Mix-and-extrude: high-viscosity sample injection towards time-resolved protein crystallography
Mohammad Vakili, Huijong Han, Christina Schmidt, Agnieszka Wrona, Marco Kloos, Iñaki de Diego, Katerina Dörner, Tian Geng, Chan Kim, F. Koua, Diogo V. M. Melo, Mathieu Rappas, Adam Round, Ekaterina Round, Marcin Sikorski, Joana Valério, Tiankun Zhou, Kristina Lorenzen, Joachim Schulz
Abstract
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 20 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.