Technical Highlight - December 2014
Short description: Time-resolved crystallography on standard synchrotron beamlines is possible with a method based on Hadamard encoding.
The classical pump-probe pulse sequence (left) versus the HATRX pulse sequence (right). 1
In assessing how a protein functions, structure determination is only part of the equation. Proteins are dynamic entities, and their movements are often crucial to their biological roles. However, fleeting intermediate conformational states are very difficult to resolve.
Time-resolved crystallography is one technique that enables high-resolution structural information to be collected for short-lived intermediate states. In the classical method, a 'pump' pulse (such as a pulse of light) is used to trigger a reaction in a protein crystal. A series of ultrashort X-ray pulses ('probe' pulses) are then used to capture diffraction snapshots at set timepoints following the initial reaction. Current time-resolved crystallography methods approach picosecond time resolution, and X-ray free-electron laser technology should enable collecting femtosecond-timescale snapshots. However, only a few highly specialized facilities in the world are currently capable of performing such time-resolved measurements. Conventional monochromatic synchrotron beamlines are not sufficiently bright to allow enough diffracted photons to build up in the required, extremely short amount of time of probe pulse applications to yield interpretable diffraction snapshots.
Pearson, Beddard, Owen and Yorke now report a clever approach based on Hadamard encoding, which opens up the possibility to perform time-resolved experiments on conventional monochromatic synchrotron beamlines. In a Hadamard time-resolved crystallographic (HATRX) experiment, a pump pulse initiates a reaction, followed by a sequence of probe pulses. However, rather than collecting diffraction data following each probe pulse, the total signal for the entire probe pulse sequence is recorded, much like taking a long-exposure photograph. After the sample is allowed to relax, a new probe pulse sequence is applied, and this process is repeated until all possible probe pulse sequences have been used. The Hadamard transform is then used on the entire dataset to extract the time-dependent information. The final effect is that the time resolution no longer depends on the brightness of the beamline, as there is sufficient time to build up an interpretable diffraction image. Thus, one can extract information about how the protein moves from the HATRX data.
The authors applied the HATRX approach to follow time-dependent changes to the protein thaumatin (PDB 4C3C) with ∼200 millisecond time resolution. They suggest various ways in which HATRX could be implemented, with further improved time-resolution, at standard monochromatic synchrotron beamlines found around the world. Finally, in addition to its application to crystallography, this transform method is of general applicability to time-resolved methods where a probe can be encoded and can, for example, be used to perform time-resolved spectroscopy.
B.A. Yorke, G.S. Beddard, R.L. Owen & A.R. Pearson Time-resolved crystallography using the Hadamard transform.
Nat. Methods. 11, 1131-34 (2014). doi:10.1038/nmeth.3139