HATRX: a new method for time-resolved structural studies

Structural biology aims to elucidate the relationship between the function of biological molecules and their structure. In addition, timeresolved methods are used to characterise the contribution of structural dynamics to biological processes, such as protein folding, force transfer, ligand binding and catalysis. Numerous time-resolved methods are available, with each being particularly suitable for the study of different set of biological processes. To observe the structural and chemical changes accompanying enzyme catalysis it is necessary to use techniques that combine atomic spatial resolution with fast-time resolution, such as Xray crystallography. Time-resolved X-ray crystallographic methods have existed for several decades, but are not widely used. A major challenge is the need for an X-ray source that is bright enough to provide a measurable signal in very short times. Laue crystallography can be used to reach 100s of pico-second time-resolutions, however, at fast time-scales, this method is only applicable to reversible reactions. More recently the use of free-electron lasers has been proposed .to directly observe atomic positions in proteins with femtosecond time-resolution. However, these experiments require the use of a free-electron laser, an expensive and limited resource. A more general approach is therefore needed to perform time-resolved crystallography on irreversible systems and increase the achievable time-resolution using flux-limited, but widely available monochromatic synchrotron beamlines, Here we propose a novel application of the Hadamard transform to time-resolved experiments and demonstrate its use for X-ray crystallography by measuring the X-ray induced breaking of the eight disulfide bonds of thaumatin. The technique is applicable to a range of diffraction and spectroscopic methods, where the probe can be encoded. The use of the Hadamard transform increases the achievable time-resolution of a time-resolved experiment using a monochromatic synchrotron beamline by orders of magnitude, enabling the observation of short-lived intermediate species in chemical and biological reactions. This will provide unprecedented information regarding the relationship between structure and function.