13C direct detected COCO-TOCSY: a tool for sequence specific assignment and structure determination in protonless NMR experiments.
A novel experiment is proposed to provide inter-residue sequential correlations among carbonyl spins in (13)C detected, protonless NMR experiments. The COCO-TOCSY experiment connects, in proteins, two carbonyls separated from each other by three, four or even five bonds. The quantitative analysis provides structural information on backbone dihedral angles phi as well as on the side chain dihedral angles of Asx and Glx residues. This is the first dihedral angle constraint that can be obtained via a protonless approach. About 75% of backbone carbonyls in Calbindin D(9K), a 75 amino acid dicalcium protein, could be sequentially connected via a COCO-TOCSY spectrum. 49(3)J(C')(C') values were measured and related to backbone phi angles. Structural information can be extended to the side chain orientation of aminoacids containing carbonyl groups. Additionally, long range homonuclear coupling constants, (4)J(CC) and (5)J(CC), could be measured. This constitutes an unprecedented case for proteins of medium and small size.
Balayssac
Stéphane
S
Magnetic Resonance Center (CERM), Department of Chemistry, University of Florence, Via Luigi Sacconi 6, 50019 Sesto Fiorentino (FI), Italy.
Jiménez
Beatriz
B
Piccioli
Mario
M
D064026:Calbindins; D002247:Carbon Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011487:Protein Conformation; D064030:S100 Calcium Binding Protein G / Q000737:chemistry*
D064026:Calbindins; D002247:Carbon Isotopes; D064030:S100 Calcium Binding Protein G