Time-resolved IR spectroscopy for monitoring protein dynamics in microcrystals

Analysis of protein dynamics is crucial for understanding the molecular mechanisms underlying protein function. To gain insights into the structural changes in proteins, time-resolved X-ray crystallography has been greatly advanced by the development of X-ray free-electron lasers. This tool has the potential to trace structural changes at atomic resolution; however, data interpretation and extrapolation to the solution state is often not straightforward as the in crystallo environment is not the same as it is in solution. On the other hand, time-resolved spectroscopy techniques, which have long been used for tracking protein dynamics, offer the advantage of being applicable irrespective of whether the target proteins are in crystalline or solution phase. Time-resolved IR spectroscopy is a particularly powerful technique, as it can be used on various proteins, including those that are colorless, and provides information on the chemical structures of functional sites of proteins and ligands which complements X-ray crystallography. This chapter presents the protocol for time-resolved IR microspectroscopic measurements of protein microcrystals. It includes an overview of the measurement system assembly, sample preparation, setting of experimental conditions, and time-resolved data analysis. It also describes, with examples, the usefulness of time-resolved IR measurements for comparing the dynamics between crystalline and solution conditions.