Methyl TROSY: explanation and experimental verification

Abstract

In TROSY experiments, relaxation interference effects are exploited to produce spectra with improved resolution and signal-to-noise. Such experiments cannot be explained using the standard product operator formalism, but must instead be analyzed at the level of individual density matrix elements. Herein we illustrate this point using an example from our recent work on a TROSY 1 H-13 C correlation experiment for methyl groups in large proteins. Methyl groups are useful spectroscopic probes of protein structure and dynamics because they are found throughout the critical core region of a folded protein and their resonances are intense and well dispersed. Additionally, it is relatively easy to produce highly deuterated protein samples that are 1 H, 13 C labeled at selected methyl positions, facilitating studies of high molecular weight systems. Methyl groups are relaxed by a network of 1 H-1 H and 1 H-13 C dipolar interactions, and in the macromolecular limit the destructive interference of these interactions leads to unusually slow relaxation for certain density matrix elements. It is this slow relaxation that forms the basis for TROSY experiments. We present a detailed analysis of evolution and relaxation during HSQC and HMQC pulse schemes for the case of a 13 C 1 H 3 spin system attached to a macromolecule. We show that the HMQC sequence is already optimal with respect to the TROSY effect, offering a significant sensitivity enhancement over HSQC at any spectrometer field strength. The gain in sensitivity is established experimentally using samples of two large proteins, malate synthase G (81.4 kDa) and ClpP protease (305 kDa), both highly deuterated and selectively 1 H, 13 C-labeled at isoleucine d methyl positions.

Quaternary Dynamics of αB-Crystallin as a Direct Consequence of Localised Tertiary Fluctuations in the C-Terminus

“…U- 2 NMR measurements 13 C-1 H correlation spectra were acquired using Varian NMR spectrometers operating at field strengths of 11.7, 14.0 and 18.8 T over a range of pHs (5-9) and temperatures (24-50°C) using experiments optimised for large-molecular-weight proteins. 8,37,39,43,61 Translational diffusion coefficients were quantified for each resonance in the two-dimensional (2D) spectrum using a 2D 13 C-1 H correlation experiment that is based on a 15 N-1 H N pulse scheme 41 with a diffusion delay of 200 ms. To determine the mobility of individual methyl groups, we obtained proton R 2 relaxation rates as described previously. 40 In addition, values of the product S axis 2 τ C were measured using the approach of Tugarinov et al in which the time dependencies of sums (I b ) and differences (I a ) of magnetization derived from methyl 1 H single-quantum transitions are quantified.…”

Ligand Modulation of Sidechain Dynamics in a Wild-Type Human GPCR

“…To apply these techniques to study the sidechains in these microswitches, one approach would entail isoleucine/leucine/valine (ILV) labeling (Goto et al, 1999;Gardner et al, 1998) and perdeuteration of a wild-type GPCR, to generate proteins with 1 H/ 13 C-labeled methyl groups within an otherwise 2 H/ 12 C-labeled background. Such samples are ideally suited for acquiring 1 H/ 13 C methyl TROSY-based (Tugarinov et al, 2003a;Ollerenshaw et al, 2003) relaxation NMR data, allowing the quantitative determination of methyl order parameters and relative motion of sidechains on the ps-ns timescale (Tugarinov et al, 2005;Ishima and Torchia, 2000). Advances in labeling methodology and pulse sequences have enabled such measurements on large macromolecular systems, such as the 80 kDa enzyme malate synthase (Tugarinov and Kay, 2003b;Sandala et al, 2007), the 670 kDa archaeal proteasome core particle (Sprangers and Kay, 2007;Religa et al, 2010), and the 1 MDa GroEL/GroES complex (Fiaux et al, 2002;Horst et al, 2005).…”

Optimum spin-state selection for all multiplicities in the acquisition dimension of the HSQC experiment

“…These reported S 3 -edited HSQC experiments only work properly for IS spin systems and they have been mainly applied to backbone NH and C a H a spin systems in labeled proteins. During the last years, a different number of methylene-specific [11][12][13][14][15][16] and methyl-specific [17][18][19][20] spin-edited HSQC-type pulse [4][5][6][7] (W = y) and in [24] (W = x). Thick and thin rectangles represent 90°and 180°pulses, respectively.…”