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J L Markley

Publications and source records attributed to J L Markley.

At least 73 records · Page 4Linked to original sources
PubMed1995

Multinuclear magnetic resonance and mutagenesis studies of the histidine residues of human mitochondrial ferredoxin.

Human mitochondrial ferredoxin is a [2Fe-2S] protein that functions to transfer electrons from NADPH-dependent ferredoxin reductase to cytochrome P450 enzymes. Two of the three histidines of human ferredoxin are strictly conserved in the sequences of all known vertebrate ferredoxins, and one of these (His56) is adjacent to Cys55, which serves as one of the ligands to the iron-sulfur cluster. All but 16 of its residues show sequence identity with those of bovine ferredoxin. It has been proposed for bovine ferredoxin that His56 hydrogen bonds with a labile sulfur and that the reduction of the iron-sulfur center is accompanied by the uptake of a proton by this histidine [Lambeth, J. D., Seybert, D. W., Lancaster, J. R., Jr., Salerno, J. C., & Kamin, H. (1982) Mol. Cell. Biochem. 45, 13-31]. In this paper, we report procedures for labeling human ferredoxin uniformly with 15N using 15NH4Cl and selectively with 13C by the incorporation of [U-13C]histidine. Most of the imidazole 1H, 13C, and 15N resonances of the three histidines have been assigned by heteronuclear two-dimensional single- and multiple-bond correlation spectroscopy. Site-directed mutagenesis was used in assigning the NMR signals from His56. The pKa values of His10 (6.5) and His62 (5.8) in oxidized human ferredoxin were found to be similar to those reported previously for the corresponding residues of bovine ferredoxin [Greenfield, N. J., Wu, X., & Jordan, F. (1989) Biochim. Biophys. Acta 995, 246-254; Miura, S., Tamita, S., & Ichikawa, Y. (1991) J. Biol. Chem. 266, 19212-19216].(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Analysis of the hyperfine-shifted nitrogen-15 resonances of the oxidized form of Anabaena 7120 heterocyst ferredoxin.

Hyperfine-shifted nitrogen signals have been detected in one-dimensional 15N NMR spectra of oxidized Anabaena 7120 heterocyst ferredoxin labeled uniformly with 15N. Several of these have been classified by amino acid type by reference to results from selective 15N-labeling studies. Remarkable agreement is seen between a dipole-dipole analysis of the 15N T1 relaxation and the distances of several of the nitrogens from the irons of the cluster as derived from the X-ray structure of this protein [Jacobson, B. L., Chae, Y. K., Markley, J. L., Rayment, I., & Holden, H. M. (1993) Biochemistry 32, 6788-6793]. The agreement is within experimental error for hyperfine-shifted nitrogens that are at least 4.2 A distant from either of the irons of the cluster; however, the simple model appears to fail for hyperfine-shifted nitrogens that are closer to the cluster. The failure of the model for short distances may stem either from a breakdown of the point-dipole approximation and/or from neglect of delocalization of unpaired electron density from the iron ions to other atoms. Even with the above limitations, dipolar analysis of 15N relaxation should provide useful distance constraints for solution-state studies of iron-sulfur proteins.

Anabaena↗
PubMed1995

Mutations of surface residues in Anabaena vegetative and heterocyst ferredoxin that affect thermodynamic stability as determined by guanidine hydrochloride denaturation.

The stability properties of oxidized wild-type (wt) and site-directed mutants in surface residues of vegetative (Vfd) and heterocyst (Hfd) ferredoxins from Anabaena 7120 have been characterized by guanidine hydrochloride (Gdn-HCl) denaturation. For Vfd it was found that mutants E95K, E94Q, F65Y, F65W, and T48A are quite similar to wt in stability. E94K is somewhat less stable, whereas E94D, F65A, F65I, R42A, and R42H are substantially less stable than wt. R42H is a substitution found in all Hfds, and NMR comparison of the Anabaena 7120 Vfd and Hfd showed the latter to be much less stable on the basis of hydrogen exchange rates (Chae YK, Abildgaard F, Mooberry ES, Markley JL, 1994, Biochemistry 33:3287-3295); we also find this to be true with respect to Gdn-HCl denaturation. Strikingly, the Hfd mutant H42R is more stable than the wt Hfd by precisely the amount of stability lost in Vfd upon mutating R42 to H (2.0 kcal/mol). On the basis of comparison of the X-ray crystal structures of wt Anabaena Vfd and Hfd, the decreased stabilities of F65A and F65I can be ascribed to increased solvent exposure of interior hydrophobic groups. In the case of Vfd mutants E94K and E94D, the decreased stabilities may result from disruption of a hydrogen bond between the E94 and S47 side chains. The instability of the R42 mutants is also most probably due to decreased hydrogen bonding capabilities.(ABSTRACT TRUNCATED AT 250 WORDS)

Anabaena↗
PubMed1995

Comparison of the accuracy of protein solution structures derived from conventional and network-edited NOESY data.

Network-editing experiments are variants of the basic NOESY experiment that allow more accurate direct measurement of interproton distances in macromolecules by defeating specific spin-diffusion pathways. Two network-editing approaches, block-decoupled NOESY and complementary-block-decoupled-NOESY, were applied as three-dimensional, heteronuclear-edited experiments to distance measurement in a small protein, turkey ovomucoid third domain (OMTKY3). Two-hundred and twelve of the original 655 distance constraints observed in this molecule (Krezel AM et al., 1994, J Mol Biol 242:203-214) were improved by their replacement by distances derived from network-edited spectra, and distance geometry/simulated annealing solution structure calculations were performed from both the unimproved and improved distance sets. The resulting two families of structures were found to differ significantly, the most important differences being the hinge angle of a beta-turn and an expansion of the sampled conformation space in the region of the reactive-site loop. The structures calculated from network-editing data are interpreted as a more accurate model of the solution conformation of OMTKY3.

Animals↗
PubMed1995

Theory and application of the maximum likelihood principle to NMR parameter estimation of multidimensional NMR data.

A general theory has been developed for the application of the maximum likelihood (ML) principle to the estimation of NMR parameters (frequency and amplitudes) from multidimensional time-domain NMR data. A computer program (ChiFit) has been written that carries out ML parameter estimation in the D-1 indirectly detected dimensions of a D-dimensional NMR data set. The performance of this algorithm has been tested with experimental three-dimensional (HNCO) and four-dimensional (HN(CO)-CAHA) data from a small protein labeled with 13C and 15N. These data sets, with different levels of digital resolution, were processed using ChiFit for ML analysis and employing conventional Fourier transform methods with prior extrapolation of the time-domain dimensions by linear prediction. Comparison of the results indicates that the ML approach provides superior frequency resolution compared to conventional methods, particularly under conditions of limited digital resolution in the time-domain input data, as is characteristic of D-dimensional NMR data of biomolecules. Close correspondence is demonstrated between the results of analyzing multidimensional time-domain NMR data by Fourier transformation, Bayesian probability theory [Chylla, R.A. and Markley, J.L. (1993) J. Biomol. NMR, 3, 515-533], and the ML principle.

Algorithms↗
PubMed1995

1H, 13C and 15N chemical shift referencing in biomolecular NMR.

A considerable degree of variability exists in the way that 1H, 13C and 15N chemical shifts are reported and referenced for biomolecules. In this article we explore some of the reasons for this situation and propose guidelines for future chemical shift referencing and for conversion from many common 1H, 13C and 15N chemical shift standards, now used in biomolecular NMR, to those proposed here.

Carbon Isotopes↗
PubMed1995

NMR spectroscopic studies of paramagnetic proteins: iron-sulfur proteins.

Newer NMR methods, particularly in conjunction with stable isotope labeling, offer exciting approaches to structure-function studies of paramagnetic proteins. This review examines progress in NMR spectroscopy of iron-sulfur proteins. The application of multidimensional multinuclear NMR spectroscopy to iron-sulfur proteins and the optimization of NMR pulse sequences for rapidly relaxing spins have allowed investigators to determine sequence-specific assignments for numerous NMR signals in rubredoxins, ferredoxins, and high-potential iron proteins, including those from the cysteine residues that ligate iron ions or iron-sulfur clusters. These advances enable one to interpret the wealth of information derived from NMR parameters, such as the temperature and pH dependence of chemical shifts and the relaxation properties of the resonances that report on interactions between nuclei and between nuclei and unpaired electron density. This information is being used to test theoretical descriptions of electron distribution within these molecules and to model the structures and dynamic properties of the proteins in solution. Mutagenesis of these proteins, in conjunction with NMR studies, is beginning to reveal which residues are important for cluster formation and stability and which residues play a role in electron transfer to and from redox partner proteins.

Bacterial Proteins↗
PubMed1994

Solution structure of turkey ovomucoid third domain as determined from nuclear magnetic resonance data.

The solution structure of the 56 amino acid residue turkey ovomucoid third domain was determined by n.m.r. methods. Of the 661 distance constraints used in the calculations, 120 were determined by quadratic approximation of the cross-relaxation rates. The remaining constraints were crudely estimated from a more standard analysis of NOESY spectra. Additionally, 29 torsion angle constraints, 17 hydrogen bonds, and three disulfide bridges were used in the structure calculations. Stereospecific assignments were accomplished for 24 beta-methylene groups and six isopropyl methyl groups (43% chiral assignments). The addition of more accurate distance constraints to the distance geometry/simulated annealing approach resulted in a significant reduction in the dispersion of calculated backbone torsion angles and root-mean-square deviations between structures. Detailed comparisons have been made between the n.m.r. structures of OMTKY3 and published X-ray structures of the same protein and of closely related avian ovomucoid third domains. The refinement with more accurate distance constraints reduced differences between families of the n.m.r. and the X-ray structures.

Amino Acids↗
PubMed1994

Solution structure of reactive-site hydrolyzed turkey ovomucoid third domain by nuclear magnetic resonance and distance geometry methods.

The solution structure of reactive-site hydrolyzed turkey ovomucoid third domain (OMTKY3*) was determined by n.m.r. methods. A total of 655 distance constraints was applied in a distance geometry/simulated annealing approach to calculate a family of structures consistent with the n.m.r. data. The input data included 24 torsion angle constraints, 14 hydrogen bonds, 611 constraints derived from two-dimensional nuclear Overhauser enhancement spectroscopy data, and three disulfide bridges. Stereospecific assignments were included for the hydrogens of 26 beta-methylene groups and for seven isopropyl methyl groups (46% chiral assignments). OMTKY3* in solution retains the global fold and overall secondary structure of the intact inhibitor (OMTKY3) but exhibits local structural differences at and adjacent to the clip site. In particular, the hydrogen-bonding network observed at the reactive-site of the intact inhibitor is disrupted, and the position of Tyr20 is altered in the modified inhibitor. No evidence was found for ion pairing between the oppositely charged termini at the clip site. Surprisingly, in light of numerous changes indicating that OMTKY3* is less stable than OMTKY3, rotation of the Tyr31 ring was found to be slow in OMTKY3* at 30 degrees C. In OMTKY3, slow rotation of the Tyr31 ring was observed only at temperatures below 15 degrees C. The n.m.r. structures of OMTKY3* are compared here with the similarly calculated structures of OMTKY3. This represents the first comparison of an intact and modified (reactive-site clipped) proteinase inhibitor under identical conditions. On comparison with published X-ray structures of modified avian ovomucoid third domains from two other species, the present structure of OMTKY3* in solution was found to resemble that of the Japanese quail protein (OMJPQ3*) more closely than that of the more closely homologous silver pheasant protein (OMSVP3*).

Amino Acids↗
PubMed1994

Optical, EPR, and 1H NMR spectroscopy of serine-ligated [2Fe-2S] ferredoxins produced by site-directed mutagenesis of cysteine residues in recombinant Anabaena 7120 vegetative ferredoxin.

Anabaena 7120 vegetative ferredoxin is a plant-type [2Fe-2S] ferredoxin that contains only four cysteine residues. The four cysteines (Cys41, Cys46, Cys49, and Cys79), which ligate the iron-sulfur cluster, were mutated individually to serine. The wild-type and mutant apoprotein genes were overexpressed in Escherichia coli, and the iron-sulfur cluster was assembled in vitro by adding iron and sulfide. UV-vis, EPR, and 1H NMR spectra were recorded on the wild-type ferredoxin and mutants. The optical spectra of all mutant proteins, in the oxidized state, differed from that of wild-type ferredoxin. Three of the mutant proteins (Cys46Ser, Cys49Ser, and Cys79Ser) exhibited a rhombic EPR spectrum in the reduced state, but one (Cys41Ser) showed a near-axial EPR spectrum. The 1H NMR spectra of each of the four oxidized mutants contained a group of broad, hyperfine-shifted peaks between 20 and 30 ppm with anti-Curie temperature dependence. The pattern of these peaks was different for each mutant, and all were distinct from that of the wild-type ferredoxin. Because of problems with protein stability, it was possible to obtain NMR spectra of only two of the mutants when reduced. The downfield hyperfine 1H NMR spectrum of the reduced Cys46Ser mutant resembled that of wild-type ferredoxin, but that of the Cys49Ser mutant did not. The hyperfine-shifted resonances of the 1H NMR spectrum of the reduced Cys46Ser mutant were assigned on the basis of results from temperature dependence studies, measurements of nuclear Overhauser effect, and 1H NMR spectra of the mutant labeled with [beta-2H]cysteine. Four hyperfine-shifted peaks of reduced Cys49Ser at 298 K were observed at 173, 120, 32, and 18 ppm. These peaks exhibited Curie-type temperature dependence and were tentatively assigned to protons from residues coordinated to Fe(III). The reduced Cys49Ser mutant showed an additional 1H NMR peak at -15 ppm (at 298 K) with Curie-type temperature dependence whose origin is unknown at present. [2Fe-2S] clusters can be placed into three different classifications according to their EPR lines shapes, NMR spectra, and reduction potentials: plant type, vertebrate type, and Rieske type. The EPR and NMR results obtained here reveal that mutant Cys46Ser has a "plant-type" cluster but that mutant Cys49Ser has a "vertebrate-type" cluster. Cysteine to serine mutations have been employed in the past to probe whether particular cysteine residues participate as iron-sulfur ligands.(ABSTRACT TRUNCATED AT 400 WORDS)

Anabaena↗
PubMed1994

Multinuclear, multidimensional NMR studies of Anabaena 7120 heterocyst ferredoxin. Sequence-specific resonance assignments and secondary structure of the oxidized form in solution.

Sequence-specific assignments were determined for the diamagnetic proton resonances from recombinant Anabaena 7120 heterocyst ferredoxin (M(r) = 11,000) produced in Escherichia coli. Several samples selectively labeled with nitrogen-15 were prepared for use in two-dimensional heteronuclear multiple quantum coherence (HMQC) [Müller, L. (1979) J. Am. Chem. Soc. 101, 4481-4484] experiments. A sample uniformly labeled with nitrogen-15 was also prepared and used in two three-dimensional experiments: NOESY-HMQC and TOCSY-HMQC [Zuiderweg, E. R. P., & Fesik, S. W. (1989) Biochemistry 28, 2387-2391; Marion, D., Ikura, M., Tsuchudin, R., & Bax, A. (1989) J. Magn. Reson. 85, 393-399]. The sequential assignment strategy relied on the detection of 15N-edited interresidue 1H alpha i/1HNi+1 NOE connectivities. Starting points and checks were provided by HMQC spectra of the selectively labeled samples. A sample doubly labeled with carbon-13 and nitrogen-15 was also prepared and used in three triple-resonance experiments: HNCA, HNCO, and HN(CO)CA [Ikura, M., Kay, L. E., & Bax, A. (1990) Biochemistry 29, 4659-4667; Kay, L. E., Ikura, M., Tschudin, R., & Bax, A. (1990) J. Magn. Reson. 89, 496-514]. The HNCA and HN(CO)CA spectra, which were used to confirm assignments from NOE connectivities, provided independent sequential assignments from spin couplings. Resonances from 18 residues were not seen in the diamagnetic region of the NMR spectrum. Several of these residues are very close to the [2Fe-2S] cluster, and their absence is explained by paramagnetic broadening and/or shifting.(ABSTRACT TRUNCATED AT 250 WORDS)

Anabaena↗
PubMed1994

Hydrogen bonding in proteins as studied by amide hydrogen D/H fractionation factors: application to staphylococcal nuclease.

The D/H fractionation factor (sigma) is the extent to which a hydrogen at a particular site becomes enriched in 2H over 1H relative to the solvent. A growing body of experimental evidence suggest that there is a correlation between the value of the fractionation factor and hydrogen-bond strength, with a lower sigma value reflecting a stronger hydrogen bond. Fractionation factors of 60% of the individual backbone amide hydrogens in the staphylococcal nuclease V8 variant (H124L) have been measured for the enzyme in the presence and absence of bound ligands (the activating ion Ca2+ and the inhibitor thymidine 3',5'-bisphosphate). The method used employed two-dimensional 1H-15N nuclear magnetic resonance analysis of uniformly 15N-labeled protein in mixed H2O/D2O solvents. Fractionation factors of individual residues were found to range from 0.3 (T120) to 1.5 (L38). The sigma value of 0.3 for the NH of T120, which is the lowest fractionation factor reported for any system yet studied, suggests that the hydrogen bond between T120 HN and D77 O delta 1 is unusually strong. The results of previous site-directed mutagenesis experiments [Hinck, A. P. (1993) Ph.D. Thesis, University of Wisconsin-Madison, Madison, WI] support the notion that formation of this hydrogen bond is important to maintain the stability and conformation of the native state. The sigma value averaged over all residues was approximately 0.85 for both the unligated and ligated enzymes. Residues in alpha-helices displayed a slightly lower average sigma value (0.79), whereas residues with solvent-exposed amide hydrogens exhibited a slightly higher average figure (0.98).(ABSTRACT TRUNCATED AT 250 WORDS)

Amides↗
PubMed1994

Near-microscopic magnetic resonance imaging of the brains of phenylalanine hydroxylase-deficient mice, normal littermates, and of normal BALB/c mice at 9.4 Tesla.

The near-microscopic resolution of the mouse brain, by magnetic resonance imaging (MRI) at 9.4 T, permits in situ examination of the entire brain and longitudinal studies of neural development. MRI can be utilized to reveal brain structure at a resolution of 100 microns in the X, Y, and Z planes of brain, to differentiate the gray from white (myelin-rich) matter, and to reveal the ventricular compartments. The present report describes the structure of normal BALB/c mouse brain as revealed by imaging at 9.4 T and by histological stains; the structure of normal brain is compared with that from a phenylalanine hydroxylase-deficient mouse mutant line (Pah(enu2)) and those from normal littermates. The brains of patients with phenylketonuria (PKU) were reported to have demyelination and other structural abnormalities revealed by magnetic resonance imaging (MRI). Therefore, high-resolution MRI was used to examine the brain of this mutant, an animal model for the study of human phenylketonuria. Our study revealed no evidence of demyelination or other abnormalities in the brains of Pah(enu2) mice. Histologically, the mutant and normal mouse brains appear similar. This is consistent with a recent study from our laboratory which demonstrated that the histology of the brain of an untreated male patient, who died with PKU at the age of 29, was similar to control brain with the exception of changes directly related to visual blindness and seizures experienced by the patient.

Adult↗
PubMed1994

Calculations of one-, two- and three-bond nuclear spin-spin couplings in a model peptide and correlations with experimental data.

We present ab initio calculations of the Fermi contact term and experimental correlations of six coupling constants, 3JHNH alpha, 1JC alpha H alpha, 2JC'H alpha, 1JC alpha N, 2JC alpha N and 1JC'N, in a peptide as functions of the backbone dihedral angles, phi and psi. Given estimates of experimental uncertainties, we find semiquantitative experimental correlations for 3JHNH alpha, 1JC alpha N and 2JC alpha N, qualitative correlations for 1JC alpha H alpha and 2JC'H alpha, but no experimental correlations of practical utility for 1JC'N, owing to its complex dependence on at least four dihedral angles. Errors in the estimation of dihedral angles from X-ray crystallographic data for proteins, which result from uncertainties in atom-to-atom distances, place substantial limitations on the quantitative reliability of coupling constant calculations fitted to such data. In the accompanying paper [Edison, A.S. et al., J. Biomol. NMR, 4, 543-551] we apply the results of the coupling constant calculations presented here to the estimation of phi and psi angles in staphylococcal nuclease from experimental coupling constants.

Alanine↗
PubMed1994

Estimates of phi and psi torsion angles in proteins from one-, two- and three-bond nuclear spin-spin couplings: application to staphylococcal nuclease.

Calculated coupling constants (3JHNH alpha, 1JC alpha H alpha, 2JC'H alpha, 1JC alpha N and 2JC alpha N) from our accompanying paper [Edison, A.S. et al. (1994) J. Biomol. NMR, 4, 519-542] have been used to generate error surfaces that can provide estimates of the phi and psi angles in proteins. We have used experimental coupling data [3JHNH alpha: Kay, L.E. et al. (1989) J. Am. Chem. Soc., 111, 5488-5490; 1JC alpha H alpha: Vuister, G. W. et al. (1993) J. Biomol. NMR, 3, 67-80; 2JC'H alpha: Vuister, G.W. and Bax, A. (1992) J. Biomol. NMR, 2, 401-405; 1JC alpha N and 2JC alpha N: Delaglio, F. et al. (1991) J. Biomol. NMR, 1, 439-446] to create error surfaces for selected residues of the protein staphylococcal nuclease. The residues were chosen to include all those with five experimental couplings, as well as some with four experimental couplings, to demonstrate the relative importance of 3JHNH alpha and 1JC alpha H alpha. For most of the cases, we obtained good agreement between the X-ray structure [Loll, P.J. and Lattman, E.E. (1989) Protein Struct. Funct. Genet., 5, 183-201] and the NMR data.

Magnetic Resonance Spectroscopy↗
PubMed1994

Evaluation of an algorithm for the automated sequential assignment of protein backbone resonances: a demonstration of the connectivity tracing assignment tools (CONTRAST) software package.

The peptide sequential assignment algorithm presented here was implemented as a macro within the CONnectivity TRacing ASsignment Tools (CONTRAST) computer software package. The algorithm provides a semi- or fully automated global means of sequentially assigning the NMR backbone resonances of proteins. The program's performance is demonstrated here by its analysis of realistic computer-generated data for IIIGlc, a 168-residue signal-transducing protein of Escherichia coli [Pelton et al. (1991) Biochemistry, 30, 10043-10057]. Missing experimental data (19 resonances) were generated so that a complete assignment set could be tested. The algorithm produces sequential assignments from appropriate peak lists of nD NMR data. It quantifies the ambiguity of each assignment and provides ranked alternatives. A 'best first' approach, in which high-scoring local assignments are made before and in preference to lower scoring assignments, is shown to be superior (in terms of the current set of CONTRAST scoring routines) to approaches such as simulated annealing that seek to maximize the combined scores of the individual assignments. The robustness of the algorithm was tested by evaluating the effects of imposed frequency imprecision (scatter), added false signals (noise), missing peaks (incomplete data), and variation in user-defined tolerances on the performance of the algorithm.

Algorithms↗