The solution structure of parsley [2Fe-2S]ferredoxin

The properties of Helicobacter pylori arginase activity in metabolically competent cells and lysates were investigated with the aim of obtaining a better understanding of the nitrogen metabolism of the bacterium. One-dimensional 1H- and 13C-nuclear magnetic resonance spectroscopy, spectrophotometry, radio tracer analysis and protein purification techniques were employed to characterize in situ the first step in the utilization of l-arginine by the bacterium. Arginase activity was associated with the cell-envelope fraction obtained by centrifugation of lysates. A Km of 22+/-3 mM was determined for the enzyme activity, and differences of Vmax were observed between strains. Divalent cations stimulated arginase activity, and the most potent activators were Co2+>Ni2+>Mn2+. The activity was highly specific for l-arginine and did not catabolize analogs recognized by other arginases of prokaryote and eukaryote origin. The Ki of several inhibitors was measured and served also to characterize the enzyme activity. The presence of bicarbonate enhanced the hydrolysis of l-arginine in cell suspensions, but not in lysates or semi-purified enzyme preparations. Amino acid sequence analyses revealed important differences between the deduced structures of H. pylori arginase and those of other organisms. This finding was consistent with experimental data which showed that H. pylori arginase has unique properties.     

Solution structure of the oxidized 2[4Fe-4S] ferredoxin from Clostridium pasteurianum

Following the recently developed approach to the solution structure of paramagnetic high-potential iron-sulfur proteins, the three-dimensional structure in solution of the oxidized Clostridium pasteurianum ferredoxin has been solved by 1H-NMR. The X-ray structure is not available. The protein contains 55 amino acids and two [4Fe-4S] clusters. In the oxidized state, the clusters have S = 0 ground states, but are paramagnetic because of thermal population of excited states. Due to the somewhat small size of the protein and to the presence of two clusters, approximately 55% of the residues have at least one proton with a non-selective T1 smaller than 25 ms. The protein has thus been used as a test system to challenge the present paramagnetic NMR methodology both in achieving an extended assignment and in obtaining a suitable number of constraints. 79% of protein protons have been assigned. Analogy with other ferredoxins of known structure has been of help to speed up the final stages of the assignment, although we have shown that this independent information is not necessary. In addition to dipolar connectivities, partially detected through tailored experiments, 3JHN-H alpha, H-bond constraints and dihedral angle constraints on the Cys chi 2 angles have been generated by using a recently derived Karplus-type relationship for the hyperfine shifts of cysteine beta CH2 protons. In total, 456 constraints have been used in distance geometry calculations. The final quality of the structures is satisfactory, with root-mean-square deviation values of 66 pm and 108 pm for backbone and heavy atoms, respectively. The resulting structure is compared with that of Clostridium acidi urici ferredoxin [Duée, E. D., Fanchon, E., Vicat, J., Sieker, L. C., Meyer, J. & Moulis, J.-M. (1994) J. Mol. Biol. 243, 683-695]. The two proteins are very similar in the overall folding, secondary structure elements and side-chain orientations. The C alpha root-mean-square deviation values between the X-ray-determined C. acidi urici ferredoxin structure and the conformer with lowest energy of the C. pasteurianum ferredoxin family is 78 pm (residues 3-53). Discrepancies in residues 26-28 may arise from the disorder observed in the X-ray structure in that region.     

Sequential assignment of 1H, 15N, 13C resonances and secondary structure of human calmodulin-like protein determined by NMR spectroscopy

Human calmodulin-like protein (CLP) is closely related to vertebrate calmodulin, yet its unique cell specific expression pattern, overlapping but divergent biochemical properties, and specific target proteins suggest that it is not an isoform of calmodulin. To gain insight into the structural differences that may underlie the difference target specificities and biochemical properties of CLP when compared to calmodulin, we determined the sequential backbone assignment and associated secondary structure of 144 out of the 148 residues of Ca2+-CLP by using multinuclear multidimensional NMR spectroscopy. Despite a very high overall degree of structural similarity between CLP and calmodulin, a number of significant differences were found mainly in the length of alpha-helices and in the central nonhelical flexible region. Interestingly, the regions of greatest primary sequence divergence between CLP and calmodulin in helices III and VIII displayed only minor secondary structure differences. The data suggest that the distinct differences in target specificity and biochemical properties of CLP and calmodulin result from the sum of several minor structural and side-chain changes spread over multiple domains in these proteins.     

Identification of the amino acids involved in the functional interaction between photosystem I and ferredoxin from Synechocystis sp. PCC 6803 by chemical cross-linking

Ferredoxin isolated from the cyanobacterium Synechocystis sp. PCC 6803 has been chemically cross-linked to purified photosystem I from the same organism. The reaction was catalyzed by N-ethyl-3-(3-dimethylaminopropyl)carbodiimide in the presence of N-hydroxysulfosuccinimide. A short reaction time and neutral pH values can be used in the presence of the two reagents, ensuring the integrity of both of the proteins and the iron-sulfur cluster of the ferredoxin. The only covalent complex detected comprised ferredoxin and the photo-system I (PSI)-D subunit, as identified by antibodies probing after electrophoresis. Electron paramagnetic resonance measurements of this covalent complex have shown that the cross-linked ferredoxin was entirely photoreducible by photosystem I and that the molar ratio of ferredoxin to PSI was close to 1. Extensive sequencing of the peptides obtained after proteolysis of the purified cross-linked product led to the identification of a covalent bond between glutamic acid 93 of ferredoxin and lysine 106 of the PSI-D subunit.     

Molecular structure of the oxidized, recombinant, heterocyst [2Fe-2S] ferredoxin from Anabaena 7120 determined to 1.7-A resolution

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. The three-dimensional structure of this ferredoxin has now been determined and refined to a crystallographic R value of 16.7%, with all measured X-ray data from 30.0 to 1.7 A. The molecular motif of this ferredoxin is similar to that of other plant-type ferredoxins with the iron-sulfur cluster located toward the outer edge of the molecule and the irons tetrahedrally coordinated by both inorganic sulfurs and sulfurs provided by protein cysteinyl residues. The overall secondary structure of the molecule consists of seven strands of beta-pleated sheet, two alpha-helices, and seven type I turns. It is of special interest that 4 of the 22 amino acid positions thought to be absolutely conserved in nonhalophilic ferredoxins are different in the heterocyst form of the protein. Three of these positions are located in the metal-cluster binding loop.     

1H-15N NMR signal assignment and secondary structure of bacteriorhodopsin (1-231) in solution

15N-Labeled de-(232-248)-bacteriorhodopsin [BR(1-231)] was solubilized in 1:1 chloroform-methanol solvent mixture that contained 1.0 M 2HCO2N2H4 and mimic membrane medium. Resonances in the 1H-15N heteronuclear multiple-quantum coherence (HMQC) spectrum of BR (1-231) were assigned using the data of two- and three-dimensional NMR experiments. Of 117 cross-peaks present in the 1H-15N HMQC spectrum, 98 were assigned to residues in 1-75 and 193-231 segments of the protein. Almost all cross-peaks that correspond to the 76-192 segment were absent in the HMQC spectrum (except for six cross-peaks from the side chains and 14 cross-peaks from the backbone). Deuterium exchange rates of amide protons and cross-peaks of nuclear Overhauser effect helped to localize helices A (residues 8-30), B (residues 40-65), and G (residues 198-226). The periodicity in the rates of deuterium exchange of NH protons of helices A, B, and G was explained by the compact arrangement of these helices in the protein globule. The broadening of signals from six residues in helix G, which, according to the electron cryomicroscopy model of bacteriorhodopsin, is in contact with the NMR-unobservable bundle of helices CDEF, indicates specific interactions of the helices in BR(1-231). These data suggest that BR(1-231) solubilized in an organic medium has a spatial structure similar to that in the electron cryomicroscopy model of BR.     

Ferredoxin reduction by photosystem I from Synechocystis sp. PCC 6803: toward an understanding of the respective roles of subunits PsaD and PsaE in ferredoxin binding

The process of ferredoxin reduction by photosystem I has been extensively investigated by flash-absorption spectroscopy in psaD and psaE deleted mutants from Synechocystis sp. PCC 6803. In both mutants, the dissociation constant for the photosystem I/ferredoxin complex at pH 8 is considerably increased as compared to the wild type: approximately 25- and 100-fold increases are found for PsaD-less and PsaE-less photosystem I, respectively. However, at high ferredoxin concentrations, submicrosecond and microsecond kinetics of electron transfer similar to that observed in the wild type are present in both mutants. The presence of these fast kinetic components indicates that the relative positions of ferredoxin and of the terminal photosystem I acceptor are not significantly disturbed by the absence of either PsaD or PsaE. The second-order rate constant of ferredoxin reduction is lowered 10- and 2-fold for PsaD-less and PsaE-less photosystem I, respectively. Assuming a simple binding equilibrium between photosystem I and ferredoxin, PsaD appears to be important for the guiding of ferredoxin to its binding site (main effect on the association rate) whereas PsaE seems to control the photosystem I/ferredoxin complex lifetime (main effect on the dissociation rate). The properties of electron transfer from photosystem I to ferredoxin were also studied at pH 5. 8. In the psaE deleted mutant as in the wild type, the change of pH from 8 to 5.8 induces a 10-fold increase in affinity of ferredoxin for photosystem I. In the absence of PsaD, this pH effect is not observed, in favor of this subunit being mostly responsible for the low pH increased affinity.     

1H and 15N resonance assignment of neural cell adhesion molecule module-2

This article presents a simple, inexpensive method for precisely locating the floor of the maxillary sinus, as well as the presence of any septa, at the time of sinus augmentation surgery. Using an anesthesia light wand placed transnasally to illuminate the sinus, the surgeon can reliably elevate the lateral maxillary wall overlying the sinus with relative ease without fear of placing the osteotomy cuts too far from the sinus floor. The same procedure can be used postoperatively to evaluate the density of the bone graft placed into the sinus prior to closure.     

Reduced turnover of the D1 polypeptide and photoactivation of electron transfer in novel herbicide resistant mutants of Synechocystis sp. PCC 6803

Two missense mutants, A263P and S264P, and a deletion mutant des-Ala263, Ser264, have been constructed in the D1 protein of the cyanobacterium Synechocystis sp PCC 6803. All were expected to induce a significant conformational change in the QB-binding region of photosystem II (PSII). Although the des-Ala263, Ser264-D1 mutant accumulated some D1 protein in the thylakoid membrane it was unable to grow photoautotrophically or evolve oxygen. Thermoluminescence and chlorophyll fluorescence studies confirmed that this deletion mutant did not show any functional PSII activity. In contrast, [S264P]D1 was able to grow photoautotrophically and give light-saturated rates of oxygen evolution at 60% of the rate of the wild-type control strain, TC31. The A263P missense mutant was also able to evolve oxygen at 50% of TC31 rates although it did not readily grow photoautotrophically. Thermoluminescence, flash oxygen yield and chlorophyll fluorescence measurements indicated that in both missense mutants electron transfer from QA to QB was significantly impaired in dark adapted cells. However, QA to QB electron transfer could be photoactivated in the mutants by background illumination. Both the A263P and S264P mutants also showed an increase in resistance to the s-triazine family of herbicides although this feature did not hold for the phenolic herbicide, ioxynil. Of particular interest was that the two missense mutants, especially S264P, possessed a slower rate of turnover of the D1 protein compared with TC31 and in vivo contained detectable levels of a 41-kDa adduct consisting of D1 and the alpha subunit of cytochrome b559. When protein synthesis was blocked by the addition of lincomycin, D1 degradation was again slower in S264P than TC31. The results are discussed in terms of structural changes in the QB-binding region which affect herbicide and plastoquinone binding and perturb the normal regulatory factors that control the degradation of the D1 protein and its synchronisation with the synthesis of a replacement D1 protein.     

Coordination of the [2Fe-2S] cluster in wild type and molecular variants of Clostridium pasteurianum ferredoxin, investigated by ESEEM spectroscopy

The [2Fe-2S] ferredoxin from Clostridium pasteurianum contains five cysteine residues in positions 11, 14, 24, 56, and 60. This pattern is unique, and a combination of site-directed mutagenesis and spectroscopy is therefore being implemented to identify the ligands of the [2Fe-2S] cluster. The possible involvement of ligands other than cysteine in some molecular variants of this ferredoxin has been considered, histidines being likely candidates. Therefore, the three histidine residues in positions 6, 7, and 90 of the amino acid sequence have been individually and collectively replaced by alanine or valine. The mutated ferredoxins have been purified and were all found to contain [2Fe-2S] clusters of which the UV-visible absorption spectra were identical to that of the wild-type protein. The H6A/H7A/ H90A triply mutated ferredoxin was further characterized by EPR and by ESEEM spectroscopy and was found to differ only marginally from the wild-type protein. The ESEEM spectra of wild-type ferredoxin displayed weak 14N hyperfine interactions at the three principal g-factors of the [2Fe-2S] center. The estimated 14N coupling constants (Aiso = 0.6 MHz; e2qQ approximately 3.3 MHz) indicate that the ESEEM effect is most likely due to 14N from the polypeptide backbone. 2H2O ESEEM spectra showed that the [2Fe-2S] cluster is accessible for exchange with solvent deuterons. ESEEM spectra of the previously characterized C24A and C14A/C24A variants have been recorded and were also found to be very similar to those of the wild-type protein. There was no evidence for coordination of the [2Fe-2S] cluster by [14N]histidine or other 14N nuclei, in either wild-type or mutant forms of the ferredoxin. By these criteria, the environment of the [2Fe-2S] center is not distinguishable from those in plant-type ferredoxins. Non-cysteinyl coordination most probably occurs only in the C14A/C24A variant, which contains no more than three cysteine residues. The data shown here indicate that the fourth ligand of the [2Fe-2S] cluster is neither a histidine residue nor another nitrogenous ligand. The possibility of oxygenic coordination for this molecular variant is discussed.     

Detection and classification of hyperfine-shifted 1H, 2H, and 15N resonances of the Rieske ferredoxin component of toluene 4-monooxygenase

T4MOC is a 12.3 kDa soluble Rieske ferredoxin that is obligately required for electron transfer between the oxidoreductase and diiron hydroxylase components of toluene 4-monooxygenase from Pseudomonas mendocina KR1. Our preliminary 1H NMR studies of oxidized and reduced T4MOC [Markley, J. L., Xia, B., Chae, Y. K., Cheng, H., Westler, W. M., Pikus, J. D., and Fox, B. G. (1996) in Protein Structure Function Relationships (Zaidi, Z., and Smith, D., Eds.) pp 135-146, Plenum Press, London] revealed the presence of hyperfine-shifted 1H resonances whose short relaxation times made it impractical to use nuclear Overhauser effect (NOE) measurements for assignment purposes. We report here the use of selective isotopic labeling to analyze the hyperfine-shifted 1H, 2H, and 15N signals from T4MOC. Selective deuteration led to identification of signals from the four Hbeta atoms of cluster ligands C45 and C64 in the oxidized and reduced forms of T4MOC. In the reduced state, the Curie temperature dependence of the Hbeta protons corresponded to that predicted from the simple vector spin-coupling model for nuclei associated with the localized ferric site. The signal at 25.5 ppm in the 1H spectrum of reduced T4MOC was assigned on the basis of selective 2H labeling to the His Hepsilon1 atom of one of the cluster ligands (H47 or H67). This assignment was corroborated by a one bond 1H-13C correlation (at 25.39 ppm 1H and 136.11 ppm 13C) observed in spectra of [U-13C]T4MOC with a 1H-13C coupling constant of approximately 192 Hz. The carbon chemical shift and one bond coupling constant are those expected for 1Hepsilon1-13Cepsilon1 in the imidazolium ring of histidine and are inconsistent with values expected for cysteine 1Halpha-13Calpha. The His Hepsilon1 proton exhibited weak Curie temperature dependence from 283 to 303 K, contrary to the anti-Curie temperature dependence predicted from the spin coupling model for nuclei associated with the localized ferrous site. A 1H peak at -12.3 ppm was observed in spectra of reduced T4MOC; this signal was found to correspond to a hydrogen (probably in an H-bond to the cluster) that exchanged with solvent with a half-time of about 2 days in the oxidized state but with a much longer (undetectable) half-time in the reduced state. These results with T4MOC call into question certain 1H assignments recently reported on the basis of NOE measurements for the comparable Rieske ferredoxin component of an evolutionarily related alkene monooxygenase from Xanthobacter sp. Py2 [Holz, R. C., Small, F. J., and Ensign, S. A, (1997) Biochemistry 36, 14690-14696]. Selective 15N labeling was used to identify hyperfine-shifted 15N NMR signals from the backbone nitrogens of all four cluster ligands (C45, H47, C64, and H67), from the Nepsilon2 atoms of the two histidine ligands (H47 and H67), and from nonligand Gln and Ala residues (Q48 and A66) present in the cluster-binding motif of T4MOC in the oxidized and reduced states. The results indicate that the Ndelta1 of each of the two ligand histidines of T4MOC are ligated to an iron atom and reveal a pattern of H-bonding to the Rieske [2Fe-2S] center involving four (H47, Q48, A66, and H67 of T4MOC) of the five backbone amide H-bonds expected on the basis of comparison with the crystal structures of other related Rieske proteins; the fifth backbone amide (I50 of T4MOC) failed to exhibit a hyperfine shift. This anomaly may arise from the lack of an associated disulfide in T4MOC, a fundamental structural difference between the three types of Rieske proteins that may be related to functional diversity in this protein family.     

Sucrose-phosphate synthase from Synechocystis sp. strain PCC 6803: identification of the spsA gene and characterization of the enzyme expressed in Escherichia coli

The first identification and characterization of a prokaryotic gene (spsA) encoding sucrose-phosphate synthase (SPS) is reported for Synechocystis sp. strain PCC 6803, a unicellular non-nitrogen-fixing cyanobacterium. Comparisons of the deduced amino acid sequence and some relevant biochemical properties of the enzyme with those of plant SPSs revealed important differences in the N-terminal and UDP-glucose binding site regions, substrate specificities, molecular masses, subunit compositions, and regulatory properties.     

1H, 13C, 15N nuclear magnetic resonance backbone assignments and secondary structure of human calcineurin B

The calmodulin- and calcium-stimulated protein phosphatase calcineurin, PP2B, consists of two subunits: calcineurin B, which binds Ca2+, and calcineurin A, which contains the catalytic site and a calmodulin binding site. Heteronuclear 3D and 4D NMR experiments were carried out on a recombinant human calcineurin B which is a 170-residue protein of molecular mass 19.3 kDa, uniformly labeled with 15N and 13C. The nondenaturing detergent CHAPS was used to obtain a monomeric form of calcineurin B. Three-dimensional triple resonance experiments yielded complete sequential assignment of the backbone nuclei (1H, 13C, and 15N). This assignment was verified by a 4D HN(COCA)NH experiment carried out with 50% randomly deuteriated and uniformly 15N- and 13C-enriched calcineurin B. The secondary structure of calcineurin B has been determined on the basis of the 13C alpha and 13C beta secondary chemical shifts, J(HNH alpha) couplings, and NOE connectivities obtained from 3D 15N-separated and 4D 13C/15N-separated NOESY spectra. Calcineurin B has eight helices distributed in four EF-hand, helix-loop-helix [Kretsinger, R. H. (1980) CRC Crit. Rev. Biochem. 8, 119-174] calcium binding domains. The secondary structure of calcineurin B is highly homologous to that of calmodulin. In comparison to calmodulin, helices B and C are shorter while helix G is considerably longer. As was observed for calmodulin in solution, calcineurin B does not have a single long central helix; rather, helices D and E are separated by a six-residue sequence in a flexible nonhelical conformation.     

Unusual helix-containing greek keys in development-specific Ca(2+)-binding protein S. 1H, 15N, and 13C assignments and secondary structure determined with the use of multidimensional double and triple resonance heteronuclear NMR spectroscopy

Multidimensional heteronuclear NMR spectroscopy has been used to determine almost complete backbone and side-chain 1H, 15N, and 13C resonance assignments of calcium loaded Myxococcus xanthus protein S (173 residues). Of the range of constant-time triple resonance experiments recorded, HNCACB and CBCA(CO)NH, which correlate C alpha and C beta with backbone amide resonances of the same and the succeeding residue respectively, proved particularly useful in resolving assignment ambiguities created by the 4-fold internal homology of the protein S amino acid sequence. Extensive side-chain 1H and 13C assignments have been obtained by analysis of HCCH-TOCSY and 15N-edited TOCSY-HMQC spectra. A combination of NOE, backbone amide proton exchange, 3JNH alpha coupling constant, and chemical shift data has been used to show that each of the protein S repeat units consists of four beta-strands in a Greek key arrangement. Two of the Greek keys contain a regular alpha-helix between the third and fourth strands, resulting in an unusual and possibly unique variation on this common folding motif. Despite similarity between two nine-residue stretches in the first and third domains of protein S and one of the Ca(2+)-binding sequences in bovine brain calmodulin [Inouye, S., Franceschini, T., & Inouye, M. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 6829-6833], the protein S topology in these regions is incompatible with an EF-hand calmodulin-type Ca(2+)-binding site.