Two-dimensional NMR lineshape analysis of single, multiple, zero and double quantum correlation experiments

Abstract

NMR spectroscopy provides a powerful approach for the characterisation of chemical exchange and molecular interactions by analysis of series of experiments acquired over the course of a titration measurement. The appearance of NMR resonances undergoing chemical exchange depends on the frequency difference relative to the rate of exchange, and in the case of one-dimensional experiments chemical exchange regimes are well established and well known. However, two-dimensional experiments present additional complexity, as at least one additional frequency difference must be considered. Here we provide a systematic classification of chemical exchange regimes in two-dimensional NMR spectra. We highlight important differences between exchange in HSQC and HMQC experiments, that on a practical level result in more severe exchange broadening in HMQC spectra, but show that complementary alternatives to the HMQC are available in the form of HZQC and HDQC experiments. We present the longitudinal relaxation optimised SOFAST-H(Z/D)QC experiment for the simultaneous acquisition of sensitivity-enhanced HZQC and HDQC spectra, and the longitudinal and transverse relaxation optimised BEST-ZQ-TROSY for analysis of large molecular weight systems. We describe the application of these experiments to the characterisation of the interaction between the Hsp90 N-terminal domain and a small molecule ligand, and show that the independent analysis of HSQC, HMQC, HZQC and HDQC experiments provides improved confidence in the fitted dissociation constant and dissociation rate. Joint analysis of such data may provide improved sensitivity to detect and analyse more complex multi-state interaction mechanisms such as induced fit or conformational selection.

A weakened interface in the P182L variant of HSP27 associated with severe Charcot‐Marie‐Tooth neuropathy causes aberrant binding to interacting proteins

“…Despite the small 15 N dispersions, the V111 and S155 resonances both become broadened during the peptide titration (Fig 3A, Appendix Figs S5 and S8A), indicative of slow‐to‐intermediate exchange. In a 2D 1 H‐ 15 N HSQC spectrum, chemical exchange‐induced broadening can arise from chemical shift changes to either the 15 N or 1 H nucleus or both (Waudby et al, 2020). Therefore, the broadening of V111 and S155 presumably arises from large 1 H chemical shift changes upon peptide binding, with Δω H V111 = 0.3 ppm and Δω H S155 = 0.5 ppm, respectively, corresponding to 1,131 rad/s and 1,885 rad/s at 14.1 T. By contrast, peptide binding does not significantly affect the 15 N chemical shifts of V111 and S155 (Δω N V111 = 0.2 ppm, Δω N S155 = 0.1 ppm; 90 rad/s and 45 rad/s at 14.1 T).…”

“…Despite the small 15 N dispersions, the V111 and S155 resonances both become broadened during the peptide titration (Fig 3A, Appendix Figs S5 and S8A), indicative of slow-to-intermediate exchange. In a 2D 1 H-15 N HSQC spectrum, chemical exchangeinduced broadening can arise from chemical shift changes to either the 15 N or 1 H nucleus or both(Waudby et al, 2020). Therefore, the broadening of V111 and S155 presumably arises from large 1 H chemical shift changes upon peptide binding, with Δω…”

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

“…2A). This approach, in which individual phase cycle steps are stored during acquisition and re-combined during processing, was also recently applied to the simultaneous measurement of HZQC and HDQC correlation spectra 11 . In both instances, the total time required to acquire the pair of experiments is halved without any compromise to the quality of the individual measurements.…”

“…2A). As demonstrated for the simultaneous acquisition of HZQC and HDQC correlation experiments 11 , by storing individual increments of the phase program and applying receiver phase cycling postacquisition it is possible to acquire P-and N-type pathways for both DQ' and QQ relaxation measurements within a single super-experiment 46 , without loss of sensitivity. Shaded grey pulses represent consecutive 5 kHz 2 ms Hx and 3 ms Hy purge pulses before the recycle delay, B!9 (with low power presaturation applied, if required).…”

“…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 .…”