Contribution Of Chemical Shift Anisotropy Research Articles

Multinuclear solid state nuclear magnetic resonance for studying CsPbBr3 nanocubes

Abstract Cesium lead bromide perovskite (CsPbBr3) nanocrystals have raised impressive interest as efficient and stable optoelectronic materials. Size and morphology play important roles in the final performances of these materials and advanced characterization studies are needed to elucidate structural and surface properties. In this work, CsPbBr3 cubic nanocrystals were obtained by colloidal synthesis and characterized by multinuclear Solid State NMR (SSNMR), complemented by X-Ray Diffraction (XRD), Transmission Electron Microscopy (TEM) and optical spectroscopy. The multinuclear NMR approach allowed the different components of the nanocubes to be separately observed. In particular, the surface ligands and their interactions with the nanocubes surface were investigated by 1H and 13C NMR experiments, while the structural investigation of the perovskite nanocubes was addressed by exploiting 207Pb and 133Cs spectral properties in comparison with bulk CsPbBr3. Static 207Pb NMR spectra indicated a possible contribution of chemical shift anisotropy from the 207Pb nuclei of the outer layer. The 133Cs NMR spectra showed signals with different chemical shifts for cesium atoms in at least three regions of the nanocubes, from the inner core to the surface, which were interpreted in terms of cubic layers with different distances from the surface using a simple geometrical model. This interpretation was also supported by 133Cs longitudinal relaxation time measurements.

Ca2+-independent Binding of Anionic Phospholipids by Phospholipase C δ1 EF-hand Domain

Recombinant EF-hand domain of phospholipase C δ1 has a moderate affinity for anionic phospholipids in the absence of Ca(2+) that is driven by interactions of cationic and hydrophobic residues in the first EF-hand sequence. This region of PLC δ1 is missing in the crystal structure. The relative orientation of recombinant EF with respect to the bilayer, established with NMR methods, shows that the N-terminal helix of EF-1 is close to the membrane interface. Specific mutations of EF-1 residues in full-length PLC δ1 reduce enzyme activity but not because of disturbing partitioning of the protein onto vesicles. The reduction in enzymatic activity coupled with vesicle binding studies are consistent with a role for this domain in aiding substrate binding in the active site once the protein is transiently anchored at its target membrane.

Dynamics of Xenon inside Hydrophobic Cavities As Probed by NMR Relaxation of Dissolved Laser-Polarized Xenon

Longitudinal relaxation times T 1 of laser-polarized xenon in water solutions containing α-cylodextrin and wheat nonspecific lipid transfer protein have been measured. By monitoring the xenon signal decay curves at different solute concentrations and different xenon pressures, we have been able to estimate the binding constant of xenon in the molecular hydrophobic cavities and to extract the xenon longitudinal self-relaxation time of the pure complex form T l c o m p l e x . By studying the variation of T l c o m p l e x with the static magnetic field, we have estimated the correlation times of the xenon-proton dipolar interactions as well as the contribution of chemical shift anisotropy to the xenon relaxation. It appears that in the two studied cases the correlation time of xenon-proton dipolar interactions is between the overall correlation time of the host molecule and the translational correlation time of xenon diffusing freely around solute protons. In particular in the case of the protein, it is typically one-tenth of the overall rotational correlation time, which explains the success of magnetization transfer from dissolved laser-polarized xenon to protein protons. It also appears that the contribution from chemical shift anisotropy to relaxation is very limited. The influence of these results on the SPINOE experiments and on the energetic mechanisms inducing xenon binding is discussed.

Thallium(III) coordination compounds: chemical information from 205Tl NMR longitudinal relaxation times

Abstract205Tl longitudinal relaxation rate measurements were performed on several thallium(III) complexes with the composition Tl(OH)n(H2O)6−n(3−n)+ (n = 1,2), Tl(Cl)n(H2O)m−n(3−n)+, Tl(Br)n(H2O)m−n(3−n)+ (m = 6 for n = 1–2, m = 5 for n = 3, m = 4 for n = 4), Tl(CN)n(H2O)m−n(3−n)+ (m = 6 for n = 1–2, m = 4 for n = 3–4) in aqueous solution, at different magnetic fields and temperatures. 13C and 2D isotopic labelling and 1H decoupling experiments showed that the contribution of the dipolar relaxation path is negligible. The less symmetric lower complexes (n < 4) had faster relaxation rate dominantly via chemical shift anisotropy contribution which depended on the applied magnetic field: T1 values are between 20 and 100 ms at 9.4 T and the shift anisotropy is Δσ = 1000–2000 ppm. The tetrahedral complexes, n = 4, relax slower; their T1 is longer than 1 s and the spin–rotation mechanism is probably the dominant relaxation path as showed by a temperature dependence study. In the case of the TlCl4− complex, presumably a trace amount of TlCl52− causes a large CSA contribution, 300 ppm. Since the geometry and the bond length for the complexes in solution are known from EXAFS data, it was possible to establish a correlation between the CSA parameter and the symmetry of the complexes. The relaxation behaviour of the Tl–bromo complexes is not in accordance with any known relaxation mechanism. Copyright © 2002 John Wiley & Sons, Ltd.

31P NMR investigation of the backbone conformation and dynamics of the hexamer duplex d(5′-GCATGC) 2 in its complex with the antibiotic nogalamycin

Heteronuclear chemical shift correlation experiments confirm that the two down-field shifted 31P resonances in the spectrum of the (nogalamycin) 2-d(GCATGC) 2 complex correspond to the phosphodiesters CpA and TpG at the intercalation sites. 31P relaxation measurements (R 1, R 2 and { 1H}- 31P NOE) at 4.7 and 9.4 T permit the correlation time of each phosphate to be determined together with their chemical shift anisotropies. Significant differences in deoxyribose H3′− 31P coupling constants and chemical shift anisotropy contributions are observed, consistent with an asymmetric DNA backbone conformation for the phosphate groups at the intercalation sites. Large amplitude internal motions of the phosphates do not appear to contribute significantly to relaxation.

Selenium-77 and phosphorus-31 nuclear spin relaxation in tri-(tert-butyl) phosphine selenide

Selenium-77 and phosphorus-31 spin-lattice relaxation times are reported for tri-tert-butylphosphine selenide in chloroform-d, at 303 K and at several different magnetic field strengths. At moderate fields the 31P–1H dipole–dipole, spin-rotation, and chemical shift anisotropy mechanisms contribute significantly towards the 31P T1. At high fields chemical shift anisotropy dominates. The selenium-77 nuclear spin relaxes almost exclusively by spin rotation at low to moderate fields and the chemical shift anisotropy contribution only becomes significant at very high fields. This is due to an unusually small 77Se CSA. The contribution due to 31P–77Se dipole–dipole interactions is small but significant. Key words: 77Se NMR, NMR relaxation, phosphine selenide.

NMR Measurement of Identical Polymer Samples by Round Robin Method II. Reliability of Spin-Lattice Relaxation Time and Nuclear Overhauser Enhancement Factor

1H and 13C NMR spin-lattice relaxation times (T1) and 13C nuclear Overhauser enhancement (NOE) of radically prepared poly(methyl methacrylate) (Mn=28500) in CDCl3 were measured on 27 NMR spectrometers. Frequency range covered between 60 and 500 MHz for 1H NMR. Precision of 1H-T1’s of α-CH3 and CH2 protons was less than 5%. The 1H-T1’s of all sorts of protons increased linearly with increasing resonance frequency. The standard deviations for 13C-T1’s were larger than those for 1H-T1’s and exceeded 10% in some cases. The T1’s of all carbons except for the carbonyl carbon increased with frequency. The contribution of chemical shift anisotropy to the relaxation of the carbonyl carbon became significant above 25 MHz. The protons and carbons in the sequence rich in meso dyad showed longer T1’s than those in the corresponding sequence rich in racemo dyad. The precision of NOE was as good as that of 13C-T1. The NOE’s decreased as the observing frequency increased and particularly the NOE for the carbonyl carbon became almost unity at 100 and 125 MHz.

Tryptophan sidechain dynamics in hydrophobic oligopeptides determined by use of 13C nuclear magnetic resonance spectroscopy

Two oligopeptides, t-boc-LAWAL-OMe and t-boc-LALALW-OMe, were synthesized for the purpose of examining the sidechain dynamics of the tryptophan residue in hydrophobic environments by 13C nuclear magnetic resonance and fluorescence spectroscopy. In both peptides, the tryptophan sidechain was greater than 95% enriched with 13C at the C delta 1 position. Spin-lattice relaxation time (T1) and steady-state nuclear Overhauser effect (NOE) data were obtained at 50.3 and 75.4 MHz for both peptides in CD3OD, and at 75.4 MHz for t-boc-LALALW-OMe in lysolecithin-D2O micelles. We have adapted the model-free approach of G. Lipari and A. Szabo (1982, J. Am. Chem. Soc. 104:4546) to interpret the 13C-NMR data. Computer-generated curves based on experimental data obtained at a single frequency demonstrate relationships between an effective correlation time for tryptophan sidechain motion (tau e), a generalized order parameter (sigma) describing the extent of motional restriction, and an overall correlation time for the peptide (tau m). Assuming predominantly dipolar relaxation, least-squares fits of the dual frequency relaxation data provide values for these parameters for both peptides. The contribution of chemical shift anisotropy (CSA), however, is also explicitly assessed in the data analysis, and is shown to perturb the predicted sigma, tau e, and tau m values and to decrease chi(2) values observed in nonlinear least-squares analysis of the data. Because of uncertainty in the contribution of CSA to the relaxation of the indole ring 13C delta 1 atom, nonlinear least-squares analysis of the relaxation data were performed with and without inclusion of a CSA term in the appropriate relaxation equations. Neglecting CSA, an overall peptide correlation time of 0.69 ns is predicted for t-boc-LAWAL-OMe in CD3OD at 20 degrees C compared with 1.28 ns for t-boc-LALALW-OMe. Given these tau m values and taking into account the effect of measurement error in the T1 and NOE data, the internal dynamics of the tryptophan residue of t-boc-LAWAL-OMe in this isotropic environment are described by a range of tau e values from 70 to 112 ps and sigma values between 0.22 and 0.36. Similarly, for t-boc-LALALW-OMe, 68 less than or equal to tau e less than or equal to 93 ps and 0.09 less than or equal to sigma less than or equal to 0.17. The Ch-terminal position of the tryptophan residue in the hexapeptide may account for its lower order parameter. In lysolecithin micelles, the model-free approach applied tot-boc-LALALW-OMe predicts a Te between 0.87 and 1.08 ns, and an order parameter range of 0.72-0.80, assuming an average Tm of 14 ns (Saunders, L. 1966. Biochim. Biophys. Acta. 125:70) for a typical peptide-micelle complex. In this case, measurement of only two 13C relaxation parameters at a single frequency yields sufficient information, plotted in the form of a composite T1-NOE solution curve, to constrain the allowed values of the model-free motional parameters within a relatively narrow range. The predicted range of eV and Te values for the peptide-micelle complex demonstrate that both the rate and spatial mobility of the indole moiety are markedly restrained in the anisotropic micelle environment relative to free methanol solution. Steady-state fluorescence anisotropy measurements made on the peptides dissolved in methanol or with synthetic lysolecithins in water were used to calculate apparent order parameters for tryptophan motion; these values agree well with order parameters calculated from 13C NMR data. The reported results are relevant to the issue of protein dynamic events occurring on the picosecond time scale predicted by molecular dynamics simulations.

Thermolysin-inhibitor complexes examined by 31P and 113Cd NMR spectroscopy.

Complexes between phosphoramidon (N-(alpha-rhamnopyranosyloxyhydroxyphosphinyl)-L-leucyl-L-tryptoph an) and zinc thermolysin and between phosphoramidon or N-phosphoryl-L-leucineamide and 113Cd-substituted thermolysin have been examined by 31P and 113Cd NMR spectroscopy. 113Cd resonances are observed at 168 and 152 ppm for the phosphoramidon and N-phosphoryl-L-leucineamide complexes, respectively. There are large but different chemical shift anisotropy contributions to the 113Cd line widths for the two complexes, which reflect the known structural differences for the zinc-enzyme complexes. 113Cd-31P spin-spin coupling is also seen and differs for the two cadmium complexes, being larger, 28 Hz, for the bidentate N-phosphoryl-L-leucineamide ligand than for the monodentate phosphoramidon, 16 Hz. Large changes in chemical shift, 7.5-10.9 ppm, are seen for the 31P resonances of the inhibitors upon binding to the enzyme reflecting direct phosphoryl-metal ligation. Chemical shift anisotropy is the dominant relaxation mechanism for the 31P nuclei at 9.4 T, while the dipole-dipole contribution seems to be unaffected by a change of solvent from H2O to D2O.

Mobility of individual 5-fluorouridine residues in 5-fluorouracil-substituted Escherichia coli valine transfer RNA : A 19F nuclear magnetic resonance relaxation study

19F nuclear magnetic resonance (n.m.r.) relaxation parameters of 5-fluorouracil-substituted Escherichia coli tRNA 1 Val were measured and used to characterize the internal mobility of individual 5-fluorouridine (FUrd) residues in terms of several models of molecular motion. Measured relaxation parameters include the spin-lattice ( T 1) relaxation time at 282 MHz, the 19F 1H NOE at 282 MHz, and the spin-spin ( T 2) relaxation time, estimated from linewidth data at 338 MHz, 282 MHz and 84 MHz. Dipolar and chemical shift anisotropy contributions to the 19F relaxation parameters were determined from the field-dependence of T 2. The results demonstrate a large chemical shift anisotropy contribution to the 19F linewidths at 282 and 338 MHz. Analysis of chemical shift anisotropy relaxation data shows that, relative to overall tumbling of the macromolecule, negligible torsional motion occurs about the glycosidic bond of FUrd residues in 19F-labeled tRNA 1 Val, consistent with the maintenance of base-base hydrogen-bond and/or stacking interactions at all fluorouracil residues in the molecule. The dipolar relaxation data are analyzed by using the “two-state jump” and “diffusion in a cone” formalisms. Motional amplitudes (θ) are interpreted as being due to pseudorotational fluctuations within the ribose ring of the fluorinated nucleoside. These amplitudes range from approximately 30 ° to 60 °, assuming a correlation time ( τ i,2 ) of 1.6 ns. By using available 19F n.m.r. assignment data for the 14 FUrd residues in 5-fluorouracil-substituted tRNA 1 Val, these motional amplitudes can be correlated directly with the environmental domain of the residue. Residues located in tertiary and helical structural domains show markedly less motion (θ ∼- 30 to 35 °) than residues located in loops (θ ∼- 45 to 60 °). A correlation between residue mobility and solvent exposure is also demonstrated. The amplitudes of internal motion for specific residues agree quite well with those derived from X-ray diffraction and molecular dynamics data for yeast tRNA Phe.

The use of interference terms to separate dipolar and chemical-shift anisotropy contributions to nuclear spin relaxation

For numerous spin-4 nuclear isotopes such as 15N, r9F, and 3’P incorporated in bulky molecular frameworks, chemical-shift anisotropy and intramolecular dipolar interactions often provide competitive mechanisms for a&cting nuclear spin relaxation. In the past few years, there has been widespread interest in rationalizing-and exploiting-the synergistic nature of these two relaxation mechanisms. A novel investigation recently described how one could utilize this synergism to individually quantify the dipolar and chemical-shift anisotropy contributions without the need for supplemental Overhauser or variation-in-field studies (1). In the following note, we would like to clarify certain features of this proposed methodology and add some cautionary comment. For our treatment, we will consider the same system as discussed in (I): two unlike spin-4 nuclei, 19F and 31P, relaxed by dipolar and chemical-shift anisotropy interactions outside the extreme narrowing limit. The two components of the phosphorus doublet have linewidths equal to Au& + AU&A + ~CP(AV~~AV&~)~‘~ and AvEn + AU&A 2cp( Av~&&,) ‘I’, whereas the two components of the fluorine doublet have linewidths equal to AZ& + AI& + 2CF(Av~nAv&~)“’ and Agn + AU&A 2C,~(Av~nAv&~) . ‘I2 (For the special case of isotropic molecular reorientation, the correlation factor, CP(~, is defined as (3 cos2Q 1) where Q is the angle between the F-P internuclear vector and the principal axis of the phosphorus (fluorine) chemicalshift tensor.) Thus, the summed width of either doublet is independent of the interference term, 2Ci(A&A&A)“‘, whereas the differential width of either doublet depends solely upon this interference. In a study of the type outlined in (I), there are four observable.linewidths and five unknown parameters, A$&, A V&A, AU&A, Cr, and Cr. Thus, without an additional assumption or independent measurement, one cannot deduce the dipolar and chemicalshift anisotropy contributions individually. In (I), it was assumed that Cr = Cr = 1. Although it was stated, “for clarity of explanation only,” it is seen that this assumption was an integral ingredient in the subsequent development. Only in the unlikely situation where all interactions share a common principal axis or highly anisotropic motions prevail (2), will this completely correlated limit obtain. A more general interpretation of the data given in (I) is displayed in Fig. 1. This figure summarizes all possible, internally self-consistent, sets of A&, AI$~, , Au&, C,, and Cr compatible with the observed linewidths. The pertinent relationships are

Function of pyridoxal 5'-phosphate in glycogen phosphorylase: 19F NMR and kinetic studies of phosphorylase reconstituted with 6-fluoropyridoxal and 6-fluoropyridoxal phosphate.

19F NMR spectroscopic properties of glycogen phosphorylase reconstituted with 6-fluoropyridoxal (6-FPAL) and 6-fluoropyridoxal phosphate (6-FPLP) were investigated. Analysis of the contribution of chemical shift anisotropy to the line width of the 6-FPLP-enzyme signal shows that the coenzyme molecule is tightly bound to the protein. The chemical shift of the fluorine nucleus in the free 6-FPLP protein is pH independent from pH 6 to pH 9.1. When the 6-FPLP-enzyme forms complexes with AMP, AMP plus glucose-1-P, and AMP plus inorganic phosphate, signals at -11.0, -13.1, and -10.4 ppm are observed, respectively. These different chemical shifts indicate that the protein in each complex has a distinct conformation. The exchange rate between the 6-FPLP-protein-AMP complex and the same complex with bound glucose-1-P is estimated to be 3300 +/- 700 s-1, and that between the 6-FPLP-protein-AMP complex and with bound inorganic phosphate is 500 +/- 100 s-1. The former exchange rate is 13 times faster than that of the same process for the 6-FPAL-enzyme. Analysis of the effects of temperature on the 19F line shape of the 6-FPLP enzyme in the presence of ligands shows that the exchange rates between different complexes drop significantly between 20 and 10 degrees C. Within this temperature range, Arrhenius plots of the enzymatic activities of the native and 6-FPLP-enzymes at varied temperatures also show a pronounced curvature.(ABSTRACT TRUNCATED AT 250 WORDS)

31P NMR relaxation studies of the activation of the coenzyme phosphate of glycogen phosphorylase. The role of motion of the bound phosphate

Spin-lattice and spin-spin relaxation rates (1/T1 and 1/T2) have been determined for the catalytically essential coenzyme phosphate at the active site of glycogen phosphorylase in both activated (R state) and inactive (T state) conformations of the enzyme. Dipolar contributions to 31P relaxation due to exchangeable protons on the phosphate group have been determined by measurement of relaxation rates at different concentrations of H2O and D2O, and field dependence studies have been performed to estimate the contribution of chemical shift anisotropy to the remaining 31P relaxation in D2O. At 109 MHz, dipolar relaxation from exchangeable protons was found to account for 50% of the spin-lattice relaxation for activated phosphorylase in 75% H2O, the remainder being due to chemical shift anisotropy. The spin-lattice relaxation rates in D2O for R-state glycogen phosphorylase are very similar to those measured for other proteins of very different size such as actin (Brauer, M., and B. D. Sykes, 1981, Biochemistry. 20:6767-6775), alkaline phosphatase (Coleman, J. E., I. D. Armitage, J. F. Chlebowski, J. D. Otvos, and A. J. M. S. Uiterkamp, 1979), and phosphoglucomutase (Rhyu, G. I., W. J. Ray, Jr., and J. L. Markley, 1984, Biochemistry. 23:252-260). In inactive (T state) phosphorylase the spin-lattice relaxation rates were almost an order of magnitude slower, while the spin-spin relaxation rates were essentially identical. These results have been analyzed by calculating the theoretically expected 31P relaxation rates in the presence of internal motions that are included in the relaxation calculation using the model-free approach of Lipari and Szabo (1982, J. Am. Chem. Soc. 104:4564-4559). The analysis suggests the coenzyme phosphate is relatively immobilized in the activated enzymic conformation, but in the inactive (Tstate) conformation it is considerably more mobile with a rotational correlation time one to two orders of magnitude smaller. Since the spin-lattice relaxation rate for the active R-state (immobilized) phosphate is similar to that observed in other phosphoenzymes of different size it is suggested that a librational motion on the nanosecond time scale may constitute a common spin-lattice relaxation pathway for phosphates in macromolecules. The consequences of phosphate motion in terms of recent suggestions concerning the environment and the catalytic role of the coenzyme phosphate are discussed.

Relaxation of individual transitions in an AX spectrum. Use of interference terms to separate the dipolar and chemical-shift anisotropy contributions to the relaxation of 31P and 19F nuclei in macromolecules

Relaxation of individual transitions in an AX spectrum. Use of interference terms to separate the dipolar and chemical-shift anisotropy contributions to the relaxation of 31P and 19F nuclei in macromolecules

Phosphorus-31 nuclear magnetic resonance studies of the two phosphoserine residues of hen egg white ovalbumin

Ovalbumin contains two phosphoserine residues that give rise to two well-resolved resonances in a 31P NMR spectrum. Ovalbumin samples that have been digested with a variety of phosphatases may give rise to only one phosphoserine resonance, indicating that one of the two phosphorylated sites is relatively inaccessible for phosphatase action. By comparison of the amino acid sequence of the peptide containing the nonsusceptible phosphate to the overall primary structure, we have assigned the resonances observed (pH 8.3) at 5.0 and 4.75 ppm to phosphoserines-68 and -344, respectively. pH titration behavior and susceptibility of the phosphoserine residues to phosphatases indicate that both are located on the surface of the protein. Both residues have a pKa = 6.00-6.04. Analysis of the Hill coefficients measured for the pH titrations and the JPH coupling constants indicate that neither residue interacts with other charged groups on the surface of the protein. Frequency dependence of 31P NMR parameters shows that at higher magnetic field strengths the contribution of chemical shift anisotropy to the line width becomes very significant. We have calculated from the field-dependent terms that phosphoserine-344 is mobile with respect to the protein surface but that phosphoserine-68 is more restricted in its motion. The latter is also involved in a pH-dependent conformational change, since it is shielded from hydrolysis by phosphatases at higher pH. A comparison of the amino acid sequence of the phosphoserine-68 site shows that it has a striking homology to the active-site peptides of a wide variety of hydrolytic enzymes. Moreover, a comparison with the primary sequences of casein suggests that both proteins are phosphorylated by a protein kinase that specifically recognizes a Ser-X-Glu peptide.

Fluoroquinacrine binding to nucleic acids: investigation of the 19F-NMR, optical, and fluorescence properties in the presence of DNA, poly(A), and tRNA.

AbstractThe interaction of fluoroquinacrine, 3‐fluoro‐7‐chloro‐9‐(diethylamino‐1‐methylbutyl‐amino)acridine, with poly(A), DNA, and tRNA has been investigated by monitoring changes in the 19F‐nmr properties, the fluorescence, and the optical absorbance of the drug. The changes in the properties of fluoroquinacrine in the presence of nucleic acids are similar to those observed for quinacrine and suggest that the drugs bind in a similar fashion. The molecular dynamics of fluoroquinacrine bound to nucleic acids were determined by interpreting the data from a number of different nmr relaxation experiments with a two‐correlation‐time model. The two motions are the long‐range bending motion of the drug‐nucleic acid complex and the sliding of the drug between the base pairs. Both dipolar and chemical shift anisotropy contributions to the nmr relaxation parameters were taken into consideration. The binding of fluoroquinacrine to tRNA appears to be different from that observed for binding to DNA. Optical absorbance and 19F‐nmr were also used to examine the helix‐to‐coil transitions of the drug–nucleic acid complexes. In the DNA complex, the 19F chemical shift changes parallel the absorption changes that occur during the transition. 19F‐nmr and absorption show that the drug–tRNA complexes undergo a cooperative helix‐to‐coil transition, with the drug binding sites melting when the tRNA is 70% denatured.

Carbon-13 magnetic resonance of carboxymethylated human carbonic anhydrase B. Chemical shift and spin--lattice relaxation studies.

We have previously prepared Ntau-carbosymethylhistidine-200 human carbonic anhydrase B using 90% [1-13C]bromoacetate and have observed the 13C NMR resonance of the enriched carboxylate now covalently attached in the active site. We report here chemical shift studies of the zinc-free carboxymethylated enzyme and its Co2+-substituted form, as well as relaxation studies of the resonance in the zinc enzyme at three frequencies (15.04, 25.15, and 90.5 MHz). The chemical shift and relaxation data are both consistent with the immobilization of the carboxylate at pH 8 and its approach or coordination to the zinc. The relaxation data indicate that lowering the pH to 5.5 leads to internal motion of the carboxymethyl moiety, consistent with the chemical shift evidence for the disruption of the proposed zinc--carboxylate coordination. Inhibitor binding at either pH 5.5 or 8.0 eliminates whatever internal motion might be present. The relaxation data have been interpreted using theoretical calculations on dipolar and chemical shift anisotropy contributions. The combined results indicate that the catalytic consequences of the carboxymethylation may be due to the proposed zinc--carboxylate coordination and need not result from the disruption of any role that histidine-200 might play in the catalytic mechanism.

31P Magnetic Relaxation in Polynucleotides

Abstract Phosphorus-31 nuclear spin-lattice relaxation times and nuclear Overhauser enhancement upon nonselective proton saturation were measured on polyriboadenylic acid, polyribocytidylic acid, and polyribouridylic acid. A method is described to separate dipolar contribution from the contribution of chemical shift anisotropy for the phosphorus relaxation by the combined use of T1 and NOE data. Chemical shift anisotropies (δ⁄⁄−δ⊥) of phosphorus in these polynucleotides in solution are estimated to be in the range 120–165 ppm. The molecular motion of the phosphorus moiety becomes faster in the order, poly(A)<poly(C)<poly(U), the thermal activation processes being characterized by single activation energies, i.e., 5.1, 5.3–6.0, and 7.2–8.1 kcal/mol for poly(U), poly(C), and poly(A), respectively.

Fluorotyrosine alkaline phosphatase: internal mobility of individual tyrosines and the role of chemical shift anisotropy as a 19F nuclear spin relaxation mechanism in proteins.

19F nuclear spin relaxation data for fluorotyrosine alkaline phosphatase at 94 and 235 MHz are presented. The factor of 2·5 in increased chemical shift separation expected at the higher field was almost exactly matched by an increase in 19F linewidths due to the field-dependent relaxation mechanism of chemical shift anisotropy. Consequently, no significant improvement in resolution or signal/noise was gained at the higher field. At 235 MHz the chemical shift anisotropy mechanism produces about 50% of the observed linewidths but only 10 to 20% at 94 MHz. Using a density matrix formalism, complete expressions are derived for the relaxation of a nuclear spin with an asymmetric chemical shift tensor (three independent principal elements) undergoing axially symmetric (symmetric top) diffusion. With this formalism, the chemical shift anisotropy contributions to T1 and T2 for fluorine-labeled residues undergoing one link of internal motion may be calculated. Such a calculation requires detailed knowledge of both the magnitude and orientation within the molecular frame of the three principal elements of the traceless chemical shift tensor. Data from the literature on 19F chemical shift tensors are collected and used in calculations of chemical shift anisotropy relaxation for m-fluorotyrosine, p-fluorophenylalanine, and trifluoromethyl residues incorporated in proteins. The relaxation is sensitive to internal motion about the Cβ−Cγ bond in the aromatic residues and the C3 axis of the CF3 group. It is found that the contribution of chemical shift anisotropy to T1 will be negligible except when dipolar relaxation is particularly inefficient and internal motion is near the Larmor frequence ω0. The contribution of chemical shift anisotropy to the linewidth increases with ω02 and becomes significant for proteins with Mr>20,000 and fields >50 kgauss. For fluorotyrosine alkaline phosphatase the calculated chemical shift anisotropy linewidths as a function of internal motion about Cβ−Cγ span a range of values that encompasses the observed linewidths at 235 MHz for nine of the eleven fluorotyrosine residues. Two other fluorotyrosines exhibit linewidths with an even greater field dependence, suggesting the influence of chemical exchange (via slow internal rotation) between different magnetic environments. Combining dipolar T1 and linewidth data, chemical shift anisotropy linewidth data, and the nuclear Overhauser enhancement observed when irradiating protein protons, it is possible to derive the overall tumbling correlation time τc for alkaline phosphatase and place limits on the internal mobility of the tyrosine residues. Thus, for overall tumbling we find that τc=76·0±15 ns or Dmacro=2·2×106 s−1. For rotation about Cα−Cβ, Dint(α−β)≤106 s−1 for all tyrosines. Rotation about Cβ−Cγ has Dint(β−γ)≤108 s−1 for all tyrosines, with nine tyrosines probably in the range 106 to 108 s−1 and two others in the range 102 to 105 s−1. These results are compared with other literature data concerning protein mobility.

Anisotropy of Chemical Shift for Some Fluorine Compounds

The magnetic resonance spectrum of nuclei in a polycrystalline solid is broadened and in general rendered asymmetrical if the nuclei have an anisotropic chemical shift. The anisotropy of the chemical shift tensor for fluorine nuclei in 1.2.4-trifluorobenzene, CFCl2CFCl2 and CF2BrCF2Br in the solid state at 85 °K was investigated by a study of the field-dependence of the second moments of their 19F magnetic resonance spectra. With the assumption of axial symmetry of the tensor about the carbon-fluorine bond, the anisotropy (σ|| - σ⊥) was found to be 2.5 ± 0.5 × 10-4 for all three compounds. These values are found to be in reasonable agreement with estimates of the anisotropy based on the theoretical treatment of Karplus and Das. It is concluded that measurements of chemical shift anisotropy can give useful insight into bond character. The measured values can be used to estimate the contribution of chemical shift anisotropy in nuclear spin-lattice relaxation in liquids. The implications of this additional source of broadening in structural investigations is discussed; and as an example, it is concluded that a significant proportion of the solid CFCl2CFCl2 was in the trans form.