The NMR assignment of 13C, 15N-labeled proteins with the use of triple resonance experiments is limited to molecular weights below approximately 25,000 Daltons, mainly because of low sensitivity due to rapid transverse nuclear spin relaxation during the evolution and recording periods. For experiments that exclusively correlate the amide proton (1HN), the amide nitrogen (15N), and 13C atoms, th...

Nearly complete 1H, 13C and 15N NMR assignments have been obtained for a doubly labeled 14-base pair DNA duplex in solution both in the free state and complexed with the uniformly 15N-labeled Antennapedia homeodomain. The DNA was either fully 13C, 15N-labeled or contained uniformly 13C, 15N-labeled nucleotides only at those positions which form the protein-DNA interface in the previously determ...

The methylotrophic yeast Pichia pastoris is now an established expression system for the production of recombinant protein for nuclear magnetic resonance (NMR) studies. It is capable of expressing high levels of heterologous proteins and possesses the ability to perform many of the posttranslational modifications of higher eukaryotes. Here, we describe efficient methods for the production of un...

An experimental strategy for determining the extent to which multiply isotopically labeled fragments are incorporated intact into relatively complicated compounds of interest is presented. The NMR methods employed are based on isotope-filtered one-dimensional spectra and difference HSQC spectra incorporating a spin echo designed to report on the presence of a second NMR active isotope at a coup...

A protocol for high-quality structure determination based on G-matrix Fourier transform (GFT) NMR is presented. Five through-bond chemical shift correlation experiments providing 4D and 5D spectral information at high digital resolution are performed for resonance assignment. These are combined with a newly implemented (4,3)D GFT NOESY experiment which encodes information of 4D 15N/15N-, 13C(al...

The comprehensive structure determination of isotopically labeled proteins by solid-state NMR requires sequence-specific assignment of 13C and 15N spectra. We describe several 2D and 3D MAS correlation techniques for resonance assignment and apply them, at 7.0 Tesla, to 13C and 15N labeled ubiquitin to examine the extent of resonance assignments in the solid state. Both interresidue and intrare...

Here we present a novel suite of projected 4D triple-resonance NMR experiments for efficient sequential assignment of polypeptide backbone chemical shifts in 13C/15N doubly labeled proteins. In the 3D HNN[CAHA] and 3D HNN(CO)[CAHA] experiments. the 13C(alpha) and lHalpha chemical shifts evolve in a common dimension and are simultaneously detected in quadrature. These experiments are particularl...

Stable isotopes are an important tool for studying invertebrate food webs and movement of invertebrates in farmland. However, stable isotope values of farmland invertebrates have been reported for only a few crop types, and rarely for other land uses within farmland. We compared 13C and 15N of invertebrates captured in corn, soybean, hay, and hedgerows in eastern Ontario, Canada. 13C was signif...

Two types of 3D MAS NMR experiments are introduced, which combine standard (NC,CC) transfer schemes with (1H,1H) mixing to simultaneously detect connectivities and structural constraints of uniformly 15N,13C-labeled proteins with high spectral resolution. The homonuclear CCHHC and CCC experiments are recorded with one double-quantum evolution dimension in order to avoid a cubic diagonal in the ...

Several HNCO-based three-dimensional experiments are described for the measurement of 13C'(i - 1)-13Calpha(i - 1), 5N(i)-13C'(i - 1), 15N(i)-13Calpha(i), 15N(i)-13Calpha(i - 1), 1H(N)(i)-13Calpha(i), 1H(N)(i)-13Calpha(i - 1), and 13Calpha(i - 1)-13Cbeta(i - 1) scalar and dipolar couplings in 15N, 13C, (2H)-labelled protein samples. These pulse sequences produce spin-state edited spectra superfi...