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J L Markley

Publications and source records attributed to J L Markley.

At least 55 records · Page 3Linked to original sources
PubMed1997

Structure-function relationships in Anabaena ferredoxin: correlations between X-ray crystal structures, reduction potentials, and rate constants of electron transfer to ferredoxin:NADP+ reductase for site-specific ferredoxin mutants.

A combination of structural, thermodynamic, and transient kinetic data on wild-type and mutant Anabaena vegetative cell ferredoxins has been used to investigate the nature of the protein-protein interactions leading to electron transfer from reduced ferredoxin to oxidized ferredoxin:NADP+ reductase (FNR). We have determined the reduction potentials of wild-type vegetative ferredoxin, heterocyst ferredoxin, and 12 site-specific mutants at seven surface residues of vegetative ferredoxin, as well as the one- and two-electron reduction potentials of FNR, both alone and in complexes with wild-type and three mutant ferredoxins. X-ray crystallographic structure determinations have been carried out for six of the ferredoxin mutants. None of the mutants showed significant structural changes in the immediate vicinity of the [2Fe-2S] cluster, despite large decreases in electron-transfer reactivity (for E94K and S47A) and sizable increases in reduction potential (80 mV for E94K and 47 mV for S47A). Furthermore, the relatively small changes in Calpha backbone atom positions which were observed in these mutants do not correlate with the kinetic and thermodynamic properties. In sharp contrast to the S47A mutant, S47T retains electron-transfer activity, and its reduction potential is 100 mV more negative than that of the S47A mutant, implicating the importance of the hydrogen bond which exists between the side chain hydroxyl group of S47 and the side chain carboxyl oxygen of E94. Other ferredoxin mutations that alter both reduction potential and electron-transfer reactivity are E94Q, F65A, and F65I, whereas D62K, D68K, Q70K, E94D, and F65Y have reduction potentials and electron-transfer reactivity that are similar to those of wild-type ferredoxin. In electrostatic complexes with recombinant FNR, three of the kinetically impaired ferredoxin mutants, as did wild-type ferredoxin, induced large (approximately 40 mV) positive shifts in the reduction potential of the flavoprotein, thereby making electron transfer thermodynamically feasible. On the basis of these observations, we conclude that nonconservative mutations of three critical residues (S47, F65, and E94) on the surface of ferredoxin have large parallel effects on both the reduction potential and the electron-transfer reactivity of the [2Fe-2S] cluster and that the reduction potential changes are not the principal factor governing electron-transfer reactivity. Rather, the kinetic properties are most likely controlled by the specific orientations of the proteins within the transient electron-transfer complex.

Anabaena↗
PubMed1997

The second finger of Urbs1 is required for iron-mediated repression of sid1 in Ustilago maydis.

The urbs1 gene encodes a transcriptional regulator of siderophore biosynthesis in Ustilago maydis. Biological and DNA-binding activities of the two putative zinc-finger motifs of Urbs1 were studied by analyzing mutants containing altered finger domains. The mutated urbs1 alleles from three previously described N'-methyl-N'-nitro-N-nitrosoguanidine (NTG) mutants were mapped and cloned by a gap-repair procedure. Sequence analyses revealed single amino acid substitutions in two of the NTG mutants. Both mutations (G-507 to D in urbs1-1 and P-491 to L in urbs1-3), which are located in the Urbs1 C-terminal finger domain, reduced DNA-binding activity by 10-fold and were sufficient to confer a urbs1-minus phenotype. The third NTG urbs1 mutant (urbs1-2) also contained a mutation in one of the conserved amino acids (P-518 to S) in the C-terminal finger domain, but this mutation alone was not sufficient to confer a urbs1-minus phenotype. A second frame shift mutation was identified in urbs1-2 and is necessary for the urbs1-minus phenotype. In an analysis of the function of the N-terminal finger of Urbs1, the conserved amino acid Arg-350 was mutated to leucine. A Urbs1 protein with this mutation complemented a urbs1 null mutant strain. By contrast, a similar mutation in the C-terminal domain abolished the ability of Urbs1 to regulate siderophore biosynthesis and greatly reduced its ability to bind target DNA.

Amino Acid Sequence↗
PubMed1997

Effects of serpin binding on the target proteinase: global stabilization, localized increased structural flexibility, and conserved hydrogen bonding at the active site.

The binding of human alpha1-proteinase inhibitor to rat trypsin was shown by NMR spectroscopy to raise the pKa' of His57 in the active site but not to disrupt the hydrogen bond between His57 and Asp102. Similar NMR results were observed for the Asp189 to serine mutant of rat trypsin, which is much more stable than wild-type trypsin against autoproteolysis as the result of mutation of the residue at the base of the specificity pocket. This mutant was used in further studies aimed at determining the extent of the conformational transition in trypsin that accompanies serpin binding and leads to disruption of the catalytic activity of the proteinase such that the inhibitor complex is trapped at the acyl enzyme intermediate stage. The stability of rat trypsin toward thermal denaturation was found to be lower in the free enzyme than in the complex with alpha1-proteinase inhibitor. This suggests that the complex contains extensive protein-protein interactions that stabilize overall folding. On the other hand, previous investigations have shown that the proteinase in serpin-proteinase complexes becomes more susceptible to limited proteolysis, suggesting that the conformational change that accompanies binding leads to the exposure of susceptible loops in the enzyme. The existence of this type of conformational change upon complex formation has been confirmed here by investigation of the rate of cleavage of disulfide linkages by added dithiothreitol. This study revealed that, despite the increased stability of trypsin in the complex, one or more of its disulfide bridges becomes much more easily reduced. We suggest that the process of complex formation with alpha1-proteinase inhibitor converts trypsin D189S into an inactive, loose structure, which serves as a "conformational trap" of the enzyme that prevents catalytic deacylation. It is also proposed that plastic region(s) of the activation domain of trypsin may play a crucial role in this inhibitor-induced structural rearrangement.

Anilino Naphthalenesulfonates↗
PubMed1997

Solution structures of staphylococcal nuclease from multidimensional, multinuclear NMR: nuclease-H124L and its ternary complex with Ca2+ and thymidine-3',5'-bisphosphate.

The solution structures of staphylococcal nuclease (nuclease) H124L and its ternary complex, (nuclease-H124L).pdTp.Ca2+, were determined by ab initio dynamic simulated annealing using 1925 NOE, 119 phi, 20 chi 1 and 112 hydrogen bond constraints for the free protein, and 2003 NOE, 118 phi, 20 chi 1 and 114 hydrogen bond constraints for the ternary complex. In both cases, the final structures display only small deviations from idealized covalent geometry. In structured regions, the overall root-mean-square deviations from mean atomic coordinates are 0.46 (+/- 0.05) A and 0.41 (+/- 0.05) A for the backbone heavy atoms of nuclease and its ternary complex, respectively. The backbone conformations of residues in the loop formed by Arg81-Gly86, which is adjacent to the active site, are more precisely defined in the ternary complex than in unligated nuclease. Also, the protein side chains that show NOEs and evidence for hydrogen bonds to pdTp (Arg35, Lys84, Tyr85, Arg87, Tyr113, and Tyr115) are better defined in the ternary complex. As has been observed previously in the X-ray structures of nuclease-WT, the binding of pdTp causes the backbone of Tyr113 to change from an extended to a left-handed alpha-helical conformation. The NMR structures reported here were compared with available X-ray structures: nuclease-H124L [Truckses et al. (1996) Protein Sci., 5, 1907-1916] and the ternary complex of wild-type staphylococcal nuclease [Loll and Lattman (1989) Proteins Struct. Funct. Genet., 5, 183-201]. Overall, the solution structures of nuclease-H124L are consistent with these crystal structures, but small differences were observed between the structures in the solution and crystal environments. These included differences in the conformations of certain side chains, a reduction in the extent of helix 1 in solution, and many fewer hydrogen bonds involving side chains in solution.

Calcium↗
PubMed1997

Probing the influence of mutations on the stability of a ferredoxin by mass spectrometry.

Hydrogen/deuterium exchange, which depends on solvent accessibility, can be probed by mass spectrometry (MS) to get information on protein conformation or protein-ligand interaction. In this work, the conformational properties of the cyanobacterium Anabaena wild-type ferredoxin as well as of two single-site mutants (Phe 65 Ala and Arg 42 Ala) were studied. After incubation of the wild type and mutant proteins in deuterated water and quenching of the exchange at low pH, the proteins were rapidly digested at high enzyme-to-substrate ratio using immobilized pepsin, and the resulting peptides were characterized using ESI-MS. We have identified specific regions for which the H-bonding or solvent accessibility properties were perturbed by the mutations. These results show that this approach can provide local information on the influence of mutations, even for a highly structured protein like ferredoxin, and sometimes in regions distant from the mutation point.

Bacterial Proteins↗
PubMed1996

Protonation-state dependence of hydrogen bond strengths and exchange rates in a serine protease catalytic triad: bovine chymotrypsinogen A.

Hydrogen-1 nuclear magnetic resonance spectroscopy was used to measure D/H fractionation factors and the temperature dependence of the rate of hydrogen exchange at two sites in the catalytic triad of chymotrypsinogen (hydrogen bond between aspartate-102 and histidine-57 and hydrogen bond between histidine-57 and serine-195) as a function of the protonation states of the constituent residues. Connectivities in one-dimensional spectra used to assign NMR data were collected at three pH values: pH 9, at which His-57 is neutral and Asp-102 is negatively charged; pH 3.5, at which His-57 is positively charged and Asp-102 is negatively charged; and pH 1, at which His-57 is positively charged and Asp-102 is neutral. The signal from H epsilon 2 of histidine-57 was assigned by reference to 1H-1H NOE connectivities at pH 3.5 to the previously assigned signals from the H epsilon 1 and H delta 2 of the same residue. The D/H fractionation factor, phi, for the hydrogen bond between Asp-102 and His-57 changed from phi = 2 at pH 9 to phi = 0.4 at pH 3.5. From studies of model systems, it may be concluded that a change of phi of this magnitude corresponds to a large increase in hydrogen bond strength. A signal from the hydrogen bond between Ser-195 and His-57 was detected only at the lower pH values studied. The D/H fractionation factor for this hydrogen bond was phi = 0.7 at pH 3.5, indicative of a moderately strong interaction. Data obtained at pH 1 indicate that the hydrogen bond between Asp-102 and His-57 is weakened but that the hydrogen bond between His-57 and Ser-195 persists. The results are consistent with the hypothesis that changes in hydrogen bonding strength serve to lower barriers along the reaction coordinate in the catalytic mechanism. Large pH-dependent changes were found in the activation enthalpy (delta H ++) for exchange with protons from the solvent at the hydrogen bond between aspartate-102 and histidine-57: delta H ++ was approximately 10-12 kcal.mol-1 higher at pH 3.5 than at pH 1 or 9.

Animals↗
PubMed1996

Engineered disulfide bonds in staphylococcal nuclease: effects on the stability and conformation of the folded protein.

Efforts to enhance the stability of proteins by introducing engineered disulfide bonds have resulted in mixed success. Most approaches to the prediction of the energetic consequences of disulfide bond formation in proteins have considered only the destabilizing effects of cross-links on the unfolded state (chain entropy model) [Pace, C. N., Grimsley, G. R., Thomson, J. A., & Barnett, B. J. (1988) J. Biol. Chem. 263, 11820-11825: Doig, A. J., & Williams, D. H. (1991) J. Mol. Biol. 217, 389-398]. It seems clear, however, that disulfide bridges also can influence the stability of the native state. In order to assess the importance of the latter effect, we have studied four variants of staphylococcal nuclease (V8 strain) each containing one potential disulfide bridge created by changing two wild-type residues to cysteines by site-directed mutagenesis. In each case, one of the introduced cysteines was within the type VIa beta turn containing cis Pro117, and the other was located in the adjacent extended loop containing Gly79. In all four cases, the overall loop size was kept nearly constant (the number of residues in the loop between the two cysteines varied from 37 to 42) so as to minimize differences from chain entropy effects. The objective was to create variants in which a change in the reduction state of the disulfide would be coupled to a change in the position of the equilibrium between the cis and trans forms of the Xxx116-Pro117 peptide bond in the folded state of the protein. The position of this equilibrium, which can be detected by NMR spectroscopy, has been shown previously to correlate with the stability of the native protein. Its determination provides a measure of strain in the folded state. The thermal stabilities and free energies for unfolding by elevated temperature and guanidinium chloride were measured for each of the four mutants under conditions in which the introduced cysteines were cross-linked (oxidized) and unlinked (reduced). In addition, reduction potentials were determined for each mutant. Formation of the different disulfide bridges was found to induce varying levels of folded state strain. The stabilization energy of a given disulfide bridge could be predicted from the measured perturbation energy for the peptide bond isomerization, provided that energetic effects on the unfolded state were calculated according to the chain entropy model. Undiagnosed strain in native states of proteins may explain the variability observed in the stabilization provided by engineered disulfide bridges.

Disulfides↗
PubMed1996

Human ferredoxin: overproduction in Escherichia coli, reconstitution in vitro, and spectroscopic studies of iron-sulfur cluster ligand cysteine-to-serine mutants.

Human ferredoxin, the human equivalent of bovine adrenodoxin, is a small iron-sulfur protein with one [2Fe-2S] cluster. It functions, as do other vertebrate ferredoxins, to transfer electrons during the processes of steroid hormone synthesis. A DNA fragment encoding the mature form of human ferredoxin was cloned into an expression vector under control of the T7 RNA polymerase/promoter system. The protein was overproduced in Escherichia coli, and the [2Fe-2S] cluster was incorporated into the protein by in vitro reconstitution. The overall yield was approximately 30 mg of purified, reconstituted ferredoxin per liter of culture. Four of the five cysteines in human ferredoxin are coordinated to the iron-sulfur cluster. First, the non-ligand cysteine (cysteine-95) was mutated to alanine, and then double mutants were created in which each of the other four cysteines (at positions 46, 52, 55, and 92) were mutated individually to serine. The wild-type ferredoxin and each of the five mutant proteins were studied by UV-visible spectroscopy and electron paramagnetic resonance spectroscopy. The EPR gav values of all five mutants were very similar to that of wild-type human ferredoxin. In the reduced state, three of the cysteine-to-serine mutants exhibited axial EPR spectra similar to that of wild-type, but one of the double mutants (C52S/C95A) exhibited a rhombic EPR spectrum. The UV-visible spectroscopic properties of the wild-type and the C95A mutant ferredoxins were identical, but those of the other cysteine-to-serine mutant proteins of human ferredoxin were quite different from those of the wild-type protein and each other. These results, along with those from cysteine-to-serine mutations in other ferredoxins, provide the basis for a more comprehensive theoretical and practical understanding of the features important to the ligation of [2Fe-2S] clusters, although they do not yet permit determination of which two cysteines ligate Fe(II) and which ligate Fe(III) in the reduced protein.

Amino Acid Sequence↗
PubMed1996

Effects of experimentally achievable improvements in the quality of NMR distance constraints on the accuracy of calculated protein structures.

New methods for collecting cross-relaxation data from proteins and nucleic acids make it possible to improve the accuracy and precision of interproton distance measurements used as input for NMR solution structure determinations. It thus is of interest to determine whether such experimentally achievable improvements in input distance constraints have significant effects on the precision and accuracy of the resulting structures. To answer this question, we have turned to a computational procedure involving the use of data simulated from a known structure, in order to allow unambiguous assessments of accuracy. The approach to improved distances evaluated here is that afforded by magnetization exchange network editing (MENE); MENE pulse sequences break the network of cross-relaxation interactions into regions that are manipulated so as to defeat certain spin-diffusion terms. A target structure was prepared from the X-ray structure of a small protein, turkey ovomucoid third domain (OMTKY3). A normal NOESY spectrum and two varieties of MENE spectra, BD-NOESY and CBD-NOESY, were simulated by means of complete relaxation matrix analysis. These results were used to create different input data sets with the same number of constraints (perfectly accurate distances derived from the target structure, more accurate distances derived from the MENE simulations, and less accurate distances derived from the NOESY simulation), and these, interpreted at different levels of precision, were used as input for solution structure calculations. The results showed that the use of more precise input data measurably improves the local precision and accuracy of calculated structures, but only if the more precise data include the actual target distance. Incorporation of the experimentally achievable, accurate distances with higher precision afforded by the MENE pulse sequences into the set of input distances was found to improve the accuracy of the resulting structures, particularly in terms of side-chain conformation.

Animals↗
PubMed1996

High-pressure denaturation of staphylococcal nuclease proline-to-glycine substitution mutants.

Our recently reported pressure-jump relaxation kinetics experiments on staphylococcal nuclease folding and unfolding [Vidugiris et al. (1995) Biochemistry 34, 4909] demonstrated that both transitions exhibit positive activation volumes, with that of folding being much larger than that of unfolding. Thus high pressure denatures proteins by slowing the rate of folding more than that of unfolding. In the present work, we take advantage of the very slow folding and unfolding rates under pressure to examine the kinetics and volume changes along the reaction coordinate for protein folding-unfolding for an interesting set of mutants of staphylococcal nuclease: P42G, P47G, P117G, and the double mutant, P47G+P117G. Previous studies have shown that replacement of an individual proline residue at position 42, 47, or 117 by glycine leads to paradoxical protein stabilization against denaturation by guanidine chloride, high temperature, or high pressure. In order to observe unfolding over an attainable pressure range, guanidine hydrochloride was employed. Within experimental error, the activation volumes and equilibrium volume changes were independent of the concentration of this denaturant and our analysis of the rate constants is consistent with the generally accepted hypothesis that this denaturant acts both by increasing the rate of unfolding and decreasing the rate of folding. We show that the stabilization resulting from each of the proline-to-glycine substitutions arises primarily from a decrease in the unfolding rate, and to a small degree, from an increase in the folding rate. The changes in rate constants upon proline-to-glycine substitution can be modeled in terms of small stabilization of the unfolded state, a greater stabilization of the transition state, and a still greater stabilization of the folded state. Although the rates were found to change for all of the mutants in the set, no changes greater than experimental error were found in the corresponding equilibrium volume changes and activation volumes for folding and unfolding. At low pressures (well below the onset of unfolding) the pressure-jump relaxation profiles for wild type proteins (both Foggi and V8) showed kinetic complexity. Although the effect was attenuated somewhat in pressure-jump profiles of one proline-to-glycine mutant (P42G), its persistence in data from all the mutants studied leads us to conclude that its origin is not cis/trans peptide bond isomerization at proline 117, 47, or 42.

Enzyme Stability↗
PubMed1996

Coupling between trans/cis proline isomerization and protein stability in staphylococcal nuclease.

The nucleases A produced by two strains of Staphylococcus aureus, which have different stabilities, differ only in the identity of the single amino acid at residue 124. The nuclease from the Foggi strain of S. aureus (by convention nuclease WT), which contains His124, is 1.9 kcal.mol-1 less stable (at pH 5.5 and 20 degrees C) than the nuclease from the V8 strain (by convention nuclease H124L), which contains Leu124. In addition, the population of the trans conformer at the Lys116-Pro117 peptide bond, as observed by NMR spectroscopy, is different for the two variants: about 15% for nuclease WT and 9% for nuclease H124L. In order to improve our understanding of the origin of these differences, we compared the properties of WT and H124L with those of the H124A and H124I variants. We discovered a correlation between effects of different residues at this position on protein stability and on stabilization of the cis configuration of the Lys116-Pro117 peptide bond. In terms of free energy, approximately 17% of the increase in protein stability manifests itself as stabilization of the cis configuration at Lys116-Pro117. This result implies that the differences in stability arise mainly from structural differences between the cis configurational isomers at Pro117 of the different variants at residue 124. We solved the X-ray structure of the cis form of the most stable variant, H124L, and compared it with the published high-resolution X-ray structure of the cis form of the most stable variant, WT (Hynes TR, Fox RO, 1991, Proteins Struct Funct Genet 10:92-105). The two structures are identical within experimental error, except for the side chain at residue 124, which is exposed in the models of both variants. Thus, the increased stability and changes in the trans/cis equilibrium of the Lys116-Pro117 peptide bond observed in H124L relative to WT are due to subtle structural changes that are not observed by current structure determination technique. Residue 124 is located in a helix. However, the stability changes are too large and follow the wrong order of stability to be explained simply by differences in helical propensity. A second site of conformational heterogeneity in native nuclease is found at the His46-Pro47 peptide bond, which is approximately 80% trans in both WT and H124L. Because proline to glycine substitutions at either residue 47 or 117 remove the structural heterogeneity at that position and increase protein stability, we determined the X-ray structures of H124L + P117G and H124L + P47G + P117G and the kinetic parameters of H124L, H124L + P47G, H124L + P117G, and H124L + P47G + P117G. The individual P117G and P47G mutations cause decreases in nuclease activity, with kcat affected more than Km, and their effects are additive. The P117G mutation in nuclease H124L leads to the same local conformational rearrangement described for the P117G mutant of WT (Hynes TR, Hodel A, Fox RO, 1994, Biochemistry 33:5021-5030). In both P117G mutants, the loop formed by residues 112-117 is located closer to the adjacent loop formed by residues 77-85, and residues 115-118 adopt a type I' beta-turn conformation with the Lys116-Gly117 peptide bond in the trans configuration, as compared with the parent protein in which these residues have a typeVIa beta-turn conformation with the Lys116-Pro117 peptide bond in the cis configuration. Addition of the P47G mutation appears not to cause any additional structural changes. However, the electron density for part of the loop containing this peptide bond was not strong enough to be interpreted.

Chemical Phenomena↗
PubMed1996

Continuous probability distribution (CUPID) analysis of potentials for internal rotations.

The continuous probability distribution (CUPID) approach for analyzing the rotamer populations from NMR spin-spin couplings and nuclear Overhauser enhancements [Z. Dzakula, W.M. Westler, A.S. Edison, and J.L. Markley, J. Amer. Chem. Soc. 114, 6195 (1992)] can be expanded to allow computation of the rotational potential from the Fourier coefficients of the angular probability distribution. This approach provides a general solution to the nonnegativity problem, which appears when lack of data causes a serious truncation in the Fourier series that defines the probability distribution. In favorable cases, this approach also allows thermodynamic characterization of internal rotation. Use of this extension of the CUPID method is illustrated by the analysis of internal rotations in an amino acid, two peptides, and an oligosaccharide from published experimental data. Three strategies have been devised for dealing with cases where the experimental input data do not provide enough information for complete reconstruction of the potential: (1) two-dimensional grid search for the undetermined third-order Fourier coefficients of the potential, (2) transfer of these coefficients from related model compounds, and (3) restriction of the magnitudes of the Fourier coefficients as required by the assumption of fast-exchange averaging of the input parameters. In addition, equations for translating uncertainties in experimental NMR input data into errors in calculated continuous probability distributions of rotamers are presented. The dependence of errors on various features of the distributions has been studied systematically from simulations. The results show that, typically, the confidence intervals are +/- 30-40 degrees for dihedral angles and +/- 0.2 for rotamer populations. For chi 1 rotamers of amino acids, the analysis is most sensitive to the uncertainties in C'-H beta couplings. A critical reexamination of the use of Gaussian functions to reconstruct a probability distribution is presented. In particular, the simplifying assumption of identical widths for all Gaussian probability peaks has been justified by showing that it does not lead to large errors in other CUPID parameters. finally, the angular dependencies of cross-relaxation rates, their uncertainties, and the potential for their use in studying chi 1 internal rotations in amino acids are discussed.

Electron Spin Resonance Spectroscopy↗
PubMed1995

Protein binding chiral discrimination of HPLC stationary phases made with whole, fragmented, and third domain turkey ovomucoid.

Individual protein domains and two domains in combination were prepared by enzymatic and chemical cleavage of turkey ovomucoid followed by isolation and purification by size-exclusion and ion-exchange chromatography. Silica bonded-phase HPLC columns were made from either whole or isolated domains of turkey ovomucoid. The protein columns were tested for chiral recognition by their abilities to resolve enantiomers among a wide range of racemates. The columns made from whole turkey ovomucoid displayed chiral activity toward many racemates, where as a combination of the first and second domain resolved only a selected number of aromatic weak bases. The first and second domains independently gave no appreciable chiral activity. The turkey ovomucoid third domain exhibited enantioselective protein binding for fused-ring aromatic weak acids. Glycosylation of the third domain did not affect chiral recognition. Titration of the third domain with model compounds in conjunction with NMR measurements enabled the identification of the amino acids responsible for binding. Molecular modeling of the ligand-protein complexation provided insights into the ability of a protein surface to discriminate enantiomers on the basis of multiple intermolecular interactions.

Amino Acid Sequence↗
PubMed1995

Structure of the trigonal form of recombinant oxidized flavodoxin from Anabaena 7120 at 1.40 A resolution.

The oxidized recombinant flavodoxin from the cyanobacterium Anabaena 7120 has been crystallized in a trigonal form. The recombinant protein has an identical primary structure to that purified directly from Anabaena, which functions as a substitute for ferredoxin in an iron-deficient environment for electron transfer from photosystem I to ferredoxin-NADP(+) reductase. X-ray data to 1.40 A were collected on a Siemens area detector. Of the 311 379 reflections collected, 36069 reflections were unique in space group P3(1)21 (a = 55.36, c = 102.59 A) with an R(merge) of 3.8%. The structure was solved by molecular replacement using coordinates from the wild-type monoclinic structure previously solved in this laboratory [Rao, Shaffie, Yu, Satyshur, Stockman & Markley (1992). Protein Sci. 1, 1413-1427]. The structure was refined with X-PLOR and SHELXL93 to a crystallographic R-factor of 13.9% for 32963 reflections with I> 2sigma(I). The final structure contains 2767 atoms including 31 flavin mononucleotide (FMN) atoms, 299 water molecules, and one sulfate ion. The protein is comprised of a central five-stranded beta-sheet surrounded by five helices and binds a single molecule of FMN at the C-terminus of the sheet. The trigonal protein structure and the crystal packing are compared with the monoclinic wild-type protein. Helix alpha3 in this structure is less distorted than in the monoclinic structure and shows additional hydrogen bonds in the N-terminal portion of the helix. The trigonal structure is extensively hydrogen bonded in three major areas with neighboring molecules compared with five regions in the monoclinic structure, but using significantly fewer hydrogen bonds to stabilize the lattice. There are several hydrogen bonds to the amide groups from water molecules several of which stabilize and extend the ends of the beta-sheet.

Journal Article↗
PubMed1995

Evidence for a molten globule-like transition state in protein folding from determination of activation volumes.

One of the most important, yet elusive, aspects of the protein folding question lies in the nature of the transition state. Direct information about the structural properties of the transition state can be obtained from determination of the activation volumes for the folding and unfolding transitions. The present pressure-jump relaxation study on the folding/unfolding of staphylococcal nuclease reveals that the volume of the protein-solvent system is larger in the transition state than in either the folded or unfolded states. Moreover, the activation volume of folding is much larger than that of unfolding. These results support a molten globule-like model for the transition state of nuclease in which the polypeptide chain is in a collapsed, loosely packed, solvent-excluded structure. In this model, hydrophobic collapse with concomitant desolvation is the rate-limiting step in the folding of the polypeptide chain, and solvent-excluded expansion of the folded state is the rate-limiting step in protein unfolding.

Kinetics↗
PubMed1995

Protein expression, selective isotopic labeling, and analysis of hyperfine-shifted NMR signals of Anabaena 7120 vegetative [2Fe-2S]ferredoxin.

Two alternative T7 RNA promoter/polymerase systems have been employed for the heterologous expression of a plant-type [2Fe-2S]ferredoxin, Anabaena 7120 vegetative ferredoxin, in Escherichia coli at high levels (approximately 20 mg/liter of culture). One system was used when 15N-labeling the ferredoxin uniformly by growing E. coli with 15NH4Cl as the nitrogen source; the other was used in conjunction with auxotrophic host strains to enrich the protein selectively by incorporating 2H-, 13C-, and 15N-labeled amino acids. The labeled ferredoxin samples were studied by 1H, 2H, 13C, and 15N NMR spectroscopy. Results from 1H and 2H NMR studies of samples containing [2H alpha]Cys, [2H beta 2, beta 3]Cys, [13 C beta]-Cys, and [15N]Cys have confirmed previous cysteinyl proton resonance assignments (L. Skjeldal, W. M. Westler, B.-H. Oh, A. M. Krezel, H. M. Holden, B. L. Jacobson, I. Rayment, and J. L. Markley (1991) Biochemistry 30, 7363-7368). All four 13C NMR peaks arising from the four cysteinyl beta-carbons and all four 15N NMR peaks from the four cysteinyl nitrogens were resolved in spectra of both the oxidized and reduced ferredoxins. The nitrogen resonance of Cys46, which is located in a unique (Ala-Cys) dipeptide, was assigned by detection of 13Ci-15Ni+1 coupling in a ferredoxin sample with incorporated [13C']Ala and [15N]Cys. The nitrogen signal of Cys 41 was assigned tentatively on the basis of its chemical shift and T1 relaxation time. The cysteinyl beta-carbon resonances in the reduced state have been assigned to individual residues on the basis of correlations with their (previously assigned) beta-protons. The beta-carbons resonance from Cys46 in the oxidized state has been assigned by its correlation with the corresponding resonance in the reduced state; this was accomplished by following the progressive air oxidation of a protein sample reduced by dithionite in the presence of methyl viologen. The spin-lattice relaxation times of the beta-carbons of the two cysteines coordinated to Fe)III) were similar in the oxidized and reduced states. This suggests that the antiferromagnetic coupling present in the reduced cluster has little influence on the electronic relaxation time of the Fe(III). Studies of the temperature dependence of the 1H, 13C, and 15N signals of the cysteinyl ligands to the [2Fe-2S] cluster show that the slope of the temperature dependence (delta delta/delta T-1) can be different for different atom types within a given residue. For example, in the reduced ferredoxin, although delta delta/delta T-1 is positive for Cys49 1H beta 2 and 1H beta 3, it is negative for Cys49 13C beta. Although delta delta/delta T-1 is negative for protons of cysteines ligated to Fe(II) and positive for protons of cysteines ligated to Fe(III), it is positive for all the cysteinyl nitrogens.(ABSTRACT TRUNCATED AT 400 WORDS)

Anabaena↗