Solution H NMR characterization of substrate-free C. diphtheriae heme oxygenase: Pertinence for determining magnetic axes in paramagnetic substrate complexes

Proton 2D NMR was used to confirm in solution a highly conserved portion of the molecular structure upon substrate loss for the heme oxygenase from the pathogenic bacterium Corynebacterium diphtheriae, HmuO. The chemical shifts for the conserved portion of the structure are assessed as references for the dipolar shifts needed to determine the orientation of the paramagnetic susceptibility tensor, χ, in paramagnetic substrate complexes of HmuO. It is shown that the chemical shifts for the structurally conserved portion of substrate-free HmuO serve as excellent references for residues with only small to moderate sized dipolar shifts in the cyanide-inhibited substrate complex of HmuO, yielding an orientation of χ that is essentially the same as conventionally obtained from large dipolar shifts based on empirical estimates of the diamagnetic reference. The implications of these diamagnetic chemical shifts for characterizing the hydrogen bonding in the physiologically relevant, resting-state, high-spin aquo complex are discussed. The pattern of labile proton exchange in the distal H-bond network of substrate-free HmuO allowed comparison of changes in dynamic stability of tertiary contacts in the substrate-free and substrate-bound HmuO and with the same complexes of human heme oxygenase.     

Calculation of z-coordinates and orientational restraints using a metal binding tag

We introduce a new simple methodology allowing the measurement of (1)H-(15)N residual dipolar couplings, dipolar shifts, and unpaired electron-amide proton distances. This method utilizes a zinc finger tag fused at either the N- or the C-terminus of a protein. We have demonstrated this fusion strategy by incorporating the zinc finger of the retroviral gag protein onto the C-terminus of barnase, a ribonuclease produced by Bacillus amiloliquifaciance. We show that this tag can be substituted with cobalt and manganese. Binding of cobalt to the gag zinc finger-barnase fusion protein introduced sufficient anisotropic paramagnetic susceptibility for orientation of the molecule in the magnetic field. Partial alignment permitted measurement of (1)J(HN) scalar couplings along with dipolar couplings. Replacement of bound cobalt with diamagnetic zinc removes the paramagnetic-induced orientation of barnase, permitting the measurement of only (1)J(HN) scalar couplings. Dipolar couplings, ranging from -0.9 to 0.6 Hz, were easily measured from the difference in splitting frequencies in the presence of cobalt and zinc. The observed paramagnetic anisotropy induced by cobalt binding to the metal binding tag also permitted measurement of dipolar shifts. Substitution of manganese into the metal binding tag permitted the measurement of unpaired electron-amide proton distances using paramagnetic relaxation enhancement methodology. The availability of both amide proton dipolar shifts and unpaired electron to amide proton distances permitted the direct calculation of z-coordinates for individual amide protons. This approach is robust and will prove powerful for global fold determination of proteins identified in genome initiatives.     

1H nuclear magnetic resonance investigation of cobalt(II) substituted carbonic anhydrase.

The structure of ClO4 and NO3 adducts of cobalt(II) substituted bovine carbonic anhydrase have been investigated through 1D NOE and 2D 1H nuclear magnetic resonance (NMR) spectroscopy. For the first time two-dimensional NMR techniques are applied to paramagnetic metalloproteins other than iron-containing proteins. Several active site signals have been assigned to specific protons on the grounds of their scalar and dipolar connectivities and T1 values. The experimental dipolar shifts for the protons belonging to noncoordinated residues have allowed the identification of a plausible orientation of the magnetic susceptibility tensor around the cobalt ion as well as of the magnitude and the anisotropy of the principal susceptibility values. In turn, a few more signals have been tentatively assigned on the grounds of their predicted dipolar shifts. The two inhibitor derivatives have a very similar orientation but a different magnitude of the chi tensor, and the protein structure around the active site is highly maintained. The results encourage a more extensive use of the two-dimensional techniques for obtaining selective structural information on the active site of metalloenzymes. With this information at hand, comparisons within homologous series of adducts with various inhibitors and/or mutants of the same enzyme of known structure should be possible using limited sets of NMR data.     

One- and two-dimensional nuclear Overhauser effect studies of the electronic/molecular structure of the heme cavity of ferricytochrome b5

A nuclear Overhauser effect, NOE, study of solubilized native bovine ferricytochrome b5 has provided the complete assignment of the heme resonances as well as those of the majority of the amino acid side-chains making contact with the prosthetic group. The resonances which could not be identified are those from positions very close to the iron (less than 5 A) for which paramagnetic relaxation is sufficiently strong to significantly decrease the NOEs. The observed 1H-1H dipolar contacts generally confirm a solution structure unchanged from that described in single crystals, except for the detailed orientation of the heme side-chains. The 2-vinyl group is found in both the cis and trans in-plane orientation as opposed to exclusively cis in the crystal, and the 7-propionate group is rotated by 30 degrees in solution towards the 6-propionate group. Identification of resonances for the individual axial histidine residues indicates non-equivalent interaction with the heme iron, and the patterns of meso-H, pyrrole substituent and amino acid dipolar shifts allow the location of the principal magnetic axes in the protein coordinate system. This identifies His-39 as the dominant influence in determining the electronic ground state that orients the molecular orbital for facile electron transfer via the exposed heme edge. The complete two-dimensional NOESY map for ferricytochrome b5 is presented that yields all the cross peaks expected on the basis of the one-dimensional NOE studies, and indicates that such two-dimensional methods should have profitable extension to strongly hyperfine-shifted resonances in paramagnetic proteins.     

Solution 1H NMR study of the active site molecular structure and magnetic properties of the cyanomet complex of the isolated alpha-chain from human hemoglobin A

The solution electronic and molecular structure for the heme pocket of the cyanomet complex of the isolated alpha-chain of human adult hemoglobin (HbA) has been investigated by homonuclear two-dimensional 1H NMR in order to establish an assignment protocol for the dimeric chain that will guide similar assignments in the intact, heterotetrameric HbA complex, and to compare the structures of the alpha-chain with its subunit in HbA. The target residues are those that exhibit significant (>0.2 ppm) dipolar shifts, as predicted by a "preliminary" set of magnetic axes determined from a small set of easily assigned active site residues. All 97 target residues (approximately 70% of total) were assigned by taking advantage of the temperature dependence predicted by the "preliminary" magnetic axes for the polypeptide backbone; they include all residues proposed to play a significant role in modulating the ligand affinity in the tetramer HbA. Left unassigned are the A-helix, the end of the G-helix and the beginning of the H-helix where dipolar shifts are less than 0.2 ppm. The complete assignments allow the determination of a robust set of orientation and anisotropies of the paramagnetic susceptibility tensor that leads to quantitative interpretation of the dipolar shifts of the alpha-chain in terms of the crystal coordinates of the alpha-subunit in ligated HbA which, in turn, confirms a largely conserved molecular structure of the isolated alpha-chain relative to that in the intact HbA. The major magnetic axis, which is correlated with the tilt of the Fe-CN unit, is tilted approximately 10 degrees from the heme normal so that the Fe-CN unit is tilted toward the beta-meso-H in a fashion remarkably similar to the Fe-CO tilt in HbACO. It is concluded that a set of "preliminary" magnetic axes and the use of variable temperature two-dimensional NMR spectra are crucial to effective assignments in the cyanomet alpha-chain and that this approach should be similarly effective in HbA.     

15N-1H Residual dipolar coupling analysis of native and alkaline-K79A Saccharomyces cerevisiae cytochrome c

Residual dipolar couplings (RDCs) and pseudocontact shifts are experimentally accessible properties in nuclear magnetic resonance that are related to structural parameters and to the magnetic susceptibility anisotropy. We have determined RDCs due to field-induced orientation of oxidized-K79A and reduced cytochrome c at pH 7.0 and oxidized-K79A cytochrome c at pH 11.1 through measurements of amide (15)N-(1)H (1)J couplings at 800 and 500 MHz. The pH 7.0 RDCs for Fe(III)- and Fe(II)-cytochrome c together with available nuclear Overhauser effects were used to recalculate solution structures that were consistent with both sets of constraints. Molecular magnetic susceptibility anisotropy values were calculated for both redox states of the protein. By subtracting the residual dipolar couplings (RDCs) of the reduced form from those of the oxidized form measured at the same magnetic field (800 MHz), we found the RDC contribution of the paramagnetic metal ion in the oxidized protein. The magnetic susceptibility anisotropy, which was calculated from the structure, was found to be the same as that of the paramagnetic metal ion obtained independently from pseudocontact shifts, thereby indicating that the elements of secondary structure either are rigid or display the same mobility in both oxidation states. The residual dipolar coupling values of the alkaline-K79A form are small with respect to those of oxidized native cytochrome, whereas the pseudocontact shifts are essentially of the same magnitude, indicating local mobility. Importantly, this is the first time that mobility has been found through comparison of RDCs with pseudocontact shifts.     

NMR approaches for monitoring domain orientations in calcium-binding proteins in solution using partial replacement of Ca2+ by Tb3+.

This work shows that the partial replacement of diamagnetic Ca2+ by paramagnetic Tb3+ in Ca2+/calmodulin systems in solution allows the measurement of interdomain NMR pseudocontact shifts and leads to magnetic alignment of the molecule such that significant residual dipolar couplings can be measured. Both these parameters can be used to provide structural information. Species in which Tb3+ ions are bound to only one domain of calmodulin (the N-domain) and Ca2+ ions to the other (the C-domain) provide convenient systems for measuring these parameters. The nuclei in the C-domain experience the local magnetic field induced by the paramagnetic Tb3+ ions bound to the other domain at distances of over 40 A from the Tb3+ ion, shifting the resonances for these nuclei. In addition, the Tb3+ ions bound to the N-domain of calmodulin greatly enhance the magnetic susceptibility anisotropy of the molecule so that a certain degree of alignment is produced due to interaction with the external magnetic field. In this way, dipolar couplings between nuclear spins are not averaged to zero due to solution molecular tumbling and yield dipolar coupling contributions to, for example, the one-bond 15N-1H splittings of up to 17 Hz in magnitude. The degree of alignment of the C-domain will also depend on the degree of orientational freedom of this domain with respect to the N-domain containing the Tb3+ ions. Pseudocontact shifts for NH groups and 1H-15N residual dipolar couplings for the directly bonded atoms have been measured for calmodulin itself, where the domains have orientational freedom, and for the complex of calmodulin with a target peptide from skeletal muscle myosin light chain kinase, where the domains have fixed orientations with respect to each other. The simultaneous measurements of these parameters for systems with domains in fixed orientations show great potential for the determination of the relative orientation of the domains.     

Solution 1H NMR study of the active site molecular structure and magnetic properties of the cyanomet complex of the isolated, tetrameric beta-chain from human adult hemoglobin

The solution molecular structure and the electronic and magnetic properties of the heme pocket of the cyanomet complex of the isolated beta-chain of human adult hemoglobin, HbA, have been investigated by homonuclear 2D (1)H NMR in order to assess the extent of assignments allowed by (1)H NMR of a homo-tetrameric 65-kDa protein, to guide the future assignments of the heterotetrameric complex of HbA, and to compare the structure of the beta-chain to the crystallographically characterized complexes that contains the beta-chain. The target residues are those that exhibit significant (>|0.2| ppm) dipolar shifts, as predicted by a "preliminary" set of magnetic axes determined from a small set of easily assigned active site residues. All 104 target residues ( approximately 70% of total) were assigned by taking advantage of the temperature dependence predicted by the "preliminary" magnetic axes for the polypeptide backbone; they include all residues proposed to play a significant role in modulating the ligand affinity in the tetramer HbA. Left unassigned are the A-helix, the end of the G-helix and the beginning of the H-helix where dipolar shifts are less than |0.2| ppm. These comprehensive assignments allow the determination of a robust set of orientation and anisotropies of the paramagnetic susceptibility tensor that leads to quantitative interpretation of the dipolar shifts of the beta-chain in terms of the crystal coordinates of the beta-subunit in ligated HbA which, in turn, confirms a largely conserved molecular structure of the isolated beta-chain relative to that in the intact R-state HbA. The major magnetic axis, which is correlated with the tilt of the Fe-CN unit, is tilted approximately 10 degrees from the heme normal so that the Fe-CN unit is tilted toward the beta-meso-H in a fashion remarkably similar to the Fe-CO tilt in the beta-subunit of HbCO. It is concluded that a set of "preliminary" magnetic axes and the use of variable temperature 2D NMR spectra are crucial to effective assignments in the tetrameric cyanomet beta-chain and that this approach should be similarly effective in HbA.     

Distance between the visible copper and cytochrome a in bovine heart cytochrome oxidase

Electron paramagnetic resonance (EPR) at 15 K was used to probe the magnetic interaction between the visible copper CuA2+ and ferric cytochrome a in the carbon monoxide compound of beef heart cytochrome oxidase. At pH 8.6, the midpoint potentials (Em's) for one-electron oxidation of CuA+ and cytochrome a2+ were found to be 195 and 235 mV, respectively. Because the Em of CuA is well below that of cytochrome a under these conditions, the microwave power saturation of CuA could be measured as a function of percentage cytochrome a oxidized. Although progressive power saturation data directly provide only the product of the spin-lattice and transverse relaxation rates delta [1/(T1T2)], Castner's theory for the saturation of inhomogeneously broadened lines [Castner, T.G., Jr. (1959) Phys. Rev. 115 (6), 1506-1515], along with our own theoretical formulation of the dipolar T2, enabled us to determine the change in T1 of CuA due to dipolar relaxation by cytochrome a. The orientation of the principal g values of CuA with respect to those of cytochrome a was evaluated in partially oriented membranous multilayers. When allowance was made for uncertainties in the relative CuA-cytochrome a configuration and in the dipolar axis-magnetic field orientation, a range for the spin-spin distance r was calculated on the basis of the dipolar T1 of the gx component of CuA. This distance range was further restricted by consideration of T1 for the nonunique orientations of CuA giving rise to the gy signal. Only those values of r are possible for which the calculated T1 ratio (gx/gy) is equal to the experimentally determined ratio.(ABSTRACT TRUNCATED AT 250 WORDS)     

Breaking symmetry in the structure determination of (large) symmetric protein dimers

We demonstrate a novel methodology to disrupt the symmetry in the NMR spectra of homodimers. A paramagnetic probe is introduced sub-stoichiometrically to create an asymmetric system with the paramagnetic probe residing on only one monomer within the dimer. This creates sufficient magnetic anisotropy for resolution of symmetry-related overlapped resonances and, consequently, detection of pseudocontact shifts and residual dipolar couplings specific to each monomeric component. These pseudocontact shifts can be readily incorporated into existing structure refinement calculations and enable determination of monomer orientation within the dimeric protein. This methodology can be widely used for solution structure determination of symmetric dimers.     

Determination of the functionally important heme peripheral vinyl group orientation in paramagnetic hemoprotein by 2D NMR

2D NMR spectroscopies have been successfully used to characterize the heme peripheral vinyl groups in paramagnetic hemoprotein in spite of the difficulties from the rapid paramagnetic relaxation and the low digital resolution of the 2D NMR map. The scalar coupling network system among the vinyl protons is clearly identified in the COSY spectra from its characteristic cross-peak pattern and the dipolar coupling connectivities of the vinyl proton resonances with other heme side-chain proton resonances not only provide the specific assignment of vinyl beta-proton resonances but also allow the determination of the vinyl group orientation with respect to the heme plane.     

Mapping protein-protein interactions in solution by NMR spectroscopy.

NMR is very well suited to the study of especially weak protein-protein interactions, as no crystallization is required. The available NMR methods to this end are reviewed and illustrated with applications from the recent biochemical literature: intermolecular NOEs, cross-saturation, chemical shift perturbation, dynamics and exchange perturbation, paramagnetic methods, and dipolar orientation. Most of these methods are now routinely applied for complexes with total molecular mass of 60 kDa and can likely be applied to systems up to 1000 kDa. A substantial fraction of complexes studied show distinct effects of induced fit affecting structural and dynamical properties beyond the contact interface.     

Proton longitudinal relaxation investigation of histidyl residues in human normal adult hemoglobin.

The longitudinal relaxation of the C2 protons of surface histidyl residues as well as other aromatic protons of human normal adult deoxyhemoglobin investigated at 360 MHz is discussed in terms of the theory proposed by Kalk and Berendsen for the proton longitudinal relaxation in proteins (Kalk, A., and H.J.C. Berendsen. 1976. J. Magn. Reson. 24:343-366). The role of the four paramagnetic iron atoms of deoxyhemoglobin as fast-relaxing sinks for the overall proton longitudinal relaxation is evaluated according to the model proposed by Bloembergen for the relaxation of nuclei in crystals containing paramagnetic centers (Bloembergen, N. 1949. Physica. 15:386-426). The results suggest that the effectiveness of the paramagnetic iron atoms of deoxyhemoglobin for the overall proton longitudinal relaxation is reduced as a result of slower spin diffusion and wide distribution of methyl groups within the hemoglobin molecule. Thus, deoxyhemoglobin provides a good model for investigating the influence of cross relaxation on proton longitudinal relaxation in proteins at the slow motion limit and in the presence of paramagnetic centers. For the C2 protons of surface histidyl residues, we show that the cross relaxation resulting from the interresidue dipolar interaction makes an important contribution to their longitudinal relaxation.     

Measurement of dipolar couplings in a transducin peptide fragment weakly bound to oriented photo-activated rhodopsin

Rhodopsin-containing disks, isolated from rod outer segments of bovine retina, align at high magnetic fields with their membrane normal parallel to the magnetic field. After light-activation of rhodopsin, transient binding of the C-terminal transducin undecapeptide, selectively labeled with ¹⁵N at Leu⁵ and Gly⁹, results in residual dipolar contributions to the ¹J_NH splittings for these two residues. Both residues show ¹J_NH splittings which are smaller than in the dark-adapted or rhodopsin-free sample, and return to their isotropic values at a rate determined by the decay of the meta II state of rhodopsin. The dipolar couplings indicate that in the bound state, N-H vectors of Leu⁵ and Gly⁹ make angles of 48±4° and 40±8°, respectively, with the disk normal. These `transferred' dipolar couplings potentially offer a useful method for studying the conformation and orientation of flexible, low affinity ligands when bound to oriented integral membrane receptors.     

1H NMR study of the molecular structure and magnetic properties of the active site for the cyanomet complex of O2-avid hemoglobin from the trematode Paramphistomum epiclitum.

The solution molecular and electronic structures of the active site in the extremely O2-avid hemoglobin from the trematode Paramphistomum epiclitum have been investigated by 1H NMR on the cyanomet form in order to elucidate the distal hydrogen-bonding to a ligated H-bond acceptor ligand. Comparison of the strengths of dipolar interactions in solution with the alternate crystal structures of methemoglobin establish that the solution structure of wild-type Hb more closely resembles the crystal structure of the recombinant wild-type than the true wild-type met-hemoglobin. The distal Tyr66(E7) is found oriented out of the heme pocket in solution as found in both crystal structures. Analysis of dipolar contacts, dipolar shift and paramagnetic relaxation establishes that the Tyr32(B10) hydrogen proton adopts an orientation that allows it to make a strong H-bond to the bound cyanide. The observation of a significant isotope effect on the heme methyl contact shifts confirms a strong contact between the Tyr32(B10) OH and the ligated cyanide. The quantitative determination of the orientation and anisotropies of the paramagnetic susceptibility tensor reveal that the cyanide is tilted approximately 10 degrees from the heme normal so as to avoid van der Waals overlap with the Tyr32(B10) Oeta. The pattern of heme contact shifts with large low-field shifts for 7-CH3 and 18-CH3 is shown to arise not from the 180 degrees rotation about the alpha-gamma-meso axis, but due to the approximately 45 degrees rotation of the axial His imidazole ring, relative to that in mammalian globins.     

NMR approaches for monitoring domain orientations in calcium-binding proteins in solution using partial replacement of Ca by Tb

This work shows that the partial replacement of diamagnetic Ca²⁺ by paramagnetic Tb³⁺ in Ca²⁺/calmodulin systems in solution allows the measurement of interdomain NMR pseudocontact shifts and leads to magnetic alignment of the molecule such that significant residual dipolar couplings can be measured. Both these parameters can be used to provide structural information. Species in which Tb³⁺ ions are bound to only one domain of calmodulin (the N-domain) and Ca²⁺ ions to the other (the C-domain) provide convenient systems for measuring these parameters. The nuclei in the C-domain experience the local magnetic field induced by the paramagnetic Tb³⁺ ions bound to the other domain at distances of over 40 Å from the Tb³⁺ ion, shifting the resonances for these nuclei. In addition, the Tb³⁺ ions bound to the N-domain of calmodulin greatly enhance the magnetic susceptibility anisotropy of the molecule so that a certain degree of alignment is produced due to interaction with the external magnetic field. In this way, dipolar couplings between nuclear spins are not averaged to zero due to solution molecular tumbling and yield dipolar coupling contributions to, for example, the one-bond ¹⁵N-¹H splittings of up to 17 Hz in magnitude. The degree of alignment of the C-domain will also depend on the degree of orientational freedom of this domain with respect to the N-domain containing the Tb³⁺ ions. Pseudocontact shifts for NH groups and ¹H-¹⁵N residual dipolar couplings for the directly bonded atoms have been measured for calmodulin itself, where the domains have orientational freedom, and for the complex of calmodulin with a target peptide from skeletal muscle myosin light chain kinase, where the domains have fixed orientations with respect to each other. The simultaneous measurements of these parameters for systems with domains in fixed orientations show great potential for the determination of the relative orientation of the domains.     

Molecular distances from dipolar coupled spin-labels: the global analysis of multifrequency continuous wave electron paramagnetic resonance data.

For immobilized nitroxide spin-labels with a well-defined interprobe geometry, resolved dipolar splittings can be observed in continuous wave electron paramagnetic resonance (CW-EPR) spectra for interelectron distances as large as 30 A using perdeuterated probes. In this work, algorithms are developed for calculating CW-EPR spectra of immobilized, dipolar coupled nitroxides, and then used to define the limits of sensitivity to the interelectron distance as a function of geometry and microwave frequency. Secondly, the CW-EPR spectra of N epsilon-spin-labeled coenzyme NAD+ bound to microcrystalline, tetrameric glyceraldehyde-3-phosphate dehydrogenase (GAPDH) have been collected at 9.8, 34, and 94 GHz. These data have been analyzed, using a combination of simulated annealing and global analysis, to obtain a unique fit to the data. The values of the intermitroxide distance and the five angles defining the relative orientation of the two nitroxides are in reasonable agreement with a molecular model built from the known crystal structure. Finally, the effect of rigid body isotropic rotational diffusion on the CW-EPR spectra of dipolar coupled nitroxides has been investigated using an algorithm based on Brownian dynamics trajectories. These calculations demonstrate the sensitivity of CW-EPR spectra to dipolar coupling in the presence of rigid body rotational diffusion.     

A novel interactive tool for rigid-body modeling of multi-domain macromolecules using residual dipolar couplings

Residual dipolar couplings (RDC), measured by dissolving proteins in dilute liquid crystal media, or by studying naturally paramagnetic molecules, have rapidly become established as routine measurements in the investigation of the structure of macromolecules by NMR. One of the most obvious applications of the previously inaccessible long-range angular information afforded by RDC is the accurate definition of domain orientation in multi-module macromolecules or complexes. In this paper we describe a novel program developed to allow the determination of alignment tensor parameters for individual or multiple domains in macromolecules from residual dipolar couplings and to facilitate their manipulation to construct low-resolution models of macromolecular structure. For multi-domain systems the program determines the relative orientation of individual structured domains, and provides graphical user-driven rigid-body modeling of the different modules relative to the common tensorial frame. Translational freedom in the common frame, and equivalent rotations about the diagonalized (x,y,z) axes are used to position the different modules in the common frame to find a model in best agreement with experimentally measured couplings alone or in combination with additional experimental or covalent information.     

Varied magnetic field, multiple-pulse, and magic-angle spinning proton nuclear magnetic resonance study of muscle water.

The nuclear magnetic resonance linewidth of 1H in water of frog muscle was studied as a function of magnetic field strength and angle of orientation. The results suggest that the observed spectra are dominated by demagnetization field anisotropy and dispersion, but a small static dipolar interaction of the order of a few hertz man be present. Data from line-narrowing, multiple-pulse experiments also indicate the presence of a small dipolar broadening.