Indexed on: 01 Jul '96Published on: 01 Jul '96Published in: Journal of Biomolecular NMR
Triple-resonance experiments can be designed to provide useful information on spin-system topologies. In this paper we demonstrate optimized proton and carbon versions of PFG-CT-HACANH and PFG-CT-HACA(CO)NH ‘straight-through’ triple-resonance experiments that allow rapid and almost complete assignments of backbone Hα, 13Cα, 15N and HN resonances in small proteins. This work provides a practical guide to using these experiments for determining resonance assignments in proteins, and for identifying both intraresidue and sequential connections involving glycine residues. Two types of delay tunings within these pulse sequences provide phase discrimination of backbone Gly Cα and Hα resonances: (i) C−H phase discrimination by tuning of the refocusing period τa_f; (ii) C−C phase discrimination by tuning of the 13C constant-time evolution period 2Tc. For small proteins, C−C phase tuning provides better S/N ratios in PFG-CT-HACANH experiments while C−H phase tuning provides better S/N ratios in PFG-CT-HACA(CO)NH. These same principles can also be applied to triple-resonance experiments utilizing 13C-13C COSY and TOCSY transfer from peripheral side-chain atoms with detection of backbone amide protons for classification of side-chain spin-system topologies. Such data are valuable in algorithms for automated analysis of resonance assignments in proteins.