An on-demand, drop-on-drop method for studying enzyme catalysis by serial crystallography

Area of Science:

• Structural Biology
• Biochemistry
• Biophysics

Background:

• Serial femtosecond crystallography (SFX) advances structural biology by enabling time-resolved experiments.
• Current SFX methods often rely on light-inducible systems, limiting studies to light-dependent enzymes.
• Many crucial enzyme-catalyzed reactions involve ligand diffusion, a process not easily studied with existing SFX techniques.

Purpose of the Study:

• To develop and validate a novel sample delivery system for time-resolved SFX.
• To enable the study of enzyme-catalyzed reactions involving ligand diffusion into active sites.
• To achieve high atomic and temporal resolution for protein dynamics.

Main Methods:

• A drop-on-drop sample delivery system was designed for microcrystal slurries.
• Ligand solutions were delivered in picoliter bursts onto crystal-containing drops, inducing turbulent mixing.
• Numerical simulations, fluorescent dye mixing, and time-resolved SFX were used to assess mixing efficiency and temporal resolution.

Main Results:

• The drop-on-drop system demonstrated rapid and efficient mixing of ligands with microcrystals.
• Time-resolved SFX experiments confirmed fast ligand diffusion through microdroplets.
• The system achieved high temporal resolution for observing reaction intermediates.

Conclusions:

• The drop-on-drop method is a versatile tool for time-resolved serial crystallography.
• It significantly expands the scope of SFX to include ubiquitous enzyme-ligand interactions.
• This technique holds great potential for studying enzyme mechanisms and dynamics with small molecule substrates.