3D NMR spectroscopy for resonance assignment and structure elucidation of proteins under MAS: novel pulse schemes and sensitivity considerations.
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 spectrum. Depending on the second transfer step, spin systems or proton-proton contacts can be determined with reduced spectral overlap. The heteronuclear NHHCC experiment encodes NH-HC proton-proton interactions, which are indicative for the backbone conformation of the protein. The third dimension facilitates the identification of the amino acid spin system. Experimental results on U-[15N,13C]valine and U-[15N,13C]ubiquitin demonstrate their usefulness for resonance assignments and for the determination of structural constraints. Furthermore, we give a detailed analysis of alternative multidimensional sampling schemes and their effect on sensitivity and resolution.
Heise
Henrike
H
Department for NMR-based Structural Biology, Max-Planck-Institute for Biophysical Chemistry, Am Fassberg 11, D-37077 Göttingen, Germany. hehe@nmr.mpibpc.mpg.de
Seidel
Karsten
K
Etzkorn
Manuel
M
Becker
Stefan
S
Baldus
Marc
M
D000596:Amino Acids / Q000737:chemistry; D002247:Carbon Isotopes; D005449:Fluorenes / Q000737:chemistry; D015394:Molecular Structure; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011506:Proteins / Q000737:chemistry; D011522:Protons; D012680:Sensitivity and Specificity; D025801:Ubiquitin / Q000737:chemistry*; D014633:Valine / Q000737:chemistry*
D000596:Amino Acids; D002247:Carbon Isotopes; D005449:Fluorenes; C016456:N(alpha)-fluorenylmethyloxycarbonylamino acids; D009587:Nitrogen Isotopes; D011506:Proteins; D011522:Protons; D025801:Ubiquitin; D014633:Valine