Large Shift Changes Research Articles

Electrical and ionic conductivity effects on magic-angle spinning nuclear magnetic resonance parameters of CuI

We investigate experimentally and theoretically the effects of two different types of conductivity, electrical and ionic, upon magic-angle spinning NMR spectra. The experimental demonstration of these effects involves (63)Cu, (65)Cu, and (127)I variable temperature MAS-NMR experiments on samples of γ-CuI, a Cu(+)-ion conductor at elevated temperatures as well as a wide bandgap semiconductor. We extend previous observations that the chemical shifts depend very strongly upon the square of the spinning-speed as well as the particular sample studied and the magnetic field strength. By using the (207)Pb resonance of lead nitrate mixed with the γ-CuI as an internal chemical shift thermometer we show that frictional heating effects of the rotor do not account for the observations. Instead, we find that spinning bulk CuI, a p-type semiconductor due to Cu(+) vacancies in nonstoichiometric samples, in a magnetic field generates induced AC electric currents from the Lorentz force that can resistively heat the sample by over 200 °C. These induced currents oscillate along the rotor spinning axis at the spinning speed. Their associated heating effects are disrupted in samples containing inert filler material, indicating the existence of macroscopic current pathways between micron-sized crystallites. Accurate measurements of the temperature-dependence of the (63)Cu and (127)I chemical shifts in such diluted samples reveal that they are of similar magnitude (ca. 0.27 ppm/K) but opposite sign (being negative for (63)Cu), and appear to depend slightly upon the particular sample. This relationship is identical to the corresponding slopes of the chemical shifts versus square of the spinning speed, again consistent with sample heating as the source of the observed large shift changes. Higher drive-gas pressures are required to spin samples that have higher effective electrical conductivities, indicating the presence of a braking effect arising from the induced currents produced by rotating a conductor in a homogeneous magnetic field. We present a theoretical analysis and finite-element simulations that account for the magnitude and rapid time-scale of the resistive heating effects and the quadratic spinning speed dependence of the chemical shift observed experimentally. Known thermophysical properties are used as inputs to the model, the sole adjustable parameter being a scaling of the bulk thermal conductivity of CuI in order to account for the effective thermal conductivity of the rotating powdered sample. In addition to the dramatic consequences of electrical conductivity in the sample, ionic conductivity also influences the spectra. All three nuclei exhibit quadrupolar satellite transitions extending over several hundred kilohertz that reflect defects perturbing the cubic symmetry of the zincblende lattice. Broadening of these satellite transitions with increasing temperature arises from the onset of Cu(+) ion jumps to sites with different electric field gradients, a process that interferes with the formation of rotational echoes. This broadening has been quantitatively analyzed for the (63)Cu and (65)Cu nuclei using a simple model in the literature to yield an activation barrier of 0.64 eV (61.7 kJ/mole) for the Cu(+) ion jumping motion responsible for the ionic conductivity that agrees with earlier results based on (63)Cu NMR relaxation times of static samples.

Assignment of Ile, Leu, and Val Methyl Correlations in Supra-Molecular Systems: An Application to Aspartate Transcarbamoylase

A number of complementary approaches for the assignment of Ile, Leu, and Val methyl groups in Methyl-TROSY spectra of supra-molecular protein complexes are presented and compared. This includes the transfer of assignments from smaller fragments to the complex using a "divide-and-conquer" approach, assignment transfer via exchange spectroscopy, or, alternatively, generating assignments of the complex through the measurement of pseudocontact shifts, facilitated by the introduction of paramagnetic probes. The methodology is applied to the assignment of the regulatory chains in the 300 kDa enzyme aspartate transcarbamoylase, ATCase. The "divide-and-conquer" method that has proven to be very powerful in applications to other systems produced assignments for approximately 60% of the observed methyl groups in TROSY maps of ATCase. By contrast, the combination of all approaches led to assignments for 86% of the methyls, providing a large number of probes of structure and dynamics. The derived assignments were used to interpret chemical shift changes of ATCase upon titration with the nucleotide ATP. Large shift changes in the N-terminal tails of the regulatory chain provide the first evidence for structural perturbations in a region that is known to play a critical role on the effect of nucleotide binding on distal catalytic sites of this allosteric enzyme.

1H-NMR parameters of common amino acid residues measured in aqueous solutions of the linear tetrapeptides Gly-Gly-X-Ala at pressures between 0.1 and 200 MPa

For the interpretation of chemical shift changes induced by pressure in proteins, a comparison with random-coil data is important. For providing such a data basis, the pressure dependence of the 1H-NMR chemical shifts of the amino acids X in the random-coil model peptides Gly-Gly- X-Ala were studied for the 20 common amino acids at two pH values (pH 5.0 and 5.4) at 305 K, in the pressure range from 0.1 to 200 MPa. The largest shift changes Δδ with pressure p can be observed for the backbone amide protons. The average linear pressure coefficient δ Δp is 0.38 ppm GPa −1, with a root mean square deviation of 0.2 ppm GPa −1. In contrast to the downfield shift typical for amide protons, the H α-resonances typically shift upfield, with a pressure coefficient of −0.025 ppm GPa −1 and a root mean square deviation of 0.05 ppm GPa −1. The side chain resonances are only weakly influenced by pressure, on average they are shifted by 0.014 ppm GPa −1 , with a root mean square deviation of 0.14 ppm GPa −1. The exceptions are the side chain amide protons of asparagine and glutamine. Here, values of 0.214 (Asn H δ21), 0.417 (Asn H δ22), 0.260 (Gln H ε21) and 0.395 (Gln H ε22) ppm GPa −1 can be observed. In both cases, the pressure dependent shift is larger for the pro-E proton than for the pro-Z proton. Within the limits of error the equilibrium constant for the trans- and cis-conformers at the proline peptide bond is independent of pressure in the pressure range studied.

The hairpin structure of the (6)F1(1)F2(2)F2 fragment from human fibronectin enhances gelatin binding.

The solution structure of the (6)F1(1)F2(2)F2 fragment from the gelatin-binding region of fibronectin has been determined (Protein Data Bank entry codes 1e88 and 1e8b). The structure reveals an extensive hydrophobic interface between the non-contiguous (6)F1 and (2)F2 modules. The buried surface area between (6)F1 and (2)F2 ( approximately 870 A(2)) is the largest intermodule interface seen in fibronectin to date. The dissection of (6)F1(1)F2(2)F2 into the (6)F1(1)F2 pair and (2)F2 results in near-complete loss of gelatin-binding activity. The hairpin topology of (6)F1(1)F2(2)F2 may facilitate intramolecular contact between the matrix assembly regions flanking the gelatin-binding domain. This is the first high-resolution study to reveal a compact, globular arrangement of modules in fibronectin. This arrangement is not consistent with the view that fibronectin is simply a linear 'string of beads'.

Two-dimensional NMR Studies of Selenomethionyl Calmodulin

Calmodulin (CaM) is a ubiquitous calcium regulatory protein that can interact with almost 30 different target proteins. The majority of the CaM-binding domains of the target proteins are believed to interact with two hydrophobic surfaces on Ca2+-CaM; these two regions are very rich in Met residues. To obtain more information about the role of these residues, we have biosynthetically incorporated selenomethionine (SeMet) in place of the nine Met residues of CaM. Amino acid analysis shows that the SeMet-CaM contains 15% Met and 85% SeMet. SeMet-CaM retains many of the properties of the wild-type protein; it activates the enzyme cyclic nucleotide phosphodiesterase, it binds to phenyl-Sepharose and myosin light chain kinase (MLCK) in a calcium-dependent manner, and it experience a calcium-dependent band shift during SDS-gel electrophoresis. Moreover, by comparing the natural abundance (1H, 13C)-heteronuclear multiple quantum coherence (HMQC) spectra of the calcium, apo and target peptide-bound forms of wild-type CaM and SeMet-CaM, we have found that the two proteins have very similar, if not identical, structures. Thus, the substitution of SeMet for Met does not cause a change in the conformation and function of CaM, in agreement with the results obtained for other proteins. The apo, calcium and target peptide-bound forms of SeMet-CaM were subsequently studied by natural abundance (1H, 77 Se)-heteronuclear multiple bond correlation (HMBC) and (1H, 13C)-HMQC NMR. Nine well-resolved 77Se resonances could be observed. Substitution of SeMet for Met gave rise to the same 1H and 13C chemical shift changes for each individual Met residue, this facilitated making the assignments from known 1 H, 13C assignments of the Met residues. Some of these assignments were confirmed by studying Met→Leu mutants of CaM. With the exception of Met76, which always remains solvent exposed, all resonances experienced large 77Se chemical shift changes upon the addition of Ca2+ and the MLCK peptide. The large shift changes indicate that the electron distribution in the SeMet side-chain can be adjusted for the different states of CaM, suggesting that the polarizability of sulfur or selenium may be important for the proper functioning of CaM. This study also shows that the natural abundance (1H, 77Se)-HMBC experiment provides a sensitive approach for the study of SeMet protein

Ionisation, self association, and proton exchange studies of nicotinamide in aqueous solution using nuclear magnetic resonance spectroscopy

From an analysis of the single resonance 13C n.m.r. spectrum of nicotinamide at 25·2 MHz at high (pH > 6·0) and low (pH < 1·0) pH values the C–H spin coupling constants have been obtained. The C–H coupling constants for the charged nicotinamide (pH < 1·0) agree well with those which could be measured in NAD+. The unusually large C–H coupling constants (ca. 12 Hz) observed between C(2) and H(6) across the nitrogen atom in the uncharged nicotinamide have been observed in the pteridine ring of folic acid and the adenine ring of NAD+: in both cases aromatic carbon atoms without directly bonded protons could be assigned on the basis of such coupling constants observed in their single resonance 13C spectra. The 13C chemical shift differences observed on protonation of the nicotinamide ring were measured and compared with values calculated on the basis of the differences in the calculated total electron densities at the ring carbons: excellent agreement was found for C(3)–C(5) but C(2) and C(6) which are near the site of protonation showed no agreement probably due to intramolecular electric field effect contributions to their shielding. Carbon-13 relaxation time measurements on nicotinamide and pyridine suggest that nicotinamide is associated at high pH (ca. pH 6·0) and concentration (ca. 2M solutions). At low pH values (< 1·0) less association is observed. 1H and 13C chemical shift measurements on nicotinamide aqueous solutions at different concentrations also support this conclusion. Furthermore the largest shift changes on dilution were centred on the CONH2 group of nicotinamide. The 1H chemical shift changes on dilution were in the opposite direction to those expected from association involving parallel stacking of the nicotinamide rings. Thus there is strong evidence that the association results from interamide interactions. Interactions involving the CONH2 group are not thought to be of importance in the intramolecular stacking found for the nicotinamide and adenine rings of NAD+ at neutral pH values. From measuring the concentrations and pH dependence of the line widths of the CONH2 protons in H2O solution it was possible to estimate the separate exchange rates between the cis- and trans-NH protons with water protons. Similar measurements could be made on NAD+ where it was found that at pH values < 5·5 the exchange rates are slower than found for nicotinamide under the same conditions.