Denatured states of human carbonic anhydrase II: an NMR study of hydrogen/deuterium exchange at tryptophan-indole-H(N) sites.

Hydrogen/deuterium (H/D) exchange measurements in low and moderate concentrations of GuHCI were conducted on the side chain H(N) atoms of the seven tryptophans of pseudo wild-type human carbonic anhydrase II. Tryptophans 5, 16 and 245, situated in or close to the N-terminal domain were found to have little protection against exchange. The H/D exchange results for Trp-123, Trp-192 and Trp-209 showed that a previously identified molten globule and the native state gave a similar protection against exchange. Global unfolding of the protein is necessary for the efficient exchange at Trp-97, which is located in the central part of the beta-sheet.

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

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

NMR solution structure of the oxidized form of MerP, a mercuric ion binding protein involved in bacterial mercuric ion resistance.

Mercuric ions are toxic to living organisms because of their strong affinity for cysteine residues in proteins. Some bacteria have developed a resistance mechanism whereby Hg2+ is transported into the cytoplasm and reduced to Hg0. One of the proteins involved in the transport of mercuric ion is the periplasmic binding protein MerP, which can exist both as oxidized (disulfide) and as reduced (dithiol) forms. Only the reduced form with Cys-17 and Cys-14 residues as free thiols is a potent receptor for mercuric ion. In this work the solution structure of the oxidized form of MerP has been determined by multidimensional NMR spectroscopy and compared to the NMR structures of the previously published structures of the reduced and mercury-bound forms of MerP. The mercury-bound and oxidized forms have similar tertiary structures, whereas in the reduced form there is a large rearrangement of the mercuric ion binding loop and the nearby loop comprising residues 38-41. The structural arrangement of the latter loop seems to be important for the stabilization of the surface location of the cysteine-containing loop. In the reduced form at low pH the cysteine-containing loop adopts a conformation similar to what is observed in the oxidized and mercury-bound forms. The oxidized form also differs with respect to the other two forms in the relative positions of some of the alpha-helices and beta-strands. Structural differences between the oxidized and reduced forms may help explain why the reduced form is stable in the periplasm, which is considered to be an oxidizing environment.

The solution NMR structure of a blue-green algae hepatotoxin, microcystin-RR--a comparison with the structure of microcystin-LR.

The microcystin-RR structures are compared with the structures of microcystin-LR in solution as well as in the crystal structure of the complex with protein phosphatase. The gross structures of the two peptides are similar, but with a more accentuated and compact saddle structure for microcystin-RR. The structural differences affect the hydrogen-bond pattern in the peptides and the location of the side chain of N-methyldehydroalanine, both of which are important for the ability of the peptide to form a tight complex with protein phosphatase. These structural differences may contribute to the observed differences in toxicity of microcystin-RR and microcystin-LR.

Characterization of the heparin-binding domain of human extracellular superoxide dismutase.

The C-terminal, heparin-binding domain of human extracellular superoxide dismutase (hEC-SOD) has been studied as a fusion to human carbonic anhydrase II (HCAII). This technique allows the properties of the EC-SOD domain to be characterized. At the same time, it allows us to differentiate the contributions from the domain, from those properties originating from other parts of EC-SOD. The fusion of the 27 C-terminal amino acids of hEC-SOD to the C-terminal of HCAII (FusCC) resulted in the formation of a monomeric protein, which binds to heparin-Sepharose with approximately the same affinity as the tetrameric hEC-SOD. The structure of the fused C-terminal was characterized by CD and NMR spectroscopy and the data were compatible with the presence of alpha-helical structures as suggested by secondary structure predictions. The NMR data show that the C-terminal of FusCC moves independently from the rest of the protein and that its central part is involved in conformational exchange. The NOESY spectra demonstrate that the C-terminal in both FusCC and hEC-SOD binds to heparin, and that arginine side chains take part in the binding.

Calcium-binding mechanism of human nonerythroid alpha-spectrin EF-structures.

We have used circular dichroism and 1H- and 15N-NMR spectroscopy to investigate calcium binding to the two EF-hands of human nonerythroid or alphaII-spectrin. Comparison of the 1H-NMR spectra from the peptide containing both EF-hands to the peptides containing the single EF-I and EF-II structures showed that both the structural and calcium-binding properties are significantly different. Further studies of the 121 amino acid peptide containing both EF-hands using circular dichroism and NMR showed that the binding of calcium ions induces conformational changes. To investigate the calcium-binding mechanism, the chemical shifts changes were recorded using multidimensional NMR spectroscopy during calcium titration. A total of 25 titration curves were obtained, each corresponding to the chemical shift changes of individual amino acid residues. The shapes of these titration curves were either hyperbolic or sigmoidal. Using factor analysis, two functions were extracted, one hyperbolic and one sigmoidal, which accounted for nearly all information present in the titration curves. By fitting the two functions to binding curves based on different binding models, we found that the binding mechanism is best described as sequential. Since the sigmoidal type was more pronounced in the titration curves corresponding to residues from the first EF-hand, we suggest that calcium binding to the first EF-hand is described by the sigmoidal function, and that the hyperbolic function describes calcium binding to the second EF-hand. Therefore, is seems likely that the second EF-hand must contain bound calcium before the first EF-hand can bind.

Solution structure of phenol hydroxylase protein component P2 determined by NMR spectroscopy.

Phenol hydroxylase from Pseudomonas sp. CF600 is a member of a family of binuclear iron-center-containing multicomponent oxygenases, which catalyzes the conversion of phenol and some of its methyl-substituted derivatives to catechol. In addition to a reductase component which transfers electrons from NADH, optimal turnover of the hydroxylase requires P2, a protein containing 90 amino acids which is readily resolved from the other components. The three-dimensional solution structure of P2 has been solved by 3D heteronuclear NMR spectroscopy. On the basis of 1206 experimental constraints, including 1060 distance constraints obtained from NOEs, 70 phi dihedral angle constraints, 42 psi dihedral angle constraints, and 34 hydrogen bond constraints, a total of 12 converged structures were obtained. The atomic root mean square deviation for the 12 converged structure with respect to the mean coordinates is 2.48 A for the backbone atoms and 3.85 A for all the heavy atoms. This relatively large uncertainty can be ascribed to conformational flexibility and exchange. The molecular structure of P2 is composed of three helices, six antiparallel beta-strands, one beta-hairpin, and some less ordered regions. This is the first structure among the known multicomponent oxygenases. On the basis of the three-dimensional structure of P2, sequence comparisons with similar proteins from other multicomponent oxygenases suggested that all of these proteins may have a conserved structure in the core regions.

Sequential assignment of 1H, 13C and 15N resonances of human carbonic anhydrase I by triple-resonance NMR techniques and extensive amino acid-specific 15N-labeling.

The backbone NMR resonances of human carbonic anhydrase I (HCA I) have been assigned. This protein is one of the largest monomeric proteins assigned so far. The assignment was enabled by a combination of 3D triple-resonance experiments and extensive use of amino acid-specific 15N-labeling. The obtained resonance assignment has been used to evaluate the secondary structure elements present in solution. The solution structure appears to be very similar to the crystal structure, although some differences can be observed. Proton-deuteron exchange experiments have shown that the assignments provide probes that can be used in future studies of HCA I.

Conformational studies of microcystin-LR using NMR spectroscopy and molecular dynamics calculations.

NMR spectroscopy in aqueous and dimethyl sulfoxide/water solutions is used to determine the three-dimensional structures of microcystin-LR, a cyclic cyanobacterial heptapeptide toxin which is a potent inhibitor of type 1 and type 2A protein phosphatases. The conformations of this toxic peptide are studied using a simulated annealing (SA) protocol followed by refined SA calculations in vacuo and free MD simulations in water. Only one conformational family in each solvent is found. The peptide ring has a saddle-shaped form, essentially the same in both solvents. The structural difference observed between the two solution structures is located to the part consisting of Mdha, Ala, and Leu. This peptide segment is not present in nodularin, a cyclic pentapeptide of similar toxicity. The Arg side chain is very flexible, while the side chain of Leu is well defined. The side chain of Adda, essential for toxicity, is constrained in the vicinity of the backbone ring but appears to be flexible in the more remote part.

Solution structure of a DNA octamer containing the Pribnow box via restrained molecular dynamics simulation with distance and torsion angle constraints derived from two-dimensional nuclear magnetic resonance spectral fitting.

The DNA octamer [d(GTATAATG].[(CATATTAC)], containing the prokaryotic upstream consensus recognition sequence, has been examined via proton homonuclear two-dimensional nuclear Overhauser effect (2D NOE) and double-quantum-filtered correlation (2QF-COSY) spectra. All proton resonances, except those of H5' and H5" protons, were assigned. A temperature dependence study of one-dimensional nuclear magnetic resonance (NMR) spectra, rotating frame 2D NOE spectroscopy (ROESY), and T1 rho measurements revealed an exchange process that apparently is global in scope. Work at lower temperatures enabled a determination of structural constraints that could be employed in determination of a time-averaged structure. Simulations of the 2QF-COSY cross-peaks were compared with experimental data, establishing scalar coupling constant ranges of the individual sugar ring protons and hence pucker parameters for individual deoxyribose rings. The rings exhibit a dynamic equilibrium of N and S-type conformers with 80 to 100% populations of the latter. A program for iterative complete relaxation matrix analysis of 2D NOE spectral intensities, MARDIGRAS, was employed to give interproton distances for each mixing time. According to the accuracy of the distance determination, upper and lower distance bounds were chosen. The distance bounds define the size of a flat-well potential function term, incorporated into the AMBER force-field, which was employed for restrained molecular dynamics calculations. Torsion angle constraints in the form of a flat-well potential were also constructed from the analysis of the sugar pucker data. Several restrained molecular dynamics runs of 25 picoseconds were performed, utilizing 184 experimental distance constraints and 80 torsion angle constraints; three different starting structures were used: energy minimized A-DNA, B-DNA, and wrinkled D-DNA, another member of the B-DNA family. Convergence to similar structures obtained with root-mean-square deviations between resulting structures of 0.37 to 0.92 A for the central hexamer of the octamer. The average structure from the nine different molecular dynamics runs was subjected to final restrained energy minimization. The resulting final structure was in good agreement with the structures derived from different molecular dynamics runs and exhibited a substantial improvement in the 2D NOE sixth-root residual index in comparison with the starting structures. An approximation of the structure in the terminal base-pairs, which displayed experimental evidence of fraying, was made by maintaining the structure of the inner four base-pairs and performing molecular dynamics simulations with the experimental structural constraints observed for the termini.(ABSTRACT TRUNCATED AT 400 WORDS)