Zinc-substituted Desulfovibrio gigas desulforedoxins: resolving subunit degeneracy with nonsymmetric pseudocontact shifts

Desulfovibrio gigas desulforedoxin (Dx) consists of two identical peptides, each containing one [Fe-4S] center per monomer. Variants with different iron and zinc metal compositions arise when desulforedoxin is produced recombinantly from Escherichia coli. The three forms of the protein, the two homodimers [Fe(III)/Fe(III)]Dx and [Zn(II)/Zn(II)]Dx, and the heterodimer [Fe(III)/Zn(II)]Dx, can be separated by ion exchange chromatography on the basis of their charge differences. Once separated, the desulforedoxins containing iron can be reduced with added dithionite. For NMR studies, different protein samples were prepared labeled with (15)N or (15)N + (13)C. Spectral assignments were determined for [Fe(II)/Fe(II)]Dx and [Fe(II)/Zn(II)]Dx from 3D (15)N TOCSY-HSQC and NOESY-HSQC data, and compared with those reported previously for [Zn(II)/Zn(II)]Dx. Assignments for the (13)C(alpha) shifts were obtained from an HNCA experiment. Comparison of (1)H-(15)N HSQC spectra of [Zn(II)/Zn(II)]Dx, [Fe(II)/Fe(II)]Dx and [Fe(II)/Zn(II)]Dx revealed that the pseudocontact shifts in [Fe(II)/Zn(II)]Dx can be decomposed into inter- and intramonomer components, which, when summed, accurately predict the observed pseudocontact shifts observed for [Fe(II)/Fe(II)]Dx. The degree of linearity observed in the pseudocontact shifts for residues >/=8.5 A from the metal center indicates that the replacement of Fe(II) by Zn(II) produces little or no change in the structure of Dx. The results suggest a general strategy for the analysis of NMR spectra of homo-oligomeric proteins in which a paramagnetic center introduced into a single subunit is used to break the magnetic symmetry and make it possible to obtain distance constraints (both pseudocontact and NOE) between subunits.     

The use of pseudocontact shifts to refine solution structures of paramagnetic metalloproteins: Met80Ala cyano-cytochrome c as an example

 The availability of NOE constraints and of the relative solution structure of a paramagnetic protein permits the use of pseudocontact shifts as further structural constraints. We have developed a strategy based on: (1) determination of the χ tensor anisotropy parameters from the starting structure; (2) recalculation of a new structure by using NOE and pseudocontact shift constraints simultaneously; (3) redetermination of the χ tensor anisotropy parameters from the new structure, and so on until self-consistency. The system investigated is the cyanide derivative of a variant of the oxidized Saccharomyces cerevisiae iso-1-cytochrome c containing the Met80Ala mutation. The structure has been substantially refined. It is shown that the analysis of the deviation of the experimental pseudocontact shifts from those calculated using the starting structure may be unsound, as may the simple structure refinement based on the pseudocontact shift constraints only. Received: 11 July 1995 / Accepted: 30 October 1995     

Spectroscopic studies of cobalt and nickel substituted rubredoxin and desulforedoxin.

The single iron site of rubredoxin was replaced by nickel and cobalt. The near-infrared/visible/UV spectra of these metal derivatives show ligand-field transitions and charge-transfer bands which closely resemble those of simple tetrathiolate complexes, indicating a tetrahedral arrangement of the sulfur cysteinyl ligands around the metal core. The 1H NMR spectra of the nickel and cobalt derivatives reveal extremely low-field contact shifted resonances of one proton intensity assigned to beta-CH2 and alpha-CH cysteinyl protons. Other well resolved resonances shifted out of the main protein spectral envelope are also observed and probably arise from contact plus pseudocontact shift mechanisms. Rubredoxins from different sulfate reducers were metal substituted and assignments of aliphatic protons are tentatively proposed, taking advantage of the amino acid sequence homologies. The present data is promising in terms of structural analysis of the coordination sphere of the metal core. It was also shown that replacement of the iron atom of desulforedoxin, a close analogue of rubredoxin, by cobalt and nickel was possible.     

Direct metal ion substitution at the [M(SCys)4]2– site of rubredoxin

 The single Fe(II) in reduced rubredoxin from Clostridium pasteurianum was found to be quantitatively displaced by either Cd²⁺ or Zn²⁺ when a modest molar excess of the substituting metal salt was anaerobically incubated with the reduced rubredoxin under mild conditions, namely, room temperature, pH 5.4–8.4, and no protein denaturants. Under the same conditions, cadmium-for-zinc substitution was also achieved upon aerobic incubation of the zinc-substituted rubredoxin with a modest molar excess of Cd²⁺. Displacements of Fe(II) from the reduced rubredoxin were not observed upon anaerobic incubation with Ni²⁺, Co²⁺, or VO²⁺ salts, and no reaction with any of the divalent metal ions was observed for the oxidized [Fe(III)] rubredoxin. Fe(II) could not be re-inserted into the Zn- or Cd-substituted rubredoxins without resorting to protein denaturation. ¹H and ¹¹³Cd NMR experiments showed that the cadmium-substituted rubredoxin prepared by the non-denaturing substitution method retained the pseudotetrahedral M(SCys)₄ coordination geometry and secondary structural elements characteristic of the native rubredoxin, and that "unzipping" of the β-sheet did not occur during metal substitution. Rates of Fe(II) displacement by M²⁺ (M=Cd or Zn) increased with increasing M²⁺/rubredoxin ratio, decreasing pH, and lower ionic strength. The substitution rates were faster for M=Cd than for M=Zn. Rates of Cd²⁺ substitution into a V8A-mutated rubredoxin were significantly faster than for the wild-type protein. The side-chain of V8 is on the protein surface and close to the metal-ligating Cys42Sγ at the M(SCys)₄ site. Therefore, the rate-limiting step in the substitution process is suggested to involve direct attack of the [M(SCys)₄]^2– site by the incoming M²⁺, without global unfolding of the protein. Implications of these results for metal ion incorporation into rubredoxins in vivo are discussed. Received: 29 May 1998 / Accepted: 11 August 1998     

Redox-dependent structure change and hyperfine nuclear magnetic resonance shifts in cytochrome c

Proton nuclear magnetic resonance assignments for reduced and oxidized equine cytochrome c show that many individual protons exhibit different chemical shifts in the two protein forms, reflecting diamagnetic shift effects due to structure change, and in addition contact and pseudocontact shifts that occur only in the paramagnetic oxidized form. To evaluate the chemical shift differences (delta delta) for structure change, we removed the pseudocontact shift contribution by a calculation based on knowledge of the electron spin g tensor. The g-tensor parameters were determined from the delta delta values of a large set (64) of C alpha H protons at well-defined spatial positions in the oxidized horse protein. The g-tensor calculation, when repeated using only 12 available C alpha H proton resonances for cytochrome c from tuna, proved to be remarkably stable. The largest principal value of the g tensor (gz) falls precisely along the ligand bond between the heme iron and methionine-80 sulfur, while gx and gy closely match the natural heme axes defined by the pyrrole nitrogens. The derived g tensor was then used together with spatial coordinates for the oxidized form to calculate the pseudocontact shift contribution (delta pc) to proton resonances at 400 identifiable sites throughout the protein, so that the redox-dependent chemical shift discrepancy, delta delta-delta pc, could be evaluated. Large residual changes in chemical shift define the Fermi contact shifts, which are found as expected to be limited to the immediate covalent structure of the heme and its ligands and to be asymmetrically distributed over the heme. Smaller chemical shift discrepancies point to a concerted change, involving residues 39-43 and 50-60 (bottom of the protein), and to other changes in the immediate vicinity of the heme ligands. Also, the three internal water molecules are implicated in redox sensitivity. The residues found to change are in good but not perfect agreement with prior X-ray diffraction observations of subangstrom redox-related displacements in the tuna protein. The chemical shift discrepancies observed appear in the main to reflect structure-dependent diamagnetic shifts rather than hyperfine effects due to displacements in the pseudocontact shift field. Although 51 protons in 29 different residues exhibit significant chemical shift changes, the general impression is one of small structural adjustments to redox-dependent strain rather than sizeable structural displacements or rearrangements.     

The effect of axial ligand plane orientation on the contact and pseudocontact shifts of low-spin ferriheme proteins

 The effect of axial ligand nodal plane orientation on the contact and pseudocontact shifts of a symmetrical low-spin octamethylferriheme center has been calculated as a function of the angle of the axial ligand. Simple Hückel techniques have been used to estimate the contact contribution, and values obtained from model hemes, together with counter-rotation of the g-tensor, have been used to estimate the pseudocontact contribution, for the eight β-pyrrole methyl and four meso-H positions. It is found that the maximum and minimum contact shifts occur when the axial ligand is aligned at an angle of ±15° to the meso-H axes of the heme, rather than when the axial ligand plane lies along the porphyrin nitrogens, as assumed previously by some investigators. For systems having one planar axial ligand or two ligands in parallel planes, the contact and pseudocontact contributions at the meso-H positions are comparable in size (at least on the basis of simple Hückel estimates), while the contact contribution clearly dominates the isotropic shifts of the heme methyls. Allowing for the substituent effect of the 2,4-vinyls of protohemin, or the 2,4-thioethers of hemin c, as well as the average diamagnetic shifts of the heme methyls and meso-H, plots of the predicted shifts as a function of axial ligand nodal plane orientation have been constructed for hemin b- and c-containing proteins. Excellent agreement in the order of shifts, and reasonable agreement in the sizes of the observed shifts, is observed in the majority of the ferriheme proteins for which the methyl and meso-H resonances have been assigned and proton shifts reported. Plots have also been constructed for hemin c-containing proteins having the two axial ligand nodal planes oriented at relative angles of 40°, 70°, and 80°. Excellent agreement in the order of shifts, and reasonable agreement in the magnitudes of the observed shifts, is observed in all cases of bacterial cytochromes which do not fit the plots that assume the ligands are in parallel planes, except one – the cytochrome c-552 of Nitrosomonas europae. Except for this case, where the order of the predicted methyl shifts at any angle of the axial ligands disagrees with the observed, the reasons can usually be attributed to a large dihedral angle between two axial ligand nodal planes, to strong H-bonding interactions involving His and/or CN^– ligands, or to off-axis binding of one (or both) axial ligand(s). Ruffling of the porphyrin ring may also contribute to the contact shift in as yet undefined ways. Hence, despite the simplicity of the calculations, the agreement with observed data is highly satisfying and the concept of the importance of axial ligand plane orientation on the observed proton shifts of heme proteins is fully confirmed. Received: 15 June 1998 / Accepted: 6 August 1998     

Heme methyl 1H chemical shifts as structural parameters in some low-spin ferriheme proteins

 The different paramagnetic shifts of the four methyl groups in ferriheme proteins have been described as being due to the effect of the axial ligand nodal plane orientation. An equation, heuristically found and theoretically explained, describing the relation between contact and pseudocontact shifts and the position of the axial ligand(s) has been derived for bis-histidine ferriheme proteins and for cyanide-histidine ferriheme proteins. The values of the heuristic parameters contained in the equations were found by fitting the shifts of bovine cytochrome b ₅ and several bis-histidine cytochromes c ₃ and histidine-cyanide systems. The agreement between the observed and the calculated shifts was found to be good. Therefore, by taking advantage of this study, information on the position of the axial ligands, that can be used as a constraint for structure determination, can be obtained from the shifts of the methyl protons. Received: 13 April 1999 / Accepted: 4 June 1999     

PSEUDYANA for NMR structure calculation of paramagnetic metalloproteins using torsion angle molecular dynamics

The program DYANA, for calculation of solution structures of biomolecules with an algorithm based on simulated annealing by torsion angle dynamics, has been supplemented with a new routine, PSEUDYANA, that enables efficient use of pseudocontact shifts as additional constraints in structure calculations of paramagnetic metalloproteins. PSEUDYANA can determine the location of the metal ion inside the protein frame and allows to define a single tensor of magnetic susceptibility from a family of conformers. As an illustration, a PSEUDYANA structure calculation is provided for a metal-undecapeptide complex, where simulated pseudocontact shifts but no NOE restraints are used as conformational constraints.     

Zinc(II) modulates specifically amyloid formation and structure in model peptides

Metal ions such as zinc and copper can have dramatic effects on the aggregation kinetics of and the structures formed by several amyloidogenic peptides/proteins. Depending on the identity of the amyloidogenic peptide/protein and the conditions, Zn(II) and Cu(II) can promote or inhibit fibril formation, and in some cases these metal ions have opposite effects. To better understand this modulation of peptide aggregation by metal ions, the impact of Zn(II) binding to three amyloidogenic peptides (Aβ14-23, Aβ11-23, and Aβ11-28) on the formation and structure of amyloid-type fibrils was investigated. Zn(II) was able to accelerate fibril formation for all three peptides as measured by thioflavin T fluorescence and transmission electron microscopy. The effects of Zn(II) on Aβ11-23 and Aβ11-28 aggregation were very different compared with the effects of Cu(II), showing that these promoting effects were metal-specific. X-ray absorption spectroscopy suggested that the Zn(II) binding to Aβ11-23 and Aβ11-28 is very different from Cu(II) binding, but that the binding is similar in the case of Aβ14-23. A model is proposed in which the different coordination chemistry of Zn(II) compared with Cu(II) explains the metal-specific effect on aggregation and the difference between peptides Aβ14-23 and Aβ11-23/Aβ11-28.     

1H NMR investigation of reduced copper-cobalt superoxide dismutase

Human copper-cobalt superoxide dismutase in the reduced form has been investigated through ¹H NMR techniques. The aim is to monitor the structural properties of this derivative and to compare them with those of reduced and oxidized native superoxide dismutases. The observed signals of the cobalt ligands have been assigned as well as the signals of the histidines bound to copper(I). The latter signals experience little pseudocontact shifts which allow a rough orientation of the magnetic susceptibility tensor in the molecular frame. The connectivities indicate that, although the histidine bridge is broken in the reduced form, the interproton distances between ligands of both ions are essentially the same.Abbreviations WEFT water eliminated Fourier transform - NOE nuclear Overhauser effect - NOESY NOE spectroscopy - COSY correlation spectroscopy - TOCSY total correlation spectroscopy - SOD superoxide dismutase - E₂Co(II)SOD SOD with empty copper site (E=empty) and with cobalt(II) in the Zinc(II) site Offprint requests to: I. Bertini     

OnionTree XML: A Format to Exchange Gene-Related Probabilities

Abstract

Combined use of shielding constant computations, measurements of chemical shifts and NOE studies reveal that poly(dG-dC)?(poly)dG-dC) in low salt solutions exist as a right- handed B-DNA double helix described by Gupta, Dhingra, Sarma, Sarma, Rajagopalan and Sasisekharan, J. Biomole. Str. Dyn. 1, 395, 1983. We present a simple and direct method to determine the handedness of DNA double helices from NOE difference spectra. This method takes advantage of the NOE between base protons and the H2′H2” sugar protons; and in the difference NOE spectra in the H2′H2” region the signatures of the right and left-handed helices become imprinted.     

Pseudocontact shifts as constraints for energy minimization and molecular dynamics calculations on solution structures of paramagnetic metalloproteins

The pseudocontact shifts of NMR signals, which arise from the magnetic susceptibility anisotropy of paramagnetic molecules, have been used as structural constraints under the form of a pseudopotential in the SANDER module of the AMBER 4.1 molecular dynamics software package. With this procedure, restrained energy minimization (REM) and restrained molecular dynamics (RMD) calculations can be performed on structural models by using pseudocontact shifts. The structure of the cyanide adduct of the Met80Ala mutant of the yeast iso-1-cytochrome c has been used for successfully testing the calculations. For this protein, a family of structures is available, which was obtained by using NOE and pseudocontact shifts as constraints in a distance geometry program. The structures obtained by REM and RMD calculations with the inclusion of pseudocontact shifts are analyzed. Proteins 29:68–76, 1997. © 1997 Wiley-Liss, Inc.     

Biosorption of heavy metals from acid mine drainage onto biopolymers (chitin and α (1,3) β-D-glucan) from industrial biowaste exhausted brewer’s yeasts (Saccharomyces cerevisiae L.)

A biosorption process has been developed for the bioremediation of heavy metal-contaminated acid drainages from Merladet and Faith open-cast mines, located in western Spain. The process is based on the physico-chemical properties for the adsorption, ion exchange, and complexation of metal ions by biopolymers (chitin and α (1,3) β-D-glucan) from industrial biowaste exhausted brewer’s yeast (Saccharomyces cerevisiae L.). Firstly, the chemical composition (U, Mn, Al, Fe, Cu, Zn, and Ni) and the physico-chemical and ecological states of these acid mine drainages were characterised. Furthermore, the selectivity for Zn, Cu, Mn, Ni, and Al the first order kinetics and the performance of the metals biosorption process by exhausted brewer’s yeast were evaluated with polluted acid synthetic waters and mine drainages. The biosorption equilibria were reached in 10 ∼ 15 min following Langmuir type isotherms with higher affinity constants for metal-biosorbent binding for synthetic waters than for acid mine drainages. The efficiency of the process with real water samples was markedly lower for the case of Mn, and zero for Zn and Al. An antagonistic interference on the biosorption of a metal due to the presence of other metals is proposed. Finally, the ecotoxicity of the acid mine drainage was removed when it was incubated with brewer’s yeast trapped in polyurethane foam.     

Combined spectroscopic and calorimetric characterisation of rubredoxin reversible thermal transition

Rubredoxins are small iron proteins containing the simplest type of iron–sulphur centre, consisting of an iron atom coordinated by the thiol groups of four cysteines. Here we report studies on the conformational stability of a new type of rubredoxin from the hyperthermophile Methanocaldococcus jannaschii, having an atypical metal site geometry resulting from a modified iron-binding motif. Absorption and fluorescence spectroscopies were used in combination with differential scanning calorimetry to probe different aspects of the thermal unfolding transition: iron site degradation (absorption at 380 nm), tertiary structure unfolding (Trp emission), exposure of hydrophobic regions (1-anilinonaphalene-8-sulphonate fluorescence enhancement) and iron release. Thermal denaturation was found to be irreversible and caused by decomposition of the metal centre. The protein is hyperstable and between pH 4 and 10 it is only thermally denatured in the presence of a strong chemical denaturant. The study of the heating rate dependence of the melting temperature allowed us to determine the reaction equilibrium thermodynamic parameters. At pH 2 the protein is destabilised owing to the absence of salt bridges and it has a T _m of 65 °C. In these conditions, there is excellent agreement between the parameters determined by the different spectroscopic methods and calorimetry. The highest stability was found to be at pH 8, and a detailed study of the heating rate dependence in the presence of guanidine thiocyanate in this condition allowed the determination of a reversible T _m of 118 °C.     

NMR analysis of duplex d(CGCGATCGCG)<Subscript>2</Subscript> modified by <Emphasis Type="Italic">Λ</Emphasis>- and <Emphasis Type="Italic">Δ</Emphasis>-[Ru(bpy)<Subscript>2</Subscript>(m-GHK)]Cl<Subscript>2</Subscript> and DNA photocleavage study

The interaction of the diastereomeric complexes Λ-[Ru(bpy)₂(m-GHK)]Cl₂ and Δ-[Ru(bpy)₂(m-GHK)]Cl₂ (bpy is 2,2′-bipyridine, GHK is glycine–l-histidine–l-lysine) with the deoxynucleotide duplex d(5′-CGCGATCGCG)₂ was studied by means of ¹H NMR spectroscopy. At a Δ-isomer to DNA ratio of 1:1, significant shifts for the metal complex are observed, whereas there is negligible effect on the oligonucleotide protons and only one intermolecular nuclear Overhauser effect (NOE) is present at the 2D nuclear Overhauser enhancement spectroscopy spectrum. The ¹Η NMR spectrum at ratio 2:1 is characterized by a slight shift for the Δ-isomer’s bpy aromatic protons as well as significant shifts for the decanucleotide G₄ H1′ and Η2″, A₅ H2, G₁₀ H1′, T₆ NH and G₂ NH protons. Furthermore, at ratio 2:1, 11 intermolecular NOEs are observed. The majority of the NOEs involve the sugar Η2′ and Η2″ protons sited in the major groove of the decanucleotide. Increasing the Δ-isomer to d(CGCGATCGCG)₂ ratio to 5:1 results in noteworthy spectral changes. The Δ-isomer’s proton shifts are reduced, whereas significant shifts are observed for the decanucleotide protons, especially the sugar protons, as well as for the exchangeable protons. Interaction is characterized by the presence of only one intermolecular NOE. Furthermore, there is significant broadening of the imino proton signals as the ratio of the Δ-isomer to DΝΑ increases, which is attributed to the opening of the two strands of the duplex. The Λ-isomer, on the other hand, approaches the minor groove of the oligonucleotide and interacts only weakly, possibly by electrostatic interactions. Photocleavage studies were also conducted with the plasmid pUC19 and a 158-bp restriction fragment, showing that both diastereomers cleave DNA with similar efficiency, attacking mainly the guanines of the sequence probably by generating active oxygen species. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users. An erratum to this article can be found at     

Metal-protein interactions: structure information from Ni(2+)-induced pseudocontact shifts in a native nonmetalloprotein

Information about the structure of a native nonmetalloprotein was obtained from the pseudocontact shifts induced by a paramagnetic metal ion bound to the protein. The approach exploits the presence of metal binding sites on the surface of the protein. Using Escherichia coli thioredoxin as a model protein, we show that potential binding sites can be identified using the Cu(2+) ion, and that pseudocontact shifts induced by a Ni(2+) ion bound to one of these sites can provide valuable long-range structure information about the protein.     

Molecular modeling of zinc and copper binding with Alzheimer’s amyloid β-peptide

Aggregation of the amyloid β-peptide (Aβ) into insoluble fibrils is a key pathological event in Alzheimer’s disease. Cu(II) and Zn(II) ions were reported to be able to induce Aβ aggregation at nearly physiological concentrations in vitro. In this study, the binding modes of Cu(II) and Zn(II) in this process were explored by molecular modeling. In the pre-associated Aβ, Nτ atom of imidazole ring of His14, O atom of carbonyl of main-chain and two O atoms of water occupied the four ligand positions of the complex. While in the aggregated form of Aβ, the His13(N)–Metals–His14(N) bridges were formed through metal cross-linking action. These results would be helpful to put insight on revealing the formation mechanism of pathogenic Aβ aggregates in brain.     

Nickel(II) biosorption by <Emphasis Type="Italic">Rhodotorula glutinis</Emphasis>

The present study reports the feasibility of using Rhodotorula glutinis biomass as an alternative low-cost biosorbent to remove Ni(II) ions from aqueous solutions. Acetone-pretreated R. glutinis cells showed higher Ni(II) biosorption capacity than untreated cells at pH values ranging from 3 to 7.5, with an optimum pH of 7.5. The effects of other relevant environmental parameters, such as initial Ni(II) concentration, shaking contact time and temperature, on Ni(II) biosorption onto acetone-pretreated R. glutinis were evaluated. Significant enhancement of Ni(II) biosorption capacity was observed by increasing initial metal concentration and temperature. Kinetic studies showed that the kinetic data were best described by a pseudo-second-order kinetic model. Among the two-, three-, and four-parameter isotherm models tested, the Fritz-Schluender model exhibited the best fit to experimental data. Thermodynamic parameters (activation energy, and changes in activation enthalpy, activation entropy, and free energy of activation) revealed that the biosorption of Ni(II) ions onto acetone-pretreated R. glutinis biomass is an endothermic and non-spontaneous process, involving chemical sorption with weak interactions between the biosorbent and Ni(II) ions. The high sorption capacity (44.45 mg g⁻¹ at 25°C, and 63.53 mg g⁻¹ at 70°C) exhibited by acetone-pretreated R. glutinis biomass places this biosorbent among the best adsorbents currently available for removal of Ni(II) ions from aqueous effluents.     

Proton NMR studies of myohemerythrin from Themiste zostericola

 One- and two-dimensional NMR experiments have been carried out on different forms of myohemerythrin (MHr), a monomeric 13.9-kDa oxygen carrier, focusing on paramagnetically shifted proton resonances. Compared to the corresponding forms of octameric hemerythrin (Hr), all of the MHr forms exhibit spectra with better resolution and signal-to-noise ratios. The metMHr spectra allow the differentiation of the signals from the N_δ-H protons of the five N_ε-coordinated His ligands and those from the bridging Asp and Glu ligands. The 1D spectra of deoxyMHr exhibit a number of relatively sharp features including three solvent-exchangeable peaks that account for five protons. One of these His N-H protons exchanges more slowly with solvent than the other four and is assigned to His 54, which, by analogy to the crystal structure of deoxyHr, is the only His ligand that is hydrogen-bonded to an amino acid residue, Glu24 in this case. One-dimensional NOE results on the non-exchangeable signals clearly show the connectivities among the α and β protons of the bridging Asp111, and the α, β, and γ protons of the bridging Glu58 ligands. One-dimensional NOE experiments performed on the N-H proton signals of the coordinated His ligands, together with the COSY results, help to identify the geminal β protons of the His ligands. Upon the binding of N₃ ^– to one of the Fe(II) sites in deoxyMHr, the overlapping His N_δ-H proton signals observed in the deoxyMHr spectrum are resolved into individual signals; these have been correlated to the corresponding signals in deoxyMHr by saturation transfer experiments. Similarly, all five His N-H protons are resolved in the ¹H NMR spectrum of the deoxy form of the single point mutant L103N MHr. However, all five N-H protons readily exchange with solvent, indicating that the mutation affects the hydrogen-bonding interaction between His54 and Glu24. Received: 20 May 1996 / Accepted: 24 October 1996     

Sequential nearest-neighbor effects on computed <Superscript>13</Superscript>C<Superscript>α</Superscript> chemical shifts

To evaluate sequential nearest-neighbor effects on quantum-chemical calculations of ¹³C^α chemical shifts, we selected the structure of the nucleic acid binding (NAB) protein from the SARS coronavirus determined by NMR in solution (PDB id 2K87). NAB is a 116-residue α/β protein, which contains 9 prolines and has 50% of its residues located in loops and turns. Overall, the results presented here show that sizeable nearest-neighbor effects are seen only for residues preceding proline, where Pro introduces an overestimation, on average, of 1.73 ppm in the computed ¹³C^α chemical shifts. A new ensemble of 20 conformers representing the NMR structure of the NAB, which was calculated with an input containing backbone torsion angle constraints derived from the theoretical ¹³C^α chemical shifts as supplementary data to the NOE distance constraints, exhibits very similar topology and comparable agreement with the NOE constraints as the published NMR structure. However, the two structures differ in the patterns of differences between observed and computed ¹³C^α chemical shifts, Δ _ca,i , for the individual residues along the sequence. This indicates that the Δ _ca,i -values for the NAB protein are primarily a consequence of the limited sampling by the bundles of 20 conformers used, as in common practice, to represent the two NMR structures, rather than of local flaws in the structures.