RESUMO
Time-resolved crystallography enables the visualization of protein molecular motion during a reaction. Although light is often used to initiate reactions in time-resolved crystallography, only a small number of proteins can be activated by light. However, many biological reactions can be triggered by the interaction between proteins and ligands. The sample delivery method presented here uses a mix-and-extrude approach based on 3D-printed microchannels in conjunction with a micronozzle. The diffusive mixing enables the study of the dynamics of samples in viscous media. The device design allows mixing of the ligands and protein crystals in 2 to 20â s. The device characterization using a model system (fluorescence quenching of iq-mEmerald proteins by copper ions) demonstrated that ligand and protein crystals, each within lipidic cubic phase, can be mixed efficiently. The potential of this approach for time-resolved membrane protein crystallography to support the development of new drugs is discussed.
RESUMO
Photolyase is an enzyme that uses light to catalyze DNA repair. To capture the reaction intermediates involved in the enzyme's catalytic cycle, we conducted a time-resolved crystallography experiment. We found that photolyase traps the excited state of the active cofactor, flavin adenine dinucleotide (FAD), in a highly bent geometry. This excited state performs electron transfer to damaged DNA, inducing repair. We show that the repair reaction, which involves the lysis of two covalent bonds, occurs through a single-bond intermediate. The transformation of the substrate into product crowds the active site and disrupts hydrogen bonds with the enzyme, resulting in stepwise product release, with the 3' thymine ejected first, followed by the 5' base.