Soft-triple resonance solid-state NMR experiments for assignments of U-13C, 15N labeled peptides and proteins.
The process of obtaining sequential resonance assignments for heterogeneous polypeptides and large proteins by solid-state NMR (ssNMR) is impeded by extensive spectral degeneracy in these systems. Even in these challenging cases, the cross peaks are not distributed uniformly over the entire spectral width. Instead, there exist both well-resolved single resonances and distinct groups of resonances well separated from the most crowded region of the spectrum. Here, we present a series of new triple resonance experiments that exploit the non-uniform clustering of resonances in heteronuclear correlation spectra to obtain additional resolution in the more crowded regions of a spectrum. Homonuclear and heteronuclear dipolar recoupling sequences are arranged to achieve directional transfer of coherence between neighboring residues in the peptide sequence. A frequency-selective (soft) pulse is applied to select initial polarization from a limited (and potentially) well-resolved region of the spectrum. The pre-existing resolution of one or more spins is thus utilized to obtain additional resolution in the more crowded regions of the spectrum. A new protocol to utilize these experiments for sequential resonance assignments in peptides and proteins is also demonstrated.
Astrof
Nathan S
NS
Francis Bitter Magnet Laboratory, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Griffin
Robert G
RG
D002247:Carbon Isotopes; D007553:Isotope Labeling; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010455:Peptides / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D020539:Sequence Analysis, Protein
D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D010455:Peptides; D011506:Proteins