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.
Highlights
The NMR lineshape of a spin undergoing chemical exchange is well known to provide a powerfulElectronic supplementary material The online version of this article contains supplementary material, which is available to authorized users.spectroscopic probe of the underlying exchange process
In this paper we have presented a systematic analysis of chemical exchange regimes within two-dimensional correlation spectra, focusing in particular on differences between SQ, MQ, ZQ and DQ-based experiments
We have previously commented upon the differences in exchange behaviour between HSQC and HMQC experiments (Waudby et al 2016), and the use of ZQ and DQ TROSY experiments to reduce chemical exchange-induced resonance broadening has been
Summary
The resulting modulations may be fitted to determine details of the exchange process such as chemical shift differences, populations of states, and the rate of exchange between them. Differences in tranverse relaxation rates, ΔR2 , may affect the observed exchange regime, and kex should strictly be compared to |Δ + iΔR2| instead of |Δ |. The effect of this additional term is to shift exchange towards a slower regime than otherwise expected, but in the majority of situations relevant to biomolecular titrations the frequency difference is likely to remain the dominant term. For simplicity hereonin we will assume that there is no significant difference in relaxation rates between states ( any such differences can still be treated exactly by numerical analyses)
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