Completely automated, highly error-tolerant macromolecular structure determination from multidimensional nuclear overhauser enhancement spectra and chemical shift assignments

The major rate-limiting step in high-throughput NMR protein structure determination involves the calculation of a reliable initial fold, the elimination of incorrect nuclear Overhauser enhancement (NOE) assignments, and the resolution of NOE assignment ambiguities. We present a robust approach to automatically calculate structures with a backbone coordinate accuracy of 1.0-1.5 A from datasets in which as much as 80% of the long-range NOE information (i.e., between residues separated by more than five positions in the sequence) is incorrect. The current algorithm differs from previously published methods in that it has been expressly designed to ensure that the results from successive cycles are not biased by the global fold of structures generated in preceding cycles. Consequently, the method is highly error tolerant and is not easily funnelled down an incorrect path in either three-dimensional structure or NOE assignment space. The algorithm incorporates three main features: a linear energy function representation of the NOE restraints to allow maximization of the number of simultaneously satisfied restraints during the course of simulated annealing; a method for handling the presence of multiple possible assignments for each NOE cross-peak which avoids local minima by treating each possible assignment as if it were an independent restraint; and a probabilistic method to permit both inactivation and reactivation of all NOE restraints on the fly during the course of simulated annealing. NOE restraints are never removed permanently, thereby significantly reducing the likelihood of becoming trapped in a false minimum of NOE assignment space. The effectiveness of the algorithm is demonstrated using completely automatically peak-picked experimental NOE data from two proteins: interleukin-4 (136 residues) and cyanovirin-N (101 residues). The limits of the method are explored using simulated data on the 56-residue B1 domain of Streptococcal protein G.     

Structures of protein-protein complexes are docked using only NMR restraints from residual dipolar coupling and chemical shift perturbations

NMR structures of protein-protein and protein-ligand complexes rely heavily on intermolecular NOEs. Recent work has shown that if no significant conformational changes occur upon complex formation residual dipolar coupling can replace most of the NOE restraints in protein-protein complexes, while restraints derived from chemical shift perturbations can largely replace intermolecular NOEs in protein-ligand structures. By combining restraints from chemical shift perturbations with orientation restraints derived from measurements of residual dipolar couplings, we show that the structure of the EIN-HPr complex can be calculated without NOE restraints. The final structure, built from the crystal structures of EIN and HPr in their uncomplexed form and docked only with NMR restraints, places HPr within 2.5 A of the position determined from the mean NMR structure of the complex.     

Eta(z)/kappa: a transverse relaxation optimized spectroscopy NMR experiment measuring longitudinal relaxation interference

NMR spin relaxation experiments provide a powerful tool for the measurement of global and local biomolecular rotational dynamics at subnanosecond time scales. Technical limitations restrict most spin relaxation studies to biomolecules weighing less than 10 kDa, considerably smaller than the average protein molecular weight of 30 kDa. In particular, experiments measuring eta(z), the longitudinal (1)H(N)-(15)N dipole-dipole (DD)/(15)N chemical shift anisotropy (CSA) cross-correlated relaxation rate, are among those least suitable for use with larger biosystems. This is unfortunate because these experiments yield valuable insight into the variability of the (15)N CSA tensor over the polypeptide backbone, and this knowledge is critical to the correct interpretation of most (15)N-NMR backbone relaxation experiments, including R(2) and R(1). In order to remedy this situation, we present a new (1)H(N)-(15)N transverse relaxation optimized spectroscopy experiment measuring eta(z) suitable for applications with larger proteins (up to at least 30 kDa). The presented experiment also yields kappa, the site-specific rate of longitudinal (1)H(N)-(1)H(') DD cross relaxation. We describe the eta(z)/kappa experiment's performance in protonated human ubiquitin at 30.0 degrees C and in protonated calcium-saturated calmodulin/peptide complex at 20.0 degrees C, and demonstrate preliminary experimental results for a deuterated E. coli DnaK ATPase domain construct at 34 degrees C.     

Docking of protein-protein complexes on the basis of highly ambiguous intermolecular distance restraints derived from 1H/15N chemical shift mapping and backbone 15N-1H residual dipolar couplings using conjoined rigid body/torsion angle dynamics

A simple and reliable method for docking protein-protein complexes using (1)H(N)/(15)N chemical shift mapping and backbone (15)N-(1)H residual dipolar couplings is presented and illustrated with three complexes (EIN-HPr, IIA(Glc)-HPr, and IIA(Mtl)-HPr) of known structure. The (1)H(N)/(15)N chemical shift mapping data are transformed into a set of highly ambiguous, intermolecular distance restraints (comprising between 400 and 3000 individual distances) with translational and some degree of orientational information content, while the dipolar couplings provide information on relative protein-protein orientation. The optimization protocol employs conjoined rigid body/torsion angle dynamics in simulated annealing calculations. The target function also comprises three nonbonded interactions terms: a van der Waals repulsion term to prevent atomic overlap, a radius of gyration term (E(rgyr)) to avoid expansion at the protein-protein interface, and a torsion angle database potential of mean force to bias interfacial side chain conformations toward physically allowed rotamers. For the EIN-HPr and IIA(Glc)-HPr complexes, all structures satisfying the experimental restraints (i.e., both the ambiguous intermolecular distance restraints and the dipolar couplings) converge to a single cluster with mean backbone coordinate accuracies of 0.7-1.5 A. For the IIA(Mtl)-HPr complex, twofold degeneracy remains, and the structures cluster into two distinct solutions differing by a 180 degrees rotation about the z axis of the alignment tensor. The correct and incorrect solutions which have mean backbone coordinate accuracies of approximately 0.5 and approximately 10.5 A, respectively, can readily be distinguished using a variety of criteria: (a) examination of the overall (1)H(N)/(15)N chemical shift perturbation map (because the incorrect cluster predicts the presence of residues at the interface that experience only minimal chemical shift perturbations; this information is readily incorporated into the calculations in the form of ambiguous intermolecular repulsion restraints); (b) back-calculation of dipolar couplings on the basis of molecular shape; or (c) the E(rgyr) distribution which, because of its global nature, directly reflects the interfacial packing quality. This methodology should be particularly useful for high throughput, NMR-based, structural proteomics.     

A new spin probe of protein dynamics: nitrogen relaxation in 15N-2H amide groups

(15)N spin relaxation data have provided a wealth of information on protein dynamics in solution. Standard R(1), R(1)(rho), and NOE experiments aimed at (15)N[(1)H] amide moieties are complemented in this work by HA(CACO)N-type experiments allowing the measurement of nitrogen R(1) and R(1)(rho) rates at deuterated (15)N[(2)D] sites. Difference rates obtained using this approach, R(1)((15)N[(1)H]) - R(1)((15)N[(2)D]) and R(2)((15)N[(1)H]) - R(2)((15)N[(2)D]), depend exclusively on dipolar interactions and are insensitive to (15)N CSA and R(ex) relaxation mechanisms. The methodology has been tested on a sample of peptostreptococcal protein L (63 residues) prepared in 50% H(2)O-50% D(2)O solvent. The results from the new and conventional experiments are found to be consistent, with respect to both local backbone dynamics and overall protein tumbling. Combining several data sets permits evaluation of the spectral density J(omega(D) + omega(N)) for each amide site. This spectral density samples a uniquely low frequency (26 MHz at a 500 MHz field) and, therefore, is expected to be highly useful for characterizing nanosecond time scale local motions. The spectral density mapping demonstrates that, in the case of protein L, J(omega(D) + omega(N)) values are compatible with the Lipari-Szabo interpretation of backbone dynamics based on the conventional (15)N relaxation data.     

Off-resonance TROSY-selected R 1rho experiment with improved sensitivity for medium- and high-molecular-weight proteins

NMR spin relaxation techniques that utilize relaxation interference phenomena (TROSY) enable chemical exchange processes to be characterized in high-molecular-weight proteins. A TROSY-selected (TS) approach for measuring off-resonance R1rho relaxation in the spin-locked rotating reference frame is developed using three principles: (i) deuteration of nonexchangeable 1H sites to minimize remote dipole-dipole interactions, (ii) selective excitation of the slowly relaxing 15N doublet component to obtain optimal initial conditions, and (iii) selective inversion of one of the 15N doublet components to suppress cross-relaxation during the spin-lock period. The method is validated using [90%-15N, 70%-2H] ubiquitin at 280 K. The TROSY-selected R1rho experiment enables characterization of backbone dynamics on the microsecond time scale in large proteins.     

Hydrogen exchange study on the hydroxyl groups of serine and threonine residues in proteins and structure refinement using NOE restraints with polar side-chain groups

We recently developed new NMR methods for monitoring the hydrogen exchange rates of tyrosine hydroxyl (Tyr-OH) and cysteine sulfhydryl (Cys-SH) groups in proteins. These methods facilitate the identification of slowly exchanging polar side-chain protons in proteins, which serve as sources of NOE restraints for protein structure refinement. Here, we have extended the methods for monitoring the hydrogen exchange rates of the OH groups of serine (Ser) and threonine (Thr) residues in an 18.2 kDa protein, EPPIb, and thus demonstrated the usefulness of NOE restraints with slowly exchanging OH protons for refining the protein structure. The slowly exchanging Ser/Thr-OH groups were readily identified by monitoring the (13)C(β)-NMR signals in an H(2)O/D(2)O (1:1) mixture, for the protein containing Ser/Thr residues with (13)C, (2)H-double labels at their β carbons. Under these circumstances, the OH groups exist in equilibrium between the protonated and deuterated isotopomers, and the (13)C(β) peaks of the two species are resolved when their exchange rate is slower than the time scale of the isotope shift effect. In the case of EPPIb dissolved in 50 mM sodium phosphate buffer (pH 7.5) at 40 °C, one Ser and four Thr residues were found to have slowly exchanging hydroxyl groups (k(ex) < ~40 s(-1)). With the information for the slowly exchanging Ser/Thr-OH groups in hand, we could collect additional NOE restraints for EPPIb, thereby making a unique and important contribution toward defining the spatial positions of the OH protons, and thus the hydrogen-bonding acceptor atoms.     

Temperature dependence of domain motions of calmodulin probed by NMR relaxation at multiple fields

Interdomain motions of Ca(2+)-ligated calmodulin were characterized by analyzing the nuclear magnetic resonance (15)N longitudinal relaxation rate R(1), transverse relaxation rate R(2), and steady-state {(1)H}-(15)N NOE of the backbone amide group at three different magnetic field strengths (18.8, 14.1, and 8.5 T) and four different temperatures (21, 27, 35, and 43 degrees C). Between 35 and 43 degrees C, a larger than expected change in the amplitude and the time scale of the interdomain motion for both N- and C-domains was observed. We attribute this to the shift in population of four residues (74-77) in the central linker from predominantly helical to random coil in this temperature range. This is consistent with the conformation of these residues in the calmodulin-peptide complex, where they are nonhelical. The doubling of the disordered region of the central helix (residues 78-81 at room temperature) when temperature is raised from 35 to 43 degrees C results in larger amplitude interdomain motion. Our analysis of the NMR relaxation data quantifies subtle changes in the interdomain dynamics and provides an additional tool to monitor conformational changes in multidomain proteins.     

Determinations of 15N chemical shift anisotropy magnitudes in a uniformly 15N,13C-labeled microcrystalline protein by three-dimensional magic-angle spinning nuclear magnetic resonance spectroscopy

Amide 15N chemical shift anisotropy (CSA) tensors provide quantitative insight into protein structure and dynamics. Experimental determinations of 15N CSA tensors in biologically relevant molecules have typically been performed by NMR relaxation studies in solution, goniometric analysis of single-crystal spectra, or slow magic-angle spinning (MAS) NMR experiments of microcrystalline samples. Here we present measurements of 15N CSA tensor magnitudes in a protein of known structure by three-dimensional MAS solid-state NMR. Isotropic 15N, 13C alpha, and 13C' chemical shifts in two dimensions resolve site-specific backbone amide recoupled CSA line shapes in the third dimension. Application of the experiments to the 56-residue beta1 immunoglobulin binding domain of protein G (GB1) enabled 91 independent determinations of 15N tensors at 51 of the 55 backbone amide sites, for which 15N-13C alpha and/or 15N-13C' cross-peaks were resolved in the two-dimensional experiment. For 37 15N signals, both intra- and interresidue correlations were resolved, enabling direct comparison of two experimental data sets to enhance measurement precision. Systematic variations between beta-sheet and alpha-helix residues are observed; the average value for the anisotropy parameter, delta (delta = delta(zz) - delta(iso)), for alpha-helical residues is 6 ppm greater than that for the beta-sheet residues. The results show a variation in delta of 15N amide backbone sites between -77 and -115 ppm, with an average value of -103.5 ppm. Some sites (e.g., G41) display smaller anisotropy due to backbone dynamics. In contrast, we observe an unusually large 15N tensor for K50, a residue that has an atypical, positive value for the backbone phi torsion angle. To our knowledge, this is the most complete experimental analysis of 15N CSA magnitude to date in a solid protein. The availability of previous high-resolution crystal and solution NMR structures, as well as detailed solid-state NMR studies, will enhance the value of these measurements as a benchmark for the development of ab initio calculations of amide 15N shielding tensor magnitudes.     

An exchange-free measure of 15N transverse relaxation: an NMR spectroscopy application to the study of a folding intermediate with pervasive chemical exchange

A series of experiments are presented that provide an exchange-free measure of dipole-dipole (15)N transverse relaxation, R(dd), that can then be substituted for (15)N R(1rho) or R(2) rates in the study of internal protein dynamics. The method is predicated on the measurement of a series of relaxation rates involving (1)H-(15)N longitudinal order, anti-phase (1)H and (15)N single-quantum coherences, and (1)H-(15)N multiple quantum coherences; the relaxation rates of all coherences are measured under conditions of spin-locking. Results from detailed simulations and experiments on a number of protein systems establish that R(dd) values are independent of exchange and systematic errors from dipolar interactions with proximal protons are calculated to be less than 1-2%, on average, for applications to perdeuterated proteins. Simulations further indicate that the methodology is rather insensitive to the exact level of deuteration so long as proteins are reasonably highly deuterated (>50%). The utility of the methodology is demonstrated with applications involving protein L, ubiquitin, and a stabilized folding intermediate of apocytochrome b(562) that shows large contributions to (15)N R(1rho) relaxation from chemical exchange.     

Evaluation of protein 15N relaxation times by inverse Laplace transformation

Relaxation times (T1, T2, T1rho) are usually evaluated from exponential decay data by least-squares fitting methods. For this procedure, the integrals or amplitudes of signals must be determined, which can be laborious with large data sets. Moreover, the fitting requires a priori knowledge of the number of exponential components responsible for the decay. We have adapted inverse Laplace transformation (ILT) for the analysis of relaxation data. Exponential components are resolved with ILT to reciprocal space on their corresponding relaxation rate values. The ILT approach was applied to 3D linewidth-resolved 15N HSQC experiments to evaluate 15N T1 and T2 relaxation times of ubiquitin. The resulting spectrum is a true 3D spectrum, where the signals are separated by their 1H and 15N chemical shifts (HSQC correlations) and by their relaxation rate values (R1 or R2). From this spectrum, the relaxation times can be obtained directly with a simple peak-picking procedure.     

Natural abundance nitrogen-15 n.m.r. spectroscopy. Spin–lattice relaxation in organic compounds

The small negative magnetogyric ratio (γ) of the ¹⁵N nucleus decreases the efficiency of ¹⁵N? ¹H dipole-dipole relaxation to about 25% of that for an analogous ¹³C nucleus. This may lead to greater competition from other relaxation mechanisms in ¹⁵N n.m.r. and consequent partial or total quenching of the negative nuclear Overhauser effect (NOE). In unfavorable circumstances nulling of the ¹⁵N resonance can occur. Previous ¹⁵N relaxation studies have examined isotopically enriched, low molecular weight compounds. The present study examines several small to intermediate size organic compounds containing nitrogen at natural isotopic abundance. In contrast to some of the earlier studies, ¹⁵N? ¹H dipolar relaxation was found to be dominant for protonated nitrogen atoms, even for two tertiary nitrogens (the tertiary amine nitrogen in 1,2,3,4,6,7,12,12b-octahydroindolo[2,3-a] quinolizine and the oxime nitrogen in 3-methyl-2-pentanone ketoxime). The magnitude of the NOE and the moderate value of T₁ indicate effective dipolar relaxation from neighboring but not directly bonded protons in these cases. Nitro groups were found, as expected, to have predominant contributions from non-dipolar mechanisms, and in one case (2-methyl-2-nitro-1, 3-propanediol) signal nulling (NOE of η = ?1) was observed. The effect of paramagnetic impurities was demonstrated for ethanolamine, which contains a basic nitrogen. In this case T₁^DD(¹⁵N? ¹H) = 4·3 s; added Ni(acac)₂ at 1 × 10^?4 Molar reduced the ¹⁵N T₁ to 0·065 s and consequently the NOE to η = 0.     

Magic angle spinning NMR spectroscopy of thioredoxin reassemblies

Differentially isotopically enriched 1-73((13)C,(15)N)/74-108((15)N) and 1-73((15)N)/74-108((13)C,(15)N) Escherichia coli thioredoxin reassemblies prepared by fragment complementation were investigated by high-resolution magic angle spinning solid-state NMR spectroscopy. Nearly complete resonance assignments, secondary and tertiary structure analysis are reported for 1-73((13)C,(15)N)/74-108((15)N) reassembled thioredoxin. Temperature dependence of the dipolar-assisted rotational resonance (DARR) spectra reveals the residues undergoing intermediate timescale motions at temperatures below - 15 degrees C. Analysis of the DARR intensity buildups as a function of mixing time in these reassemblies indicates that at long mixing times medium- and long-range cross-peaks do not experience dipolar truncation, suggesting that isotopic dilution is not required for gaining nontrivial distance restraints for structure calculations.     

Elucidation of structural restraints for phosphate residues with different hydrogen bonding and ionization states

Solid state NMR spectroscopy and gauge including atomic orbital (GIAO) theoretical calculations were employed to establish structural restraints (ionization, hydrogen bonding, spatial arrangement) for O-phosphorylated l-threonine derivatives in different ionization states and hydrogen bonding strengths. These structural restraints are invaluable in molecular modeling and docking procedures for biological species containing phosphoryl groups. Both the experimental and the GIAO approach show that 31P delta ii chemical shift tensor parameters are very sensitive to the ionization state. The negative values found for the skew kappa are typical for -2 phosphates. The distinct span Omega values reflect the change of strength of hydrogen bonding. For species in the -1 ionization state, engaged in very strong hydrogen bonds, Omega is smaller than for a phosphate group involved in weak hydrogen bonding. For phosphates in the -2 ionization state, Omega is significantly smaller compared to -1 species, although the kappa for -1 samples never reaches negative values. For -1 phosphate residues, in the case when 1H one pulse and/or combined rotation and multiple pulse spectroscopy (CRAMPS) sequences fail and assignment of proton chemical shift is ambiguous, a combination of 1H-(13)C and 1H-(31)P 2D heteronuclear correlation (HETCOR) correlations is found to be an excellent tool for the elucidation of 1H isotropic chemical shifts. In addition, a 2D strategy using 1H-(1)H double quantum filter (DQF) correlations [a back-to-back (BABA) sequence in this work] is useful for analyzing the topology of hydrogen bonding. In the case of a multicenter phosphorus domain, 2D 31P-(31)P proton driven spin diffusion experiments give information about the spatial arrangement of the phosphate residues.     

CABS‐NMR—De novo tool for rapid global fold determination from chemical shifts,residual dipolar couplings and sparse methyl‐methyl noes

Recent development of nuclear magnetic resonance (NMR) techniques provided new types of structural restraints that can be successfully used in fast and low‐cost global protein fold determination. Here, we present CABS‐NMR, an efficient protein modeling tool, which takes advantage of such structural restraints. The restraints are converted from original NMR data to fit the coarse grained protein representation of the C‐Alpha‐Beta‐Side‐group (CABS) algorithm. CABS is a Monte Carlo search algorithm that uses a knowledge‐based force field. Its versatile structure enables a variety of protein‐modeling protocols, including purely de novo folding, folding guided by restraints derived from template structures or, structure assembly based on experimental data. In particular, CABS‐NMR uses the distance and angular restraints set derived from various NMR experiments. This new modeling technique was successfully tested in structure determination of 10 globular proteins of size up to 216 residues, for which sparse NMR data were available. Additional detailed analysis was performed for a S100A1 protein. Namely, we successfully predicted Nuclear Overhauser Effect signals on the basis of low‐energy structures obtained from chemical shifts by CABS‐NMR. It has been observed that utility of chemical shifts and other types of experimental data (i.e. residual dipolar couplings and methyl‐methyl Nuclear Overhauser Effect signals) in the presented modeling pipeline depends mainly on size of a protein and complexity of its topology. In this work, we have provided tools for either post‐experiment processing of various kinds of NMR data or fast and low‐cost structural analysis in the still challenging field of new fold predictions. © 2010 Wiley Periodicals, Inc. J Comput Chem, 2011     

Site-directed parallel spin-labeling and paramagnetic relaxation enhancement in structure determination of membrane proteins by solution NMR spectroscopy

A major challenge for the structure determination of integral membrane proteins by solution NMR spectroscopy is the limited number of NOE restraints in these systems stemming from extensive deuteration. Paramagnetic relaxation enhancement (PRE) by means of nitroxide spin-labels can provide valuable long-range distance information but, in practice, has limits in its application to membrane proteins because spin-labels are often incompletely reduced in highly apolar environments. Using the integral membrane protein OmpA as a model system, we introduce a method of parallel spin-labeling with paramagnetic and diamagnetic labels and show that distances in the range 15-24 Angstroms can be readily determined. The protein was labeled at 11 water-exposed and lipid-covered sites, and 320 PRE distance restraints were measured. The addition of these restraints resulted in significant improvement of the calculated backbone structure of OmpA. Structures of reasonable quality can even be calculated with PRE distance restraints only, i.e., in the absence of NOE distance restraints.     

Three-dimensional 13C-detected CH3-TOCSY using selectively protonated proteins: facile methyl resonance assignment and protein structure determination

Recent advances in instrumentation and isotope labeling methodology allow proteins up to 100 kDa in size to be studied in detail using NMR spectroscopy. Using 2H/13C/15N enrichment and selective methyl protonation, we show that newly developed 13C direct detection methods can be used to rapidly yield proton and carbon resonance assignments for the methyl groups of Val, Leu, and Ile residues. We present a highly sensitive 13C-detected CH3-TOCSY experiment that, in combination with standard 1H-detected backbone experiments, allows the full assignment of side chain resonances in methyl-protonated residues. Selective methyl protonation, originally developed by Kay and co-workers (Rosen, M. K.; Gardner, K. H.; Willis, R. C.; Parris, W. E.; Pawson, T.; Kay, L. E. J. Mol. Biol. 1996, 263, 627-636; Gardner, K. G.; Kay, L. E. Annu. Rev. Biophys. Biomol. Struct. 1998, 27, 357-406; Goto, N. K.; Kay, L. E. Curr. Opin. Struct. Biol. 2000, 10, 585-592), improves the nuclear relaxation behavior of larger proteins compared to their fully protonated counterparts, allows significant simplification of spectra, and facilitates NOE assignments. Here, we demonstrate the usefulness of the 13C-detected CH3-TOCSY experiment through studies of (i) a medium-sized protein (CbpA-R1; 14 kDa) with a repetitive primary sequence that yields highly degenerate NMR spectra, and (ii) a larger, bimolecular protein complex (p21-KID/Cdk2; 45 kDa) at low concentration in a high ionic strength solution. Through the analysis of NOEs involving amide and Ile, Leu, and Val methyl protons, we determined the global fold of CbpA-R1, a bacterial protein that mediates the pathogenic effects of Streptococcus pneumoniae, demonstrating that this approach can significantly reduce the time required to determine protein structures by NMR.     

Zinc(II) complexes of ubiquitin: speciation, affinity and binding features

Intraneuronal inclusions consisting of hypermetallated, (poly-)ubiquitinated proteins are a hallmark of neurodegeneration. To highlight the possible role played by metal ions in the dysfunction of the ubiquitin-proteasome system, here we report on zinc(II)/ubiquitin binding in terms of affinity constants, speciation, preferential binding sites and effects on protein stability and self-assembly. Potentiometric titrations allowed us to establish that at neutral pH only two species, ZnUb and Zn(2)Ub, are present in solution, in line with ESI-MS data. A change in the diffusion coefficient of ubiquitin was observed by NMR DOSY experiments after addition of Zn(II) ions, and thus indicates metal-promoted formation of protein assemblies. Analysis of (1)H, (15)N, (13)Cα and (13)CO chemical-shift perturbation after equimolar addition of Zn(II) ions to ubiquitin outlined two different metal-binding modes. The first involves a dynamic equilibrium in which zinc(II) is shared between a region including Met1, Gln2, Ile3, Phe4, Thr12, Leu15, Glu16, Val17, Glu18, Ile61 and Gln62 residues, which represent a site already described for copper binding, and a domain comprising Ile23, Glu24, Lys27, Ala28, Gln49, Glu51, Asp52, Arg54 and Thr55 residues. A second looser binding mode is centred on His68. Differential scanning calorimetry evidenced that addition of increasing amounts of Zn(II) ions does not affect protein thermal stability; rather it influences the shape of thermograms because of the increased propensity of ubiquitin to self-associate. The results presented here indicate that Zn(II) ions may interact with specific regions of ubiquitin and promote protein-protein contacts.     

Variability of the 15N chemical shielding tensors in the B3 domain of protein G from 15N relaxation measurements at several fields. Implications for backbone order parameters

We applied a combination of 15N relaxation and CSA/dipolar cross-correlation measurements at five magnetic fields (9.4, 11.7, 14.1, 16.4, and 18.8 T) to determine the 15N chemical shielding tensors for backbone amides in protein G in solution. The data were analyzed using various model-independent approaches and those based on Lipari-Szabo approximation, all of them yielding similar results. The results indicate a range of site-specific values of the anisotropy (CSA) and orientation of the 15N chemical shielding tensor, similar to those in ubiquitin (Fushman, et al. J. Am. Chem. Soc. 1998, 120, 10947; J. Am. Chem. Soc. 1999, 121, 8577). Assuming a Gaussian distribution of the 15N CSA values, the mean anisotropy is -173.9 to -177.2 ppm (for 1.02 A NH bond length) and the site-to-site CSA variability is +/-17.6 to +/-21.4 ppm, depending on the method used. This CSA variability is significantly larger than derived previously for ribonuclease H (Kroenke, et al. J. Am. Chem. Soc. 1999, 121, 10119) or recently, using "meta-analysis" for ubiquitin (Damberg, et al. J. Am. Chem. Soc. 2005, 127, 1995). Standard interpretation of 15N relaxation studies of backbone dynamics in proteins involves an a priori assumption of a uniform 15N CSA. We show that this assumption leads to a significant discrepancy between the order parameters obtained at different fields. Using the site-specific CSAs obtained from our study removes this discrepancy and allows simultaneous fit of relaxation data at all five fields to Lipari-Szabo spectral densities. These findings emphasize the necessity of taking into account the variability of 15N CSA for accurate analysis of protein dynamics from 15N relaxation measurements.