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Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy
Published on: September 17, 2017
13C direct detected COCO-TOCSY: a tool for sequence specific assignment and structure determination in protonless NMR
Stéphane Balayssac1, Beatriz Jiménez, Mario Piccioli
1Magnetic Resonance Center (CERM), Department of Chemistry, University of Florence, Via Luigi Sacconi 6, 50019 Sesto Fiorentino (FI), Italy.
Journal of Magnetic Resonance (San Diego, Calif. : 1997)
|July 18, 2006
Summary
A new COCO-TOCSY experiment links carbonyl spins in proteins, enabling novel structural insights. This protonless nuclear magnetic resonance method provides dihedral angle constraints for protein backbone and side chains.
Area of Science:
- Biophysical Chemistry
- Structural Biology
- Nuclear Magnetic Resonance (NMR) Spectroscopy
Background:
- Nuclear Magnetic Resonance (NMR) spectroscopy is crucial for determining protein structure.
- Protonless NMR experiments offer advantages in certain biological systems.
- Obtaining dihedral angle constraints, particularly for backbone and side chains, is vital for accurate protein structure determination.
Purpose of the Study:
- To introduce a novel experiment, COCO-TOCSY, for inter-residue sequential correlations among carbonyl spins.
- To enable dihedral angle constraints via a protonless NMR approach.
- To measure long-range homonuclear coupling constants for structural analysis.
Main Methods:
- Development and application of the COCO-TOCSY experiment.
- Utilizing (13)C-detected, protonless NMR techniques.
- Quantitative analysis of spectral data to measure coupling constants.
Main Results:
- The COCO-TOCSY experiment successfully connected carbonyl spins separated by 3-5 bonds in proteins.
- Structural information on backbone dihedral angles (phi) and side chain angles of Asx/Glx residues was obtained.
- Approximately 75% of backbone carbonyls in Calbindin D(9K) were sequentially connected.
- Measurement of 49 (3)J(C')(C') values and long-range (4)J(CC), (5)J(CC) couplings was achieved.
Conclusions:
- The COCO-TOCSY experiment provides the first protonless method for obtaining dihedral angle constraints.
- This technique offers valuable structural information for medium and small-sized proteins.
- The ability to measure long-range couplings is unprecedented for proteins of this size.
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