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J L Markley

Publications and source records attributed to J L Markley.

At least 37 records · Page 2Linked to original sources
PubMed1998

NMR solution structure of type II human cellular retinoic acid binding protein: implications for ligand binding.

The structure of human apo-cellular retinoic acid binding protein II (apo-CRABPII) in solution at pH 7.3 has been determined by NMR spectroscopy. The sequential assignments of the 1H, 13C, and 15N resonances of apo-CRABPII were established by multinuclear, multidimensional NMR spectroscopy. The solution structure of apo-CRABPII was derived from 2382 experimental NMR restraints using a hybrid distance geometry-simulated annealing protocol. The root-mean-square deviation of the ensemble of 25 refined conformers that represent the structure from the mean coordinate set derived from them was 0.54 +/- 0.18 and 0.92 +/- 0.20 A for the backbone atoms and all heavy atoms, respectively, of all residues except Ala32-Pro39 and Thr57-Glu62, which are in disordered regions. The solution structure of apo-CRABPII is similar to the crystal structure of holo-CRABPII [Kleywegt, G. J., Bergfors, T., Senn, H., Le Motte, P., Gsell, B., Shudo, K., and Jones, T. A. (1994) Structure 2, 1241-1258] except the ligand entrance, which is sufficiently enlarged in the apoprotein to be readily accessible to retinoic acid. The enlargement of the ligand entrance of apo-CRABPII relative to that of holo-CRABPII is due mainly to a concerted conformational change in three structural elements, namely, the second helix, the betaC-betaD loop, and the betaE-betaF loop. Furthermore, the ligand-binding pocket of apo-CRABPII showed evidence of dynamic disorder; among the 21 residues that constitute this pocket, 16 residues had weak or no detectable cross-peaks in the two-dimensional 1H-15N HSQC spectrum recorded under conditions of minimal water saturation or dephasing. Apo-CRABPII is largely monomeric in solution, with no evidence for the dimeric structure shown in the crystal structure of apo-CRABPI which was suggested to be a prerequisite for ligand entry [Thompson, J. R., Bratt, J. M., and Banaszak, L. J. (1995) J. Mol. Biol. 252, 433-446]. Thus, the widening of the ligand entrance required for entry of retinoic acid appears to be a property of monomeric apo-CRABPII.

Crystallization↗
PubMed1998

Recommendations for the presentation of NMR structures of proteins and nucleic acids--IUPAC-IUBMB-IUPAB Inter-Union Task Group on the standardization of data bases of protein and nucleic acid structures determined by NMR spectroscopy.

The recommendations presented here are designed to support easier communication of NMR data and NMR structures of proteins and nucleic acids through unified nomenclature and reporting standards. Much of this document pertains to the reporting of data in journal articles; however, in the interest of the future development of structural biology, it is desirable that the bulk of the reported information be stored in computer-accessible form and be freely accessible to the scientific community in standardized formats for data exchange. These recommendations stem from an IUPAC-IUBMB-IUPAB inter-union venture with the direct involvement of ICSU and CODATA. The Task Group has reviewed previous formal recommendations and has extended them in the light of more recent developments in the field of biomolecular NMR spectroscopy. Drafts of the recommendations presented here have been examined critically by more than 50 specialists in the field and have gone through two rounds of extensive modification to incorporate suggestions and criticisms.

Amino Acid Sequence↗
PubMed1998

Solution structure and backbone dynamics of component IV Glycera dibranchiata monomeric hemoglobin-CO.

The solution structure and backbone dynamics of the recombinant, ferrous CO-ligated form of component IV monomeric hemoglobin from Glycera dibranchiata (GMH4CO) have been characterized by NMR spectroscopy. Distance geometry and simulated annealing calculations utilizing a total of 2550 distance and torsion angle constraints yielded an ensemble of 29 structures with an overall average backbone rmsd of 0.48 A from the average structure. Differences between the solution structure and a related crystal structure are confined to regions of lower precision in either the NMR or X-ray structure, or in regions where the amino acid sequences differ. 15N relaxation measurements at 76.0 and 60.8 MHz were analyzed with an extended model-free approach, and revealed low-frequency motions in the vicinity of the heme, concentrated in the F helix. Amide proton protection factors were obtained from H-D amide exchange measurements on 15N-labeled protein. Patterns in the backbone dynamics and protection factors were shown to correlate with regions of heterogeneity and disorder in the ensemble of NMR structures and with large crystallographic B-factors in the X-ray structures. Surprisingly, while the backbone atoms of the F helix have higher rmsds and larger measures of dynamics on the microsecond to millisecond time scale than the other helices, amide protection factors for residues in the F helix were observed to be similar to those of the other helices. This contrasts with H-D amide exchange measurements on sperm whale myoglobin which indicated low protection for the F helix (S. N. Loh and B. F. Volkman, unpublished results). These results for GMH4 suggest a model in which the F helix undergoes collective motions as a relatively rigid hydrogen-bonded unit, possibly pivoting about a central position near residue Val87.

Amino Acid Sequence↗
PubMed1998

Recommendations for the presentation of NMR structures of proteins and nucleic acids.

The recommendations presented here are designed to support easier communication of NMR data and NMR structures of proteins and nucleic acids through unified nomenclature and reporting standards. Much of this document pertains to the reporting of data in journal articles; however, in the interest of the future development of structural biology, it is desirable that the bulk of the reported information be stored in computer-accessible form and be freely accessible to the scientific community in standardized formats for data exchange. These recommendations stem from an IUPAC-IUBMB-IUPAB inter-union venture with the direct involvement of ICSU and CODATA. The Task Group has reviewed previous formal recommendations and has extended them in the light of more recent developments in the field of biomolecular NMR spectroscopy. Drafts of the recommendations presented here have been examined critically by more than 50 specialists in the field and have gone through two rounds of extensive modification to incorporate suggestions and criticisms.

Databases as Topic↗
PubMed1998

An electrochemical, kinetic, and spectroscopic characterization of [2Fe-2S] vegetative and heterocyst ferredoxins from Anabaena 7120 with mutations in the cluster binding loop.

Residues within the cluster binding loops of plant-type [2Fe-2S] ferredoxins are highly conserved and serve to structurally stabilize this unique region of the protein. We have investigated the influence of these residues on the thermodynamic reduction potentials and rate constants of electron transfer to ferredoxin:NADP+ reductase (FNR) by characterizing various single and multiple site-specific mutants of both the vegetative (VFd) and the heterocyst (HFd) [2Fe-2S] ferredoxins from Anabaena. Incorporation of residues from one isoform into the polypeptide backbone of the other created hybrid mutants whose reduction potentials either were not significantly altered or were shifted, but did not reconcile the 33-mV potential difference between VFd and HFd. The reduction potential of VFd appears relatively insensitive to mutations in the binding loop, excepting nonconservative variations at position 78 (T78A/I) which resulted in approximately 40- to 50-mV positive shifts compared to wild type. These perturbations may be linked to the role of the T78 side chain in stabilizing an ordered water channel between the iron-sulfur cluster and the surface of the wild-type protein. While no thermodynamic barrier to electron transfer to FNR is created by these potential shifts, the electron-transfer reactivities of mutants T78A/I (as well as T48A which has a wild-type-like potential) are reduced to approximately 55-75% that of wild type. These studies suggest that residues 48 and 78 are involved in the pathway of electron transfer between VFd and FNR and/or that mutations at these positions induce a unique, but unproductive orientation of the two proteins within the protein-protein complex.

Anabaena↗
PubMed1998

Solution structure of the transmembrane H+-transporting subunit c of the F1F0 ATP synthase.

Subunit c is the H+-translocating component of the F1F0 ATP synthase complex. H+ transport is coupled to conformational changes that ultimately lead to ATP synthesis by the enzyme. The properties of the monomeric subunit in a single-phase solution of chloroform-methanol-water (4:4:1) have been shown to mimic those of the protein in the native complex. Triple resonance NMR experiments were used to determine the complete structure of monomeric subunit c in this solvent mixture. The structure of the protein was defined by >2000 interproton distances, 64 (3)JN alpha, and 43 hydrogen-bonding NMR-derived restraints. The root mean squared deviation for the backbone atoms of the two transmembrane helices was 0.63 A. The protein folds as a hairpin of two antiparallel helical segments, connected by a short structured loop. The conserved Arg41-Gln42-Pro43 form the top of this loop. The essential H+-transporting Asp61 residue is located at a slight break in the middle of the C-terminal helix, just prior to Pro64. The C-terminal helix changes direction by 30 +/- 5 degrees at the conserved Pro64. In its protonated form, the Asp61 lies in a cavity created by the absence of side chains at Gly23 and Gly27 in the N-terminal helix. The shape and charge distribution of the molecular surface of the monomeric protein suggest a packing arrangement for the oligomeric protein in the F0 complex, with the front face of one monomer packing favorably against the back face of a second monomer. The packing suggests that the proton (cation) binding site lies between packed pairs of adjacent subunit c.

Amino Acid Sequence↗
PubMed1998

Pressure denaturation of proteins: evaluation of compressibility effects.

One of the key pieces of information from pressure denaturation experiments is the standard volume change for unfolding (Delta V(o)). The pressure dependence of the volume change, the standard compressibility change (Delta K(o)T), is typically assumed to be zero in the analysis of these experiments. We show here that this assumption can be incorrect and that the neglect of compressibility differences can skew the interpretation of experimental results. Analysis of experimental, variable-pressure NMR data for bovine pancreatic ribonuclease A in 2H2O at pH 2.0 and 295 K yielded the following statistically significant, non-zero values: Delta K(o) T = 0.015 +/- 0.002 mL mol-1 bar-1, Delta V(o) = -21 +/- 2 mL mol-1, and Delta G(o) = 2.8 +/- 0.3 kcal mol-1. The experimental protein stability is in good agreement with one (Delta G(o) = 2.5 kcal mol-1) determined independently for the same protein by calorimetry at atmospheric pressure under equivalent conditions [Makhatadze, G. I., Clore, G. M., and Gronenborn, A. M. (1995) Nat. Struct. Biol. 2, 852-855]. The positive value for Delta K(o)T indicates that the denatured form of ribonuclease A is more compressible than the native form; this is explained in terms of an interplay between the intrinsic compressibility of the protein and solvation effects. When the same data were fitted to a model that assumes a zero compressibility change, the Delta G(o) value of 4. 0 +/- 0.1 kcal mol-1 returned by the model no longer agreed with the independent measurement, and the Delta V(o) returned by the model was a very different -59 +/- 1 mL mol-1. By contrast, it was not possible to carry out a similar thermodynamic analysis of fluorescence spectroscopic data for the denaturation of staphylococcal nuclease to yield well-defined values of Delta G(o), Delta V(o), and Delta K(o)T. This limitation was shown by evaluation of synthetic data to be intrinsic to spectroscopic data whose analysis requires fitting of the plateaus at either side of the transition. Because NMR data do not have this requirement, they can be analyzed more rigorously.

Animals↗
PubMed1998

Evidence for oxidation-state-dependent conformational changes in human ferredoxin from multinuclear, multidimensional NMR spectroscopy.

Human ferredoxin belongs to the vertebrate ferredoxin family which includes bovine adrenodoxin. It is a small (13.8 kDa) acidic protein with a [2Fe-2S] cluster. It functions as an electron shuttle in the cholesterol side-chain cleavage reaction which is the first step of steroid hormone biosynthesis. The protein studied here was produced in Escherichia coli and doubly labeled with 13C and 15N. The diamagnetic 15N, 13C', 13C alpha, 13C beta, 1H alpha, and 1HN resonances from about 70% of the 124 amino acid residues for oxidized human ferredoxin and 80% of those for the reduced protein have been assigned primarily on the basis of results from three-dimensional, triple-resonance experiments. Secondary structure features for human ferredoxin in its oxidized and reduced states have been identified from a combination of chemical shift index and NOE data. Comparison of NMR results from the protein in its oxidized and reduced states indicates that structural changes accompany the change in the oxidation state of the [2Fe-2S] cluster. Major differences are localized at two regions: residues 29-31 and residues 109-124; the latter stretch forms the C-terminal region of the protein. The possible functional significance of these oxidation-state-dependent structural changes is discussed.

Amino Acid Sequence↗
PubMed1998

The role of aromatic and acidic amino acids in the electron transfer reaction catalyzed by spinach ferredoxin-dependent glutamate synthase.

Treatment of the ferredoxin-dependent, spinach glutamate synthase with N-bromosuccinimide (NBS) modifies 2 mol of tryptophan residues per mol of enzyme, without detectable modification of other amino acids, and inhibits enzyme activity by 85% with either reduced ferredoxin or reduced methyl viologen serving as the source of electrons. The inhibition of ferredoxin-dependent activity resulting from NBS treatment arises entirely from a decrease in the turnover number. Complex formation of glutamate synthase with ferredoxin prevented both the modification of tryptophan residues by NBS and inhibition of the enzyme. NBS treatment had no effect on the secondary structure of the enzyme, did not affect the Kms for 2-oxoglutarate and glutamine, did not affect the midpoint potentials of the enzyme's prosthetic groups and did not decrease the ability of the enzyme to bind ferredoxin. It thus appears that the ferredoxin-binding site(s) of glutamate synthase contains at least one, and possibly two, tryptophans. Replacement of either phenylalanine at position 65, in the ferredoxin from the cyanobacterium Anabaena PCC 7120, with a non-aromatic amino acid, or replacement of the glutamate at ferredoxin position 94, decreased the turnover number compared to that observed with wild-type Anabaena ferredoxin. The effect of the change at position 65 was quite modest compared to that at position 94, suggesting that an aromatic amino acid is not absolutely essential at position 65, but that glutamate 94 is essential for optimal electron transfer.

Amino Acid Oxidoreductases↗
PubMed1998

Analysis of error propagation from NMR-derived internuclear distances into molecular structure of cyclo-pro-gly.

Analytical expressions have been derived that translate uncertainties in distance constraints (obtained from NMR investigations) into uncertainties in atom positions in the maximum likelihood (ML) structure consistent with these inputs. As a test of this approach, a comparison was made between test structures reconstructed by the new ML approach, which yields a single structure and a covariance matrix for coordinates, and those reconstructed by metric matrix distance-geometry (MMDG), which yields a family of structures that sample uncertainty space. The test structures used were 560 polyhedra, with edges of arbitrary length containing up to 50 vertices, and one polyhedron, with 100 vertices; randomized distance constraints generated from these structures were used in reconstructing the polyhedra. The uncertainties derived from the two methods showed excellent agreement, and the correlation improved, as expected, with increasingly larger numbers of MMDG structures. This agreement supports the validity of the rapid analytical ML approach, which requires the calculation of only a single structure. As a second test of the ML method, the approach was applied to the determination of uncertainties in the structure of a cyclic dipeptide, cyclo(DL-Pro-Gly) (cPG), derived from NMR cross-relaxation data. The input data were interproton distances calculated from NOEs measured for a solution of the peptide in 2:1 DMSO:H2O at -40 degreesC (so as to yield large negative NOEs). In order to evaluate effects of the quality of the input spectral parameters on the precision of the resulting NMR structure, information from the covalent geometry of cPG was not used in the structure calculations. Results obtained from the analytical ML approach compared favorably with those from the much slower random-walk variant of the Monte Carlo method applied to the same input data. As a third test, the ML approach was used with synthetic structural constraints for a small protein; the results indicate that it will be feasible to use this rapid method to translate uncertainties associated with a given set of distance restraints into uncertainties in atom positions in larger molecules.

Dipeptides↗
PubMed1998

Detailed NMR analysis of the heme-protein interactions in component IV Glycera dibranchiata monomeric hemoglobin-CO.

Complete 13C, 15N, and 1H resonance assignments have been obtained for the recombinant, ferrous CO-ligated from of component IV monomeric hemoglobin from Glycera dibranchiata. This 15642 Da myoglobin-like protein contains a large number of glycine and alanine residues (47) and a heme prosthetic group. Coupling constant information has allowed the determination of chi(1) and chi(2) torsion angles, backbone phi angles, as well as 43 of 81 possible assignments to H beta 2/beta 3 pairs. The 13C alpha, 13 beta, 13C', and 1H alpha assignments yield a consensus chemical shift index (CSI) that, in combination with NOE information and backbone torsion angles, defines seven distinct helical regions for the protein's global architecture. Discrepancies between the CSI and NOE/3JHNH alpha-based secondary structure definitions have been attributed to heme ring current shifts on the basis of calculations from a model structure [Alam et al. (1994) J. Protein Chem., 13, 151-164]. The agreement can be improved by correcting the 1H alpha chemical shifts for the ring current contributions. Because the holoprotein was assembled from isotopically enriched globin and natural isotope-abundance heme, data from 13C-filtered/13C-edited and 13C-filtered/13C-filtered 2D NOESY experiments could be used to determine complete heme proton assignments and to position the heme within the protein. The results confirm the unusual presence of Phe31 (B10) and Leu58 (E7) side chains near the heme ligand binding site which may alter the polarity and steric environment and thus the functional properties of this protein.

Amino Acid Sequence↗
PubMed1998

Practical model fitting approaches to the direct extraction of NMR parameters simultaneously from all dimensions of multidimensional NMR spectra.

A maximum likelihood (ML)-based approach has been established for the direct extraction of NMR parameters (e.g., frequency, amplitude, phase, and decay rate) simultaneously from all dimensions of a D-dimensional NMR spectrum. The approach, referred to here as HTFD-ML (hybrid time frequency domain maximum likelihood), constructs a time-domain model composed of a sum of exponentially-decaying sinusoidal signals. The apodized Fourier transform of this time-domain signal is a model spectrum that represents the 'best-fit' to the equivalent frequency-domain data spectrum. The desired amplitude and frequency parameters can be extracted directly from the signal model constructed by the HTFD-ML algorithm. The HTFD-ML approach presented here, as embodied in the software package CHIFIT, is designed to meet the challenges posed by model fitting of D-dimensional NMR data sets, where each consists of many data points (10(8) is not uncommon) encoding information about numerous signals (up to 10(5) for a protein of moderate size) that exhibit spectral overlap. The suitability of the approach is demonstrated by its application to the concerted analysis of a series of ten 2D 1H-15N HSQC experiments measuring 15N T1 relaxation. In addition to demonstrating the practicality of performing maximum likelihood analysis on large, multidimensional NMR spectra, the results demonstrate that this parametric model-fitting approach provides more accurate amplitude and frequency estimates than those obtained from conventional peak-based analysis of the FT spectrum. The improved performance of the model fitting approach derives from its ability to take into account the simultaneous contributions of all signals in a crowded spectral region (deconvolution) as well as to incorporate prior knowledge in constructing models to fit the data.

Algorithms↗
PubMed1998

Recommendations for the presentation of NMR structures of proteins and nucleic acids. IUPAC-IUBMB-IUPAB Inter-Union Task Group on the Standardization of Data Bases of Protein and Nucleic Acid Structures Determined by NMR Spectroscopy.

The recommendations presented here are designed to support easier communication of NMR data and NMR structures of proteins and nucleic acids through unified nomenclature and reporting standards. Much of this document pertains to the reporting of data in journal articles; however, in the interest of the future development of structural biology, it is desirable that the bulk of the reported information be stored in computer-accessible form and be freely accessible to the scientific community in standardized formats for data exchange. These recommendations stem from an IUPAC-IUBMB-IUPAB inter-union venture with the direct involvement of ICSU and CODATA. The Task Group has reviewed previous formal recommendations and has extended them in the light of more recent developments in the field of biomolecular NMR spectroscopy. Drafts of the recommendations presented here have been examined critically by more than 50 specialists in the field and have gone through two rounds of extensive modification to incorporate suggestions and criticisms.

Amino Acid Sequence↗
PubMed1998

Solution structure of the thermostable sweet-tasting protein brazzein.

The fruit of Pentadiplandra brazzeana Baillon contains a small, sweet-tasting protein named brazzein. The structure of brazzein in solution was determined by proton nuclear magnetic resonance spectroscopy at pH 5.2 and 22 degrees C. The brazzein fold, which contains one alpha-helix and three strands of antiparallel beta-sheet, does not resemble that of either of the other two sweet-tasting proteins with known structures, monellin and thaumatin. Instead, the structure of brazzein resembles those of plant gamma-thionins and defensins and arthropod toxins. Sequence comparisons predict that members of a newly-identified family of serine proteinase inhibitors share the brazzein fold.

Amino Acid Sequence↗
PubMed1997

Iron-sulfur cluster cysteine-to-serine mutants of Anabaena -2Fe-2S- ferredoxin exhibit unexpected redox properties and are competent in electron transfer to ferredoxin:NADP+ reductase.

The reduction potentials and the rate constants for electron transfer (et) to ferredoxin:NADP+ reductase (FNR) are reported for site-directed mutants of the [2Fe-2S] vegetative cell ferredoxin (Fd) from Anabaena PCC 7120, each of which has a cluster ligating cysteine residue mutated to serine (C41S, C46S, and C49S). The X-ray crystal structure of the C49S mutant has also been determined. The UV-visible optical and CD spectra of the mutants differ from each other and from wild-type (wt) Fd. This is a consequence of oxygen replacing one of the ligating cysteine sulfur atoms, thus altering the ligand --> Fe charge transfer transition energies and the chiro-optical properties of the chromophore. Each mutant is able to rapidly accept an electron from deazariboflavin semiquinone (dRfH.) and to transfer an electron from its reduced form to oxidized FNR although all are somewhat less reactive (30-50%) toward FNR and are appreciably less stable in solution than is wt Fd. Whereas the reduction potential of C46S (-381 mV) is not significantly altered from that of wt Fd (-384 mV), the potential of the C49S mutant (-329 mV) is shifted positively by 55 mV, demonstrating that the cluster potential is sensitive to mutations made at the ferric iron in reduced [2Fe-2S] Fds with localized valences. Despite the decrease in thermodynamic driving force for et from C49S to FNR, the et rate constant is similar to that measured for C46S. Thus, the et reactivity of the mutants does not correlate with altered reduction potentials. The et rate constants of the mutants also do not correlate with the apparent binding constants of the intermediate (Fdred:FNRox) complexes or with the ability of the prosthetic group to be reduced by dRfH.. Furthermore, the X-ray crystal structure of the C49S mutant is virtually identical to that of wt Fd. We conclude from these data that cysteine sulfur d-orbitals are not essential for et into or out of the iron atoms of the cluster and that the decreased et reactivity of these Fd mutants toward FNR may be due to small changes in the mutual orientation of the proteins within the intermediate complex and/or alterations in the electronic structure of the [2Fe-2S] cluster.

Anabaena↗
PubMed1997

Microscopic pKa values of Escherichia coli thioredoxin.

Thiol:disulfide oxidoreductases have a CXXC motif within their active sites. To initiate the reduction of a substrate disulfide bond, the thiolate form of the N-terminal cysteine residue (CXXC) of this motif performs a nucleophilic attack. Escherichia coli thioredoxin [Trx (CGPC)] is the best characterized thiol:disulfide oxidoreductase. Previous determinations of the active-site pKa values of Trx have led to conflicting interpretations. Here, 13C-NMR spectroscopy, site-specific isotopic labeling, and site-directed mutagenesis were used to demonstrate that analysis of the titration behavior of wild-type Trx requires the invocation of microscopic pKa values for two interacting active-site residues: Asp26 (7.5 and 9.2) and Cys32 (CXXC; 7.5 and 9.2). By contrast, in two Trx variants, D26N Trx and D26L Trx, Cys32 exhibits a pKa near 7.5 and has a well-defined, single-pKa titration curve. Similarly, in oxidized wild-type Trx, Asp26 has a pKa near 7.5. In CVWC and CWGC Trx, Cys32 exhibits a single pKa near 6.2. In all five enzymes studied here, there is no evidence for a Cys35 (CXXC) pKa of < 11. This study demonstrates that a comprehensive approach must be used to unravel complex titration behavior of the functional groups in a protein.

Acids↗