Observing Macromolecular Dynamics
Many proteins in crystals, including many enzymes, can retain the conformational flexibility needed to perform biological processes, making crystallographic studies of protein dynamics possible.
These studies provide details of the atomic positions and motions involved in molecular recognition, transition state stabilization, and other aspects of the catalytic process. While time-resolved studies of this sort had been challenging in the past, the extremely short pulse length of 5–50 fs provided by LCLS enables dynamic crystallography on shorter time scales and using smaller, more radiation-sensitive samples, expanding these methods to investigate a wider variety of scientific questions.
The proceeding figure shows the time scales accessible to LCLS and SSRL for room temperature and near-physiological studies. The time scales are relevant for light-activated processes (Optical pump-probe experiment) as well as for rapid mixing studies serial crystallography (MISC) for SSRL and LCLS that can combine to provide capabilities spanning much of the biomedically relevant time scales of interest.
Successes at LCLS using photoexcitation at LCLS include molecular movies of conformation changes in photoactive yellow protein, CO dissociation from myoglobin, retinal isomerization in bacteriorhodopsin, and chromophore dynamics in a switchable fluorescent protein.
The SDB Resource builds on these results to develop more generalized time-resolved approaches applicable to non-photoactive systems, such as those involved in cell signaling and transport.
These methods include the use of photo-released caged compounds (Driving Biomedical Projects on RNA polymerase II with Guillermo Calero, University of Pittsburgh, and GPCRs with Vadim Cherezov, University of Southern California) and laser-pump temperature-jump techniques (Driving Biomedical Projects with Michael Thompson, University of California Merced).
The Resource will also further develop Mix and Inject Serial Crystallography (MISC) to support work on cytochrome c oxidase dynamics (Driving Biomedical Project with Denis Rousseau, Albert Einstein College of Medicine), riboswitches (Driving Biomedical Project with Yun-Xing Wang, National Cancer Institute), and enzymology (Driving Biomedical Project with Marius Schmidt, University of Wisconsin-Milwaukee).
MISC enables the examination of interaction dynamics between a molecule and a substrate and has strong implications for drug development, understanding disease mechanisms, cellular messaging, signaling, and transport.
MISC has been used to study large domain motions in an RNA riboswitch and the ring cleavage of an antibiotic by a protein involved in antibiotic resistance in bacteria. TR-SFX experiments using either MISC or photoexcitation pose numerous challenges in sample handling, injector operation, and data collection that will be addressed by the Resource developments.
Many developments for TR-SFX also benefit TR-Small and Wide-Angle X-ray Scattering (SAXS/WAXS) experiments, including experiments to follow antibiotic binding and to follow the dynamics of molecules after a temperature jump. SAXS/WAXS at LCLS can monitor transient protein structures to detect structural intermediates along reaction pathways at timescales not possible at synchrotron beamlines.
For example, the structural response of CO-bound myoglobin after photolysis was recorded at multiple time points up to 100 ps. Within 0.8 ps, clear difference signals were observed, with most structural changes completed within 10 ps.
This type of ultra-fast TR-SAXS/WAXS experiment enables observed structural entities to be correlated with markers obtained through optical spectroscopy methods of similar time resolution. Furthermore, coupled with rapid mixers, the LCLS source can mitigate radiation-induced artifacts during TR-SAXS/WAXS studies based on parameters such as temperature, pH, and ligand binding at a range of timescales.
TR-SAXS/WAXS can track both reversible and irreversible reactions, such as protein folding induced by ligand dissociation and electron transfer, expanding its applicability to many protein reactions such as drug binding to target proteins. The short X-ray pulses combined with Fluctuation X-ray Scattering (FXS) analysis offer enhanced resolution by extracting correlations in the SAXS/WAXS patterns to provide finer detail to the reconstructed objects.
The work of the Resource will drive technology and methodology developments to support all these scientific goals.