Off-resonance rotating-frame relaxation dispersion experiment for 13C in aromatic side chains using L-optimized TROSY-selection

• Published in: Journal of biomolecular NMR Vol. 59; no. 1; pp. 23 - 29
• Main Authors: Weininger, Ulrich, Brath, Ulrika, Modig, Kristofer, Teilum, Kaare, Akke, Mikael
• Format: Journal Article
• Language: English
• Published: Dordrecht Springer Netherlands 01-05-2014
• Subjects: aprotinin; Aromatic ring flip; Biochemistry; Biochemistry and Molecular Biology; Biokemi och molekylärbiologi; Biological and Medical Physics; Biomolecular; Biophysics; Bovinae; carbon; carbon 13; Carbon Isotopes; chemistry; Communication; Conformational exchange; human; ligand binding; molecular dynamics; nonhuman; nuclear magnetic resonance; nuclear magnetic resonance spectroscopy; Nuclear Magnetic Resonance, Biomolecular - methods; Physics; Physics and Astronomy; priority journal; procedures; protein; protein analysis; protein binding; Protein Conformation; protein folding; Proteins; Proteins - chemistry; proton nuclear magnetic resonance; Spectroscopy/Spectrometry; Spin-lock; Strong coupling; Strong coupling; Spin-lock; Conformational exchange; Aromatic ring flip
• ISSN: 0925-2738; 1573-5001; 1573-5001
• DOI: 10.1007/s10858-014-9826-2

Protein dynamics on the microsecond–millisecond time scales often play a critical role in biological function. NMR relaxation dispersion experiments are powerful approaches for investigating biologically relevant dynamics with site-specific resolution, as shown by a growing number of publications on enzyme catalysis, protein folding, ligand binding, and allostery. To date, the majority of studies has probed the backbone amides or side-chain methyl groups, while experiments targeting other sites have been used more sparingly. Aromatic side chains are useful probes of protein dynamics, because they are over-represented in protein binding interfaces, have important catalytic roles in enzymes, and form a sizable part of the protein interior. Here we present an off-resonance
R
1ρ
experiment for measuring microsecond to millisecond conformational exchange of aromatic side chains in selectively
13
C labeled proteins by means of longitudinal- and transverse-relaxation optimization. Using selective excitation and inversion of the narrow component of the
13
C doublet, the experiment achieves significant sensitivity enhancement in terms of both signal intensity and the fractional contribution from exchange to transverse relaxation; additional signal enhancement is achieved by optimizing the longitudinal relaxation recovery of the covalently attached
1
H spins. We validated the L-TROSY-selected
R
1ρ
experiment by measuring exchange parameters for Y23 in bovine pancreatic trypsin inhibitor at a temperature of 328 K, where the ring flip is in the fast exchange regime with a mean waiting time between flips of 320 μs. The determined chemical shift difference matches perfectly with that measured from the NMR spectrum at lower temperatures, where separate peaks are observed for the two sites. We further show that potentially complicating effects of strong scalar coupling between protons (Weininger et al. in J Phys Chem B 117: 9241–9247,
2013b
) can be accounted for using a simple expression, and provide recommendations for data acquisition when the studied system exhibits this behavior. The present method extends the repertoire of relaxation methods tailored for aromatic side chains by enabling studies of faster processes and improved control over artifacts due to strong coupling.