A simple method for measuring signs of (1)H (N) chemical shift differences between ground and excited protein states

Abstract

NMR relaxation dispersion spectroscopy is a powerful method for studying protein conformational dynamics whereby visible, ground and invisible, excited conformers interconvert on the millisecond time-scale. In addition to providing kinetics and thermodynamics parameters of the exchange process, the CPMG dispersion experiment also allows extraction of the absolute values of the chemical shift differences between interconverting states, /Delta(omega)/, opening the way for structure determination of excited state conformers. Central to the goal of structural analysis is the availability of the chemical shifts of the excited state that can only be obtained once the signs of Delta(omega) are known. Herein we describe a very simple method for determining the signs of (1)H(N) Delta(omega) values based on a comparison of peak positions in the directly detected dimensions of a pair of (1)H(N)-(15)N correlation maps recorded at different static magnetic fields. The utility of the approach is demonstrated for three proteins that undergo millisecond time-scale conformational rearrangements. Although the method provides fewer signs than previously published techniques it does have a number of strengths: (1) Data sets needed for analysis are typically available from other experiments, such as those required for measuring signs of (15)N Delta(omega) values, thus requiring no additional experimental time, (2) acquisition times in the critical detection dimension can be as long as necessary and (3) the signs obtained can be used to cross-validate those from other approaches.

Figure 1

Schematic illustrating the relation between ${\tilde{\delta }}_H$ and B₀ for a single spin exchanging between two sites with distinct chemical shifts. (a) In the limit $\Delta {\omega }_{\text{X}}\gg k_{\text{ex}}$ the system is in slow exchange and separate peaks are observed at positions that are essentially independent of exchange (${\tilde{\omega }}_{\text{X,G}}$ and ${\tilde{\omega }}_{\text{X,E}}$). (b) In the limit where $\Delta {\omega }_{\mathrm{X}}\ll k_{\text{ex}}$, a single peak is observed at the population weighted average of ${\tilde{\omega }}_{X,E}$ and ${\tilde{\omega }}_{X,G}$. (c,d) As B₀ increases $k_{ex}∕\Delta \omega$ decreases so that the exchanging system moves towards the slow exchange regime and hence ${\tilde{\delta }}_X$ decreases.

Figure 2

Correlation between $\Delta {\tilde{\delta }}_H^{\exp }$ and $\Delta {\tilde{\delta }}_H^{calc}$ for the Pfl6 I58D domain based on analysis of HSQC (a) or HMQC (b) data sets recorded at 500 and 800 MHz (5 °C). In red are $(\Delta {\tilde{\delta }}_H^{calc},\Delta {\tilde{\delta }}_H^{\exp })$ values satisfying criteria 1-4 in the text so that the sign from $\Delta {\tilde{\delta }}_H^{\exp }$ would be ‘accepted’ as correct, while in blue are data points that cannot be considered as trustworthy based on the same criteria; signs from $\Delta {\tilde{\delta }}_H^{\exp }$ would not be used in this case.

Figure 3

Correlation plot between ¹H^N chemical shift differences between the ground and excited states of the Pfl6 I58D domain as measured by relaxation dispersion experiments, $\Delta {\tilde{\omega }}_H^{disp}$, and predicted shift differences assuming that the excited state is unfolded, $\Delta {\tilde{\omega }}_H^{CSI}$, calculated as described by Wishart and coworkers (Wishart et al. 1995). Signs of $\Delta {\tilde{\omega }}_H^{disp}$ were determined using the experimentally measured exchange induced shifts, as described in the text.

Figure 4

Correlation between $\Delta {\tilde{\delta }}_H^{\text{exp}}$ and $\Delta {\tilde{\delta }}_H^{calc}$ values for the (a) FF (30 °C) and (b) A39V/N53P/V55L Fyn SH3 (20 °C) domains based on analysis of HSQC data sets recorded at 500 and 800 MHz. Other details are as in the legend to Figure 2.