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

Abstract

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 R1ρ experiment for measuring microsecond to millisecond conformational exchange of aromatic side chains in selectively 13C labeled proteins by means of longitudinal- and transverse-relaxation optimization. Using selective excitation and inversion of the narrow component of the 13C 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 1H spins. We validated the L-TROSY-selected R1ρ 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.

Site-selective 13C labeling of proteins using erythrose

“…13 C relaxation dispersion experiments both for CPMG (Weininger et al 2012b) and R 1ρ (Weininger et al 2014a) were validated for glucose labeled samples previously. These experiments can be directly applied to samples resulting from erythrose labeling, since the relaxation behaviour is identical.…”

Slow ring flips in aromatic cluster of GB1 studied by aromatic 13C relaxation dispersion methods

“…Here we investigate slow ring flips in the aromatic cluster of GB1 that have been found recently (Dreydoppel et al 2018), using 13 C aromatic relaxation dispersion methods (Weininger et al 2012(Weininger et al , 2014a in a temperature and pressure dependent way. We found that all four residues of the cluster (Y3, F30, Y45, F52) show slow ring flips.…”

“…All experiments were performed at Bruker Avance III spectrometers at a static magnetic field strength of 14.1 T. Aromatic L-optimized TROSY selected 13 C CPMG (Weininger et al 2012) and R 1ρ (Weininger et al 2014a) relaxation dispersion experiments have been acquired between 10 and 40 °C and 0.1 and 100 MPa. R 1ρ relaxation dispersion experiments have been recorded on-resonance.…”

“…Experimental measurements of ring flips, however, have been limited so far to a handful of cases since their discovery in the 1970s (Campbell et al 1975;Hull and Sykes 1975;Wagner et al 1976). Recently, new cases have been reported (Weininger et al 2014b;Yang et al 2015) enabled by methodological advances in site-selective 13 C labeling (Lundström et al 2007;Teilum et al 2006), aromatic 13 C relaxation dispersion experiments (Weininger et al 2012(Weininger et al , 2014a and the understanding of strong 1 H-1 H couplings (Weininger et al 2013). Additionally, ring flips can be studied by long scale MD simulations (Shaw et al 2010) and extremely fast ring flips are shown to affect order parameters (Kasinath et al 2015).…”

“…The spectrum at − 5 °C and 200 MPa is shown in blue, where split signals (δ1 and δ2, or ε1 and ε2, respectively) can be observed. Signals indicated as # are caused by sample impurities which can be detected at very high S/N experiments, which were needed for the − 5 °C and 200 MPa condition, where the split signals are still severely broadened experiments(Weininger et al 2014a) have been applied.…”