Structure determination of a new protein from backbone-centered NMR data and NMR-assisted structure prediction.

Targeting of proteins for structure determination in structural genomic programs often includes the use of threading and fold recognition methods to exclude proteins belonging to well-populated fold families, but such methods can still fail to recognize preexisting folds. The authors illustrate here a method in which limited amounts of structural data are used to improve an initial homology search and the data are subsequently used to produce a structure by data-constrained refinement of an identified structural template. The data used are primarily NMR-based residual dipolar couplings, but they also include additional chemical shift and backbone-nuclear Overhauser effect data. Using this methodology, a backbone structure was efficiently produced for a 10 kDa protein (PF1455) from Pyrococcus furiosus. Its relationship to existing structures and its probable function are discussed.

Investigation of the role of surface residues in the ferredoxin from Clostridium pasteurianum.

Eleven mutant forms of the ferredoxin from Clostridium pasteurianum (CpFd; 2 Fe4S4; 6200 Da) have been isolated in which six surface carboxylates are changed systematically to their uncharged but stereochemically equivalent carboxamide analogues. Such changes provide molecules which vary in overall charge and its surface distribution but vary minimally in structure and reduction potential. Glu-17 and Asp-6, -27, -33, -35, and -39 were converted providing six single mutants, four double mutants and one triple mutant. The proteins were characterised by UV-visible spectroscopy, square-wave voltammetry and 1H NMR. Their ability to mediate electron transfer between spinach NADH:ferredoxin oxidoreductase and horse heart cytochrome c was assessed. Each mutant is 30-100% as active as the recombinant protein with the triple mutant D33,35,39N being least active. Second-order rate constants k2 for the oxidation of reduced mutant ferredoxins by [Co(NH3)6]3+ were measured at 25 degrees C and I = 0.1 M by stopped-flow techniques. Each mutant displayed saturation kinetics with k2 being 30-100% of that for the recombinant protein. The rates were moderately sensitive to ionic strength. Variation in association constant K could not be detected within the confidence limits of the data. Overall the effects of the mutations were minor. In contrast to human and Anabaena 7120 [Fe2S2]-ferredoxins, electron transfer does not appear to rely on the presence of one or two specific surface carboxylate residues. It may occur from multiple sites on the surface of CpFd with recognition processes for its many physiological redox partners being controlled by relative reduction potentials, in addition to unidentified criteria. The conclusions are consistent with previous results for another series of mutant CpFd proteins interacting with physiological redox partners pyruvate: Fd oxidoreductase and hydrogenase (J.M. Moulis, V. Davasse (1995) Biochemistry 34, 16781-16788).

Light and electron microscopy localization of the 11beta-hydroxysteroid dehydrogenase type I enzyme in the rat.

The 11beta-hydroxysteroid dehydrogenase type I enzyme (11betaHSD1) converts cortisone to cortisol in humans, and 11-dehydrocorticosterone to corticosterone in rodents. In the present study we used a new immunopurified polyclonal antibody, RAH113, to localize 11betaHSD1 at the light and electron microscopy levels in a wide range of rat tissues. 11betaHSD1 staining in the liver was of highest intensity around the central vein and decreased radially. In the lung, 11betaHSD1 was found at highest levels in the interstitial fibroblast, with levels in the type II pneumocyte an order of magnitude lower. RAH113 stained proximal tubules of the renal cortex and interstitial cells of the medulla and papilla. Adrenal 11betaHSD1 was confined to the glomerulosa and medulla, whereas the glucocorticoid-inactivating hydroxysteroid dehydrogenase isoform 11betaHSD2 was present in fascilulata/reticularis. 11betaHSD1 was found in parietal cells of the fundic region of the stomach, but not in the antrum. In the heart, 11betaHSD1 was detected in cells resembling interstitial fibroblasts of the endocardium and in the adventitial fibroblasts of blood vessels. Western blot analysis confirmed the presence of an antigen of the correct size (34 kDa) and intensity consistent with levels of enzyme activity previously reported in these tissues. Brain and testis also displayed the 34-kDa protein, confirming the expression of authentic 11betaHSD1 in these tissues. Electron microscopy of lung and kidney interstitial cells showed that 11betaHSD1 was localized both to the endoplasmic reticulum and the nuclear membrane. These results show that 11betaHSD1 is present in discrete cell populations where it may facilitate intracrine and paracrine glucocorticoid action in addition to its classical role of maintaining circulating glucocorticoids via activity in the liver.

A pure S = 3/2 [Fe4S4]+ cluster in the A33Y variant of Pyrococcus furiosus ferredoxin.

The properties of the [4Fe-4S]2+/+ cluster in wild-type and the A33Y variant of Pyrococcus furiosus ferredoxin have been investigated by the combination of EPR, variable-temperature magnetic circular dichroism (VTMCD) and resonance Raman (RR) spectroscopies. The A33Y variant involves the replacement of an alanine whose alpha-C is less than 4 A from one of the cluster iron atoms by a tyrosine residue. Although the spectroscopic results give no indication of tyrosyl cluster ligation, the presence of a tyrosine residue in close proximity to the cluster results in a 38-mV decrease in the midpoint potential of the [4Fe-4S]2+/+ couple and has a marked effect on the ground state properties of the reduced cluster. The mixed spin [4Fe-4S]+ cluster in the wild-type protein, 80% S = 3/2 (E/D = 0.22, D = +3.3 cm(-1)) and 20% S = 1/2 (g = 2.10, 1.87, 1.80), is converted into a homogeneous S = 3/2 (E/D = 0.30, D = -0.7 cm(-1)) form in the A33Y variant. As the first example of a pure S = 3/2 [4Fe-4S]+ cluster in a ferredoxin, this variant affords the opportunity for detailed characterization of the excited electronic properties via VTMCD studies and demonstrates that the protein environment can play a crucial role in determining the ground state properties of [4Fe-4S]+ clusters.

GRKO mice express an aberrant dexamethasone-binding glucocorticoid receptor, but are profoundly glucocorticoid resistant.

The introduction of a targeted insertion mutation into exon 2 of the gene coding for the glucocorticoid receptor (GR) enabled production of glucocorticoid receptor knock-out (GRKO) mice. GRKO mice on a C57BL/6/129sv mixed genetic background show a variable phenotype, with 90% of -/- mice dying at birth with respiratory insufficiency but 10% of mutant mice surviving to maturity. To investigate the possibility of residual GR expression in surviving GRKO mice we have measured binding of the synthetic glucocorticoid dexamethasone in tissue extracts from adrenalectomized mice. High affinity binding of dexamethasone in protein extracts of liver, kidney, lung and brain from adult GRKO mice is found at levels 30-60% those in wild-type mice, with heterozygotes (+/-) having intermediate levels. PCR and ribonuclease protection analysis showed comparable levels of GR mRNA on the 3' side of the gene-targeted insertional mutation in exon 2 of the GR gene, with almost no GR mRNA detected from exons 1 and 2 on the 5' side of the gene-targeted insertional mutation. Western blot analysis using a C-terminal specific GR antibody detects a 39 kDa GR fragment in extracts from adult GRKO mice. Despite the evidence for expression of a ligand-binding domain fragment of the glucocorticoid receptor these mice are profoundly glucocorticoid resistant, with elevated levels of plasma ACTH and corticosterone. Thymocytes from adult and fetal GRKO mice are resistant to dexamethasone-induced apoptosis and cultured fetal hepatocytes from GRKO mice are completely refractory to glucocorticoid induction of the gluconeogenic enzyme glucose-6-phosphatase. Thus although the surviving adult homozygous GRKO mice express a dexamethasone-binding GR fragment, their classic target tissues remain profoundly glucocorticoid insensitive.

High-throughput production of Pyrococcus furiosus proteins: considerations for metalloproteins.

Free-living prokaryotic organisms contain all of the proteins required for the basic biochemical processes of life. As part of the Southeastern Collaboratory for Structural Genomics (SECSG), Pyrococcus furiosus is being used as a model system for developing a high-throughput protein expression and purification protocol. Its 1.9 million basepair genome encodes approximately 2200 putative proteins, less than 25% of which show similarity to any structurally characterized protein in the Protein Data Bank. The overall goal of the structural genomics initiative is to determine, in total, all existing protein folds. The immediate objective of this work is to obtain recombinant forms of all P. furiosus proteins in their functional states for structural determination. Proteins successfully produced by overexpression in another organism such as the bacterium Escherichia coli typically contain a single subunit, are soluble and do not contain (complex) cofactors. Analyses of the P. furiosus genome suggest that perhaps only a quarter of the genes encode proteins that would fall into this category. The hypothesis is that lack of the appropriate cofactor or of the partner protein(s) necessary to form a complex are major reasons why many recombinant proteins are insoluble. This work describes development of the production pipeline with attention to prediction and incorporation of cofactors.

Effect of iron-sulfur cluster environment in modulating the thermodynamic properties and biological function of ferredoxin from Pyrococcus furiosus.

The ferredoxin (7.5 kDa) of the hyperthermophilic archaeon, Pyrococcus furiosus, contains a single [4Fe-4S]1+,2+ cluster that is coordinated by three Cys and one Asp residue rather than the expected four Cys. The role of this Asp residue was investigated using a series of mutants, D14X, where X = C, S, H, N, V, and Y, prepared by heterologous gene expression in Escherichia coli. While the recombinant form of the wild-type and the D14S and D14C mutants contained a [4Fe-4S]1+,2+ cluster, the D14V, D14H, D14Y, and D14N proteins contained a [3Fe-4S]0,+ center, as determined by visible spectroscopy and electrochemistry. The redox potentials (at pH 7.0, 23 degrees C) of the D14C and D14S mutants were decreased by 58 and 133 mV, respectively, compared to those of the wild-type 4Fe-ferredoxin (Em -368 mV), while those of the 3Fe-protein mutants (including the 3Fe-form of the D14S, generated by chemical oxidation) were between 15 and 118 mV more positive than that of wild-type 3Fe-form (obtained by chemical oxidation, Em -203 mV). The reduction potentials of all of the 3Fe-forms, except the D14S mutant, showed a pH response over the range 3.0-10.0 with a pK of 3.3-4.7, and this was assigned to cluster protonation. The D14H mutant and the wild-type 3Fe-proteins showed an additional pK (both at 5.9) assumed to arise from protonation of the amino acid side chain. With the 4Fe-proteins, there was no dramatic change in the potentials of the wild-type or D14C form, while the pH response of the D14S mutant (pK 4.75) was ascribed to protonation of the serinate. While the ferredoxin variants exhibited a range of thermal stabilities (measured at 80 degrees C, pH 2.5), none of them showed any temperature-dependent transitions (0-80 degrees C) in their reduction potentials, and there was no correlation between the calculated DeltaS degrees' values and the absorbance maximum, reduction potential, or hydrophobicity of residue 14. In contrast, there was a linear correlation between the DeltaH degrees' value and reduction potential. Kinetic analyses were carried out at 80 degrees C using the ferredoxin as either an electron acceptor to pyruvate oxidoreductase (POR) or as an electron donor to ferredoxin:NADP oxidoreductase (FNOR, both from P. furiosus). The data showed that the reduction potential of the ferredoxin, rather than cluster type or the nature of the residue at position 14, appears to be the predominant factor in determining efficiency of electron transfer in both systems. However, compared to all the variants, the reduction potential of WT Fd makes it the most appropriate protein to both accept electrons from POR and donate them to FNOR.

Probing the stoichiometry and oxidation states of metal centers in iron-sulfur proteins using electrospray FTICR mass spectrometry.

Electrospray ionization (ESI) Fourier transform ion cyclotron resonance mass spectrometry is used to determine the stoichiometry and oxidation states of the metal centers in several iron-sulfur proteins. Samples are introduced into the ESI source under nondenaturing conditions in order to observe intact metal-containing protein ions. The stoichiometry and oxidation state of the metal or metal-sulfur cluster in the protein ion can be derived from the mass spectrum. Mononuclear metal-containing proteins and [4Fe-4S] centers are very stable and yield the molecular ion with little or no fragmentation. Proteins that contain [2Fe-2S] clusters are less stable and yield loss of one or two sulfur atoms from the molecular species, although the molecular ion is more abundant than the fragment peaks. [3Fe-4S]-containing proteins are the least stable of the species investigated, yielding abundant peaks corresponding to the loss of one to four sulfur atoms in addition to a peak representing the molecular ion. Isotope labeling experiments show that the sulfur loss originates from the [3Fe-4S] center. Negative ion mode mass spectra were obtained and found to produce much more stable [3Fe-4S]-containing ions than obtained in positive ion mode. ESI analysis of the same proteins under denaturing conditions yields mass spectra of the apo form of the proteins. Disulfide bonds are observed in the apoprotein mass spectra that are not present in the holoprotein. These result from oxidative coupling of the cysteinyl sulfur atoms that are responsible for binding the metal center. In addition, inorganic sulfide is found to incorporate itself into the apoprotein by forming sulfur bridges between cysteine residues.

Effects of mutations in aspartate 14 on the spectroscopic properties of the [Fe3S4]+,0 clusters in Pyrococcus furiosus ferredoxin.

The properties of [Fe(3)S(4)](+,0) clusters in wild-type and mutant forms of Pf Fd with Asp, Ser, Cys, Val, His, Asn, and Tyr residues occupying position 14, i.e., proximal to the three micro(2)-S atoms of the cluster, have been investigated by the combination of EPR, variable-temperature magnetic circular dichroism (VTMCD), and resonance Raman (RR) spectroscopies. Two distinct types of [Fe(3)S(4)] clusters are identified on the basis of the breadth of the S = (1)/(2) [Fe(3)S(4)](+) EPR resonances and the marked differences in the VTMCD spectra of the S = 2 [Fe(3)S(4)](0) clusters. On the basis of the available NMR data for [Fe(3)S(4)](+, 0) clusters in ferredoxins, the distinctive properties of these two types of [Fe(3)S(4)] clusters are interpreted in terms of different locations of the more strongly coupled pair of irons in the oxidized clusters and the valence-delocalized pair in the reduced clusters. Near-IR VTMCD measurements indicate the presence of S = (9)/(2) valence-delocalized pairs in both types of [Fe(3)S(4)](0) clusters, and the spin-dependent delocalization energies associated with the Fe-Fe interactions were determined to be approximately 4300 cm(-)(1) in both cases. We conclude that the nature of the residue at position 14 in Pyrococcus furiosus ferredoxin is an important determinant of the location of the reducible pair of irons in a [Fe(3)S(4)](+,0) cluster, and the redox properties of the wild-type and mutant ferredoxins are discussed in light of these new results.

Effect of serinate ligation at each of the iron sites of the [Fe4S4] cluster of Pyrococcus furiosus ferredoxin on the redox, spectroscopic, and biological properties.

Pyrococcus furiosus ferredoxin (Fd) contains a single [Fe(4)S(4)] cluster coordinated by three cysteine (at positions 11, 17, and 56) and one aspartate ligand (at position 14). In this study, the spectroscopic, redox, and functional consequences of D14C, D14C/C11S, D14S, D14C/C17S, and D14C/C56S mutations have been investigated. The four serine variants each contain a potential cluster coordination sphere of one serine and three cysteine residues, with serine ligation at each of the four Fe sites of the [Fe(4)S(4)] cluster. All five variants were expressed in Escherichia coli, and each contained a [Fe(4)S(4)](2+,+) cluster as shown by UV-visible absorption and resonance Raman studies of the oxidized protein and EPR and variable-temperature magnetic circular dichroism (VTMCD) studies of the as-prepared, dithionite-reduced protein. Changes in both the absorption and resonance Raman spectra are consistent with changing from complete cysteinyl cluster ligation in the D14C variant to three cysteines and one oxygenic ligand in each of the four serine variants. EPR and VTMCD studies show distinctive ground and excited state properties for the paramagnetic [Fe(4)S(4)](+) centers in each of these variant proteins, with the D14C and D14C/C11S variants having homogeneous S = (1)/(2) ground states and the D14S, D14C/C17S, and D14C/C56S variants having mixed-spin, S = (1)/(2) and (3)/(2) ground states. The midpoint potentials (pH 7.0, 23 degrees C) of the D14C/C11S and D14C/C17S variants were unchanged compared to that of the D14C variant (E(m) = -427 mV) within experimental error, but the potentials of D14C/C56S and D14S variants were more negative by 49 and 78 mV, respectively. Since the VTMCD spectra indicate the presence of a valence-delocalized Fe(2. 5+)Fe(2.5+) pair in all five variants, the midpoint potentials are interpreted in terms of Cys11 and Cys17 ligating the nonreducible valence-delocalized pair in D14C. Only the D14S variant exhibited a pH-dependent redox potential over the range of 3.5-10, and this is attributed to protonation of the serinate ligand to the reduced cluster (pK(a) = 4.75). All five variants had similar K(m) and V(m) values in a coupled assay in which Fd was reduced by pyruvate ferredoxin oxidoreductase (POR) and oxidized by ferredoxin NADP oxidoreductase (FNOR), both purified from P. furiosus. Hence, the mode of ligation at each Fe atom in the [Fe(4)S(4)] cluster appears to have little effect on the interaction and the electron transfer between Fd and FNOR.

Backbone solution structures of proteins using residual dipolar couplings: application to a novel structural genomics target.

Structural genomics (or proteomics) activities are critically dependent on the availability of high-throughput structure determination methodology. Development of such methodology has been a particular challenge for NMR based structure determination because of the demands for isotopic labeling of proteins and the requirements for very long data acquisition times. We present here a methodology that gains efficiency from a focus on determination of backbone structures of proteins as opposed to full structures with all sidechains in place. This focus is appropriate given the presumption that many protein structures in the future will be built using computational methods that start from representative fold family structures and replace as many as 70% of the sidechains in the course of structure determination. The methodology we present is based primarily on residual dipolar couplings (RDCs), readily accessible NMR observables that constrain the orientation of backbone fragments irrespective of separation in space. A new software tool is described for the assembly of backbone fragments under RDC constraints and an application to a structural genomics target is presented. The target is an 8.7 kDa protein from Pyrococcus furiosus, PF1061, that was previously not well annotated, and had a nearest structurally characterized neighbor with only 33% sequence identity. The structure produced shows structural similarity to this sequence homologue, but also shows similarity to other proteins, which suggests a functional role in sulfur transfer. Given the backbone structure and a possible functional link this should be an ideal target for development of modeling methods.

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.

Comparison of native and mutant proteins provides a sequence-specific assignment of the cysteinyl ligand proton NMR resonances in the 2[Fe4S4] ferredoxin from Clostridium pasteurianum.

A sequence-specific assignment is presented for the eight low-field paramagnetically shifted cysteinyl ligand proton NMR resonances in the 2[Fe4S4] ferredoxin from Clostridium pasteurianum. The assignment is based upon comparison of chemical shifts in 1D and 2D NMR spectra of native oxidized protein and those of three mutants. The mutant proteins G12A and G41A were designed to produce minor local structural changes (hence small chemical shift perturbations) in either cluster I (glycine 12 to alanine) or in cluster II (glycine 41 to alanine). Observed chemical shift changes in spectra of the double mutant G12,41A support the interpretation. The comparison is aided by structural models derived from the crystal structure of the related ferredoxin from Peptococcus aerogenes. Each of the eight low-field resonances is assigned to a beta-proton from a different cysteinyl ligand, and so connectivities established from previous TOCSY and HMQC data allow assignment of all 24 cysteinyl ligand protons.

Secondary structure extensions in Pyrococcus furiosus ferredoxin destabilize the disulfide bond relative to that in other hyperthermostable ferredoxins. Global consequences for the disulfide orientational heterogeneity.

The single cubane cluster ferredoxin (Fd) from the hyperthermophilic archaeon Pyrococcus furiosus (Pf) possesses several unique properties when compared even to Fds from other hyperthermophilic archaea or bacteria. These include an equilibrium molecular heterogeneity, a six- to seven-residue increase in size, an Asp rather than the Cys as one cluster ligand, and a readily reducible disulfide bond. NMR assignments and determination of both secondary structure and tertiary contacts remote from the paramagnetic oxidized cluster of Pf 3Fe Fd with an intact disulfide bond reported previously (Teng Q., Zhou, Z. H., Smith, E. T., Busse, S. C., Howard, J. B. Adams, M. W. W., and La Mar, G. (1994) Biochemistry 33, 6316-6328) are extended here to the 4Fe oxidized cluster WT (1H and 15N) and D14C (1H only) Fds with an intact disulfide bond and to the 4Fe oxidized WT Fd (1H and 15N) with a cleaved disulfide bond. All forms are shown to possess a long (13-member) alpha-helix, two beta-sheets (one double-, one triple-stranded), and three turns outside the cluster vicinity, each with tertiary contacts among themselves as found in other Fds. While the same secondary structural elements, with similar tertiary contacts, are found in other hyperthermostable Fds, Pf Fd has two elements, the long helix and the triple-stranded beta-sheet, that exhibit extensions and form multiple tertiary contacts. All Pf Fd forms with an intact disulfide bond exhibit a dynamic equilibrium heterogeneity which is shown to modulate a hydrogen-bonding network in the hydrophobic core that radiates from the Cys21-Cys48 disulfide bond and encompasses residues Lys36, Val24, Cys21, and Cys17 and the majority of the long helix. The heterogeneity is attributed to population of the alternate S and R chiralities of the disulfide bond, each destabilized by steric interactions with the extended alpha-helix. Comparison of the chemical shifts and their temperature gradients reveals that the molecular structure of the protein with the less stable R disulfide resembles that of the Fd with a cleaved disulfide bond. Both cluster architecture (3Fe vs 4Fe) and ligand mutation (Cys for Asp14) leave the disulfide orientational heterogeneity largely unperturbed. It is concluded that the six- to seven-residue extension that results in a longer helix and larger beta-sheet in Pf Fd, relative to other hyperthermostable Fds, more likely serves to destabilize the disulfide bond, and hence make it more readily reducible, than to significantly increase protein thermostability.