Photocage-initiated time-resolved solution X-ray scattering investigation of protein dimerization

Abstract

This work demonstrates a new method for investigating time-resolved structural changes in protein conformation and oligomerization via photocage-initiated time-resolved X-ray solution scattering by observing the ATP-driven dimerization of the MsbA nucleotide-binding domain. Photocaged small molecules allow the observation of single-turnover reactions of non-naturally photoactivatable proteins. The kinetics of the reaction can be derived from changes in X-ray scattering associated with ATP-binding and subsequent dimerization. This method can be expanded to any small-molecule-driven protein reaction with conformational changes traceable by X-ray scattering where the small molecule can be photocaged.

Keywords: X-ray solution scattering; biophysics; photocaging; structural biology.

Figure 1

Overview of the experimental setup and protein structural transitions. (a) Photoexcited decaging of the NPE group from NPE-ATP using laser irradiation at 355 nm releases free ATP into solution. The decaging reaction happens on a 10 ms timescale. The quantum yield of the reaction is 0.67 (Syberg et al., 2012 ▸). (b) Mechanism of ATP-dependent (yellow circle) dimerization of NBDs (blue). (c) Schematic diagram of the experimental set up at the beamline.

Figure 2

Time-resolved SAXS data following photo-decaging of NPE-ATP. (a) Plot of the scattering difference curves (qΔI versus q) calculated by subtracting the protein scattered with a negative laser offset (−100 µs) from all the subsequent time-point measurements. Inset shows the difference curves of the first two time points (50 and 100 ms). (b) Radius of gyration representing NBD dimerization and kinetic fit with an increase in R _g over time (black squares with corresponding standard deviations). The time points after laser excitation were fitted with a second-order kinetic function (black line). The calculated rate constant for dimerization (k _dim) from the fit is 6200 M ⁻¹ s⁻¹.

Figure 3

Single-value decomposition analysis of the TR-XSS data reveals the presence of two components. (a) Singular values (from the diagonal of matrix s) plotted on a linear scale. (b) Singular (V^T) matrix showing the relative amplitudes of the eigenvectors (component 1 and 2 in red and blue, respectively) versus time. (c) Singular (U) matrix showing the eigenvectors for the two major components.