R. A. McKay

Analysis of double cross-polarization rates in solid proteins

Abstract The double cross-polarization experiment involving the sequential transfer of nuclear polarization among 1H-, 15N-, and 13C-spin systems can be used to detect the occurrence of 13C-15N bonds in solid proteins. However, the complexity of most proteins requires simplifying approximations to make possible the determination of the concentrations of those 13C-15N bonds. This paper demonstrates that a model involving only two 13C-15N cross-polarization rates (determined from model compounds) and simple statistics is adequate to treat data from heavily 13C- and 15N-labeled proteins in soybean leaves. In the course of examining the applicability of model compounds to this problem, we have used a moment analysis of 15N NMR lineshapes to determine 13C-15N dipolar couplings in two doubly labeled amino acids, glycine and asparagine.

Cross-polarization nmr of N-15 labeled soybeans

Abstract Cross-polarization 15N nmr spectra of 15N-labeled soybean seeds, pods, and leaves have been obtained at 9.12 MHz both with and without high-speed sample rotation at the magic angle. Spectral resolution is sufficient to permit a determination of the relative concentrations of amide and amine nitrogens, as well as of a few specific amino acid residues of proteins in the solid, intact samples. Utilization by soybean of nitrogen from labeled fertilizer in the presence of dinitrogen fixation can be determined from these spectra. A double-cross polarization 13C nmr spectrum of a spinning, 15N-labeled seed has been obtained in which resonances are observed only from these carbons directly bonded to nitrogens. This technique leads to a qualitative estimate of amino-acid composition of the protein which is complementary to that obtained directly from the 15N nmr spectrum.

Dynamic nuclear polarization enhanced nuclear magnetic resonance of polymer-blend interfaces

Abstract Heterogeneous blends of [3,3′- 13 C 2 ]polycarbonate and [uniform-ring- 12 C 6 ]polystyrene were formed by serial film casting. The polystyrene phase of each blend was homogeneously doped with 2% by weight of a bis (diphenylene) phenylalyl free-radical complex with benzene. Proton polarization enhanced by dynamic nuclear polarization was generated in the polycarbonate phase by dipolar coupling to electrons in the polystyrene phase under 39 GHz microwave irradiation at the difference of the electron and proton Larmor frequencies. Proton magnetization was then transferred to carbons under matched spin-lock conditions for detection with chemical-shift selectivity by magic-angle spinning 13 C nuclear magnetic resonance. The 13 C signal from polycarbonate arises exclusively from chains which are at the polycarbonate-polystyrene interface. Signals from bulk polycarbonate were suppressed by differencing techniques. The dominant mechanism of polarization transfer from the electrons in the polystyrene phase to the protons in the polycarbonate phase is by direct polarization transfer. The interface signal arises from a 60 A region which is 2% of the film thickness. As monitored by dynamic nuclear polarization selected dipolar rotational spin-echo 13 C nuclear magnetic resonance, polycarbonate chains have less motion at the interface than in the bulk.

Magic-angle N-15 NMR study of nitrate metabolism of Neurosporacrassa

Magic-angle cross-polarization 15N nmr spectra of intact lyophilized mycelia from N.crassa cultured on media containing [15N] nitrate have been obtained at 9.12 MHz. The time development of the uptake and distribution of label into protein and amino-acid metabolites can be observed directly. Nitrate metabolism is delayed about one hour if the cells innoculating the culture are grown on nitrate-free medium.

Magic angle NMR studies of polymers

The combination of cross polarization, high—power resonant proton decoupling, and magic angle sample spinning makes possible liquid—like, high—resolution 13C and 1N NMR spectra in many kinds of organic solids. Such spectra can be particularly useful in the characteri— zation of the chemical and physical structure of solids which may be difficult to analyze by other means. Many polymers fall into this category in that they are non—crystalline, opaque, and impossible to melt or dissolve without altering their structure. Cross—linking, thermal degradation, or the energetic condensation of small molecules can produce polymeric systems which are difficult to characterize for these reasons. Biological macromolecules represent another class of systems presenting demanding analytical problems. Examples are given where either 13C or 15N high—resolution, solid—state NMR spectra provide unique structural information about such materials.

Spin-echo, double-resonance NMR with flipped spinning (SEDORFS)

Abstract A simple experiment for the measurement of rare-spin, heteronuclear dipolar couplings while sample spinning is reported here. The experiment uses a single, dephasing π pulse and requires reorientation of the sample spinning axis.