Optimal design of adaptively sampled NMR experiments for measurement of methyl group dynamics with application to a ribosome-nascent chain complex

Abstract

NMR measurements of cross-correlated nuclear spin relaxation provide powerful probes of polypeptide dynamics and rotational diffusion, free from contributions due to chemical exchange or interactions with external spins. Here, we report on the development of a sensitivity-optimized pulse sequence for the measurement of cross-correlated relaxation in methyl spin systems by analysis of the differential relaxation of transitions within the 13C multiplet. We describe the application of optimal design theory to implement a real-time 'on-the-fly' adaptive sampling scheme that maximizes the accuracy of the measured rate constants. The increase in sensitivity obtained using this approach enables, for the first time, quantitative measurements of rotational diffusion within folded states of translationally-arrested ribosome-nascent chain complexes of the FLN5 filamin domain, and can be used to place strong limits on interactions between the domain and the ribosome surface.

Analysis of Conformational Exchange Processes using Methyl-TROSY-Based Hahn Echo Measurements of Quadruple-Quantum Relaxation

“…3A). Based on our analysis of the standard 1 H-coupled HSQC experiment 6 , and fully incorporating the effects of relaxation and cross-correlated relaxation throughout the pulse sequence, the entire multiplet lineshape may be expressed as a function of the 1 H and 13 C chemical shifts (determined from a regular HMQC spectrum), 1 H and 13 C relaxation rates, the scalar coupling :5 + ≈ 125 Hz, and the parameters of interest, 6"78 # 9 and Δ : . This expression may be used to fit 2D spectra obtained at multiple relaxation delays, to estimate the parameters 6"78 # 9 and Δ : simultaneously.…”

“…A list of peak positions was prepared from a 1 H, 13 C HMQC experiment, and parsed to determine clusters of non-overlapping resonances based on a 0.05 ppm strip width in the 1 H dimension and a 2.4 ppm strip width in the 13 C dimension. For each peak within a cluster, 2D spectra of 13 C quartets were simulated as recently described 6 , incorporating the additional relaxation period shown in Fig. 2a where is the line broadening applied in Hz, and max is the acquisition time.…”

“…Solution NMR spectroscopy is a powerful tool for the characterization of macromolecular dynamics over a range of timescales relevant to biological function 1 : from backbone and sidechain disorder on ps-ns timescales, characterized by S 2 order parameters [2][3][4] ; rotational diffusion and domain motions, characterized by rotational correlation times, τc 5,6 ; through to real-time measurements of kinetics on timescales of seconds and beyond, following rapid mixing, temperature or pressure jumps [7][8][9] . NMR is also particularly well suited to the analysis of reversible chemical exchange on timescales of the order of microseconds to milliseconds, via lineshape analysis across a titration series [10][11][12] or using sophisticated pulse sequences such as ZZ-exchange spectroscopy, chemical exchange saturation transfer (CEST), and Carr-Purcell-Meiboom-Gill (CPMG) and R1ρ relaxation dispersion 13,14 .…”