly with nitrogen-15 (95% ul 15N), and specifically by incorporating nitrogen-15-labeled leucine ([98% 15N]Leu) or carbon-13-labeled lysine ([26% ul 13C]Lys), arginine ([26% ul 13C]Arg), or methionine ([26% ul 13C]Met). Solutions of the ternary complexes of these analogues (nuclease H124L-pdTp-Ca2+) at pH 5.1 (H2O) or pH* 5.5 (2H2O) at 45 degrees C were analyzed as appropriate to the labeling pattern by multinuclear two-dimensional (2D) NMR experiments at spectrometer fields of 14.09 and 11.74 T: 1H-13C single-bond correlation (1H[13C]SBC); 1H-13C single-bond correlation with NOE relay (1H[13C]SBC-NOE); 1H-13C single-bond correlation with Hartmann-Hahn relay (1H-[13C]SBC-HH); 1H-13C multiple-bond correlation (1H[13C]MBC); 1H-15N single-bond correlation (1H-[15N]SBC); 1H-15N single-bond correlation with NOE relay (1H[15N]SBC-NOE). The results have assisted in spin system assignments and in identification of secondary structural elements. Nuclear Overhauser enhancements (NOE's) characteristic of antiparallel beta-sheet (d alpha alpha NOE's) were observed in the 1H [13C]-SBC-NOE spectrum of the nuclease ternary complex labeled uniformly with 13C. NOE's characteristic of alpha-helix (dNN NOE's) were observed in the 1H[15N]SBC-NOE spectrum of the complex prepared from protein labeled uniformly with 15N. The assignments obtained from these multinuclear NMR studies have confirmed and extended assignments based on 1H[1H] 2D NMR experiments [Wang, J., LeMaster, D. M., & Markley, J. L. (1990) Biochemistry (preceding paper in this issue)].     

Solution NMR characterization of the thermodynamics of the disulfide bond orientational isomerism and its effect of cluster electronic properties for the hyperthermostable three-iron cluster ferredoxin from the archaeon Pyrococcus furiosus

The thermodynamics and dynamics of the Cys21-Cys48 disulfide "S" if "R" conformational isomerism in the three-iron, single cubane cluster ferredoxin (Fd) from the hyperthermophilic archaeon Pyrococcus furiosus (Pf) have been characterized by (1)H NMR spectroscopy in both water and water/methanol mixed solvents. The mean interconversion rate at 25 degrees C is 3 x 10(3) s(-1) and DeltaG(298) = -0.2 kcal/mol [DeltaH = 4.0 kcal/mol; DeltaS = 14 cal/(mol.K)], with the S orientation as the more stable form at low temperature (< 0 degrees C) but the R orientation predominating at >100 degrees C, where the organism thrives. The distinct pattern of ligated Cys beta-proton contact shifts for the resolved signals and their characteristic temperature behavior for the forms of the 3Fe Fd with alternate disulfide orientations have been analyzed to determine the influences of disulfide orientation and methanol cosolvent on the topology of the inter-iron spin coupling in the 3Fe cluster. The Cys21-Cys48 disulfide orientation influences primarily the spin couplings involving the iron ligated to Cys17, whose carbonyl oxygen is a hydrogen bond acceptor to the Cys21 peptide proton. Comparison of the Cys beta-proton contact shift pattern for the alternate disulfide orientations with the pattern exhibited upon cleaving the disulfide bridge confirms an earlier [Wang, P.-L., Calzolai, L., Bren, K. L., Teng, Q., Jenney, F. E., Jr., Brereton, P. S., Howard, J. B., Adams, M. W. W., and La Mar, G. N. (1999) Biochemistry 38, 8167-8178] proposal that the structure of the same Fd with the R disulfide orientation resembles that of the Fd upon cleaving the disulfide bond.     

Application of automated NOE assignment to three-dimensional structure refinement of a 28 kDa single-chain T cell receptor

An automated procedure for NOE assignment and three-dimensional structure refinement is presented. The input to the procedure consists of (1) an ensemble of preliminary protein NMR structures, (2) partial sequence-specific assignments for the protein and (3) the positions and volumes of unassigned NOESY cross peaks. Chemical shifts for unassigned side chain protons are predicted from the preliminary structures. The chemical shifts and unassigned NOESY cross peaks are input to an automated procedure for NOE assignment and structure calculation (ARIA) [Nilges et al. (1997) J. Mol. Biol., 269, 408–422]. ARIA is optimized for the task of structure refinement of larger proteins. Errors are filtered to ensure that sequence-specific assignments are reliable. The procedure is applied to the 27.8 kDa single-chain T cell receptor (scTCR). Preliminary NMR structures, nearly complete backbone assignments, partial assignments of side chain protons and more than 1300 unassigned NOESY cross peaks are input. Using the procedure, the resonant frequencies of more than 40 additional side chain protons are assigned. Over 400 new NOE cross peaks are assigned unambiguously. Distances derived from the automatically assigned NOEs improve the precision and quality of calculated scTCR structures. In the refined structures, a hydrophobic cluster of side chains on the scTCR surface that binds major histocompatibility complex (MHC)/antigen is revealed. It is composed of the side chains of residues from three loops and stabilizes the conformation of residues that interact with MHC.     

Two-dimensional NMR studies of staphylococcal nuclease: evidence for conformational heterogeneity from hydrogen-1, carbon-13, and nitrogen-15 spin system assignments of the aromatic amino acids in the nuclease H124L-thymidine 3',5'-bisphosphate-Ca2+ ternary complex

A combination of multinuclear two-dimensional NMR experiments served to identify and assign the combined 1H, 13C, and 15N spin systems of the single tryptophan, three phenylalanines, three histidines, and seven tyrosines of staphylococcal nuclease H124L in its ternary complex with calcium and thymidine 3',5'-bisphosphate at pH 5.1 (H2O) or pH 5.5 (2H2O). Samples of recombinant nuclease were labeled with 13C or 15N as appropriate to individual NMR experiments: uniformly with 15N (all sites to greater than 95%), uniformly with 13C (all sites to 26%), selectively with 13C (single amino acids uniformly labeled to 26%), or selectively with 15N (single amino acids uniformly labeled to greater than 95%). NMR data used in the analysis included single-bond and multiple-bond 1H-13C and multiple-bond 1H-15N correlations, 1H-13C single-bond correlation with Hartmann-Hahn relay (1H[13C]SBC-HH), and 1H-13C single-bond correlation with NOE relay (1H[13C]SBC-NOE). The aromatic protons of the spin systems were identified from 1H[13C]SBC-HH data, and the nonprotonated aromatic ring carbons were identified from 1H-13C multiple-bond correlations. Sequence-specific assignments were made on the basis of observed NOE relay connectivities between assigned 1H alpha-13C alpha or 1H beta-13C beta direct cross peaks in the aliphatic region [Wang, J., LeMaster, D. M., & Markley, J. L. (1990) Biochemistry 29, 88-101] and 1H delta-13C delta direct cross peaks in the aromatic region of the 1H[13C]SBC-NOE spectrum. The His121 1H delta 2 resonance, which has an unusual upfield shift (at 4.3 ppm in the aliphatic region), was assigned from 1H[13C]SBC, 1H[13C]MBC, and 1H[15N]MBC data. Evidence for local structural heterogeneity in the ternary complex was provided by doubled peaks assigned to His46, one tyrosine, and one phenylalanine. Measurement of NOE buildup rates between protons on different aromatic residues of the major ternary complex species yielded a number of interproton distances that could be compared with those from X-ray structures of the wild-type nuclease ternary complex with calcium and thymidine 3',5'-bisphosphate [Cotton, F. A., Hazen, E. E., Jr., & Legg, M. J. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 2551-2555; Loll, P. J., & Lattman, E. E. (1989) Proteins: Struct., Funct., Genet. 5, 183-201]. The unusual chemical shift of His121 1H delta 2 is consistent with ring current calculations from either X-ray structure.