RIDME distance measurements using Gd(iii) tags with a narrow central transition.

Methods based on pulse electron paramagnetic resonance allow measurement of the electron-electron dipolar coupling between two spin labels. Here we compare the most popular technique, Double Electron-Electron Resonance (DEER or PELDOR), with the dead-time free 5-pulse Relaxation-Induced Dipolar Modulation Enhancement (RIDME) method for Gd(iii)-Gd(iii) distance measurements at W-band (94.9 GHz, ≈3.5 T) using Gd(iii) tags with a small zero field splitting (ZFS). Such tags are important because of their high EPR sensitivity arising from their narrow central transition. Two systems were investigated: (i) a rigid model compound with an inter-spin distance of 2.35 nm, and (ii) two mutants of a homodimeric protein, both labeled with a DOTA-based Gd(iii) chelate and characterized by an inter-spin distance of around 6 nm, one having a narrow distance distribution and the other a broad distribution. Measurements on the model compound show that RIDME is less sensitive to the complications arising from the failure of the weak coupling approximation which affect DEER measurements on systems characterized by short inter-spin distances between Gd(iii) tags having a narrow central transition. Measurements on the protein samples, which are characterized by a long inter-spin distance, emphasize the complications due to the appearance of harmonics of the dipolar interaction frequency in the RIDME traces for S > 1/2 spin systems, as well as enhanced uncertainties in the background subtraction. In both cases the sensitivity of RIDME was found to be significantly better than DEER. The effects of the experimental parameters on the RIDME trace are discussed.

Dynamic deuterium magic angle spinning NMR of a molecule grafted at the inner surface of a mesoporous material.

Deuterium magic angle spinning (MAS) NMR is used to study the dynamics of an organic molecule, N-[triethoxysilylpropyl]acetamide-d3, grafted at the inner surface of the mesoporous silica material, MCM-41. The grafted molecule has a deuterated methyl group at its free terminus to monitor its local mobility through changes in its dynamic (2)H-MAS NMR spectrum. Different spectra were recorded as a function of temperature from two different water containing samples. Observation shows that a major part of the grafted molecule remains static, irrespective of the temperature and hydration state of the sample, whereas the rest shows spectral changes indicative of a two-site jump motion of the methyl groups. Experimental observations were substantiated using molecular dynamic (MD) simulations of the grafted molecule. Subsequently, the MD results corroborate a model for the grafted molecules experiencing an exchange between two conformations consistent with the analysis of the (2)H-MAS NMR spectra.

Solid and liquid phase 59Co NMR studies of cobalamins and their derivatives.

We describe the application of 59Co NMR to the study of naturally occurring cobalamins. Targets of these investigations included vitamin B12, the B12 coenzyme, methylcobalamin, and dicyanocobyrinic acid heptamethylester. These measurements were carried out on solutions and powders of different origins, and repeated at a variety of magnetic field strengths. Particularly informative were the solid-state central transition NMR spectra, which when combined with numerical line shape analyses provided a clear description of the cobalt coupling parameters. These parameters showed a high sensitivity to the type of ligands attached to the metal and to the crystallization history of the sample. 59Co NMR determinations also were carried out on synthetic cobaloximes possessing alkyl, cyanide, aquo, and nitrogenated axial groups, substituents that paralleled the coordination of the natural compounds. These analogs displayed coupling anisotropies comparable to those of the cobalamins, as well as systematic up-field shifts that can be rationalized in terms of their stronger binding affinity to the cobalt atom. Cobaloximes also displayed a higher regularity in the relative orientations of their quadrupole and shielding coupling tensors, reflecting a higher symmetry in their in-plane coordination. For the cobalamines, poor correlations were observed between the values measured for the quadrupole couplings in the solid and the line widths observed in the corresponding solution 59Co NMR resonances.

Interactions between natural polyamines and tRNA: an 15N NMR analysis.

15N NMR spectroscopy was used to explore the interactions between natural polyamines and Escherichia coli tRNA. It was found that when tRNA is added to solutions of 15N-labeled spermine or spermidine, there is a considerable decrease in the relative heights of the -NH(2+)--resonances with respect to the signals arising from the -NH3+ groups. The presence of tRNA was also found to reduce the longitudinal relaxation times T1 of the nitrogens, mainly those of the -NH(2+)- groups. The longitudinal relaxation times of the nitrogens were used to characterize the temperature dependence of the binding, and they allowed us to calculate the activation energies that determine the correlation times of amino groups in the presence of tRNA. Both the thermodynamic and the relaxation results indicate that (i) spermine binds more strongly to tRNA than spermidine does and (ii) within each of these molecules the -NH(2+)- groups bind more strongly to tRNA than the more electropositive -NH3+ moieties. This specificity suggests that the interaction between polyamines and tRNA cannot be described exclusively in terms of electrostatic forces and that other interactions (most likely, hydrogen bonding) are very important for establishing the polyamine-tRNA link. Some of the factors that may conspire against the binding of -NH3+ groups to tRNA are briefly discussed.

The metabolism of gibberellins A9, A 20 and A 29 in immature seeds of Pisum sativum cv. Progress No. 9.

Gibberellin A9, GA20 and GA29, all known to be endogenous to immature seeds of Pisum sativum cv. Progress No. 9, were fed in tritiated form to seeds of this cultivar cultured in vitro. [(3)H]GA9 was metabolised to [(3)H]GA20, 2ß-OH-[(3)H]GA9, H2-[(3)H]GA31 and a H2-[(3)H]GA31 conjugate. The percentage conversion to each metabolite varied from feed to feed but in each instance [(3)H]GA20 was, at most, a minor product, suggesting that in vivo GA9 may not be the principal precursor of GA20. [(3)H]GA20 was converted in very high yield to [(3)H]GA29 under conditions which suggest very strongly that this is a normal metabolic sequence in developing seeds of this cultivar. In addition, conjugates of [(3)H]GA20 and [(3)H] GA29 were formed in low yield. It was presumed that the latter was a metabolite of [(3)H]GA29. All metabolites, in each instance, were identified by combined gas chromatography-radiocounting and combined gas chromatography-mass spectrometry, and criteria for the unequivocal identification of radioactive products in feeds to plant tissues are put forward.

Qualitative and quantitative analyses of gibberellins throughout seed maturation in Pisum sativum cv. Progress No. 9.

In addition to the previously identified GA20 and GA29 in immature seeds of Pisum sativum L. cv. Progress No. 9, GA9, GA17, GA38, GA44, abscisic acid and dihydrophaseic acid have been identified. The levels of GA9, GA17, GA20 and GA29 have been determined throughout seed maturation by GC-MS. GA20 and GA29 are the major gibberellins in terms of quantity, the other gibberellins remain at very low levels throughout development of the seed.

Identification of gibberellins A20 and A 29 in seed of Pisum sativum cv. Progress No. 9 by combined gas chromatography-mass spectrometry.

The gibberellin A1 (GA1)-like and GA5-like fractions from immature seeds of Pisum sativum cv. Progress No. 9 were identified by combined gas chromatography-mass spectrometry as GA29 and GA20 respectively.