Room-temperature crystallography using a microfluidic protein crystal array device and its application to...

Area of Science:

• Structural biology
• Biophysics
• Drug discovery

Background:

• Room-temperature (RT) protein crystallography offers insights into protein function under physiological conditions.
• Analyzing protein-ligand complexes via X-ray crystallography determines 3D binding site configurations, crucial for structure-based and fragment-based drug design (FBDD).
• Current RT crystallography methods face challenges in simultaneous crystal preparation and sequential X-ray diffraction measurement due to radiation damage.

Purpose of the Study:

• To develop an advanced RT crystallography technique for protein-ligand complex structure analysis.
• To overcome the limitations of preparing and measuring numerous crystals required for RT crystallography.
• To enable high-throughput analysis and obtain more natural protein-ligand complex structures.

Main Methods:

• A microfluidic protein crystal array device was developed for RT crystallography.
• Microfluidic sorting efficiently immobilizes protein crystals into microwells for sequential X-ray diffraction measurement.
• High-throughput protein-ligand complex preparation was achieved by replacing microchannel contents with ligand solutions.

Main Results:

• The microfluidic device facilitated the sorting and sequential measurement of protein crystals.
• Eight trypsin-ligand complex structures were determined, demonstrating the technique's proof of concept.
• Differences were observed between RT and conventional cryogenic structures in ligand coordination.

Conclusions:

• The microfluidic RT crystallography technique simplifies protein-ligand complex analysis.
• This methodology allows for the acquisition of more biologically relevant protein structures.
• The approach has the potential to enhance the effectiveness of fragment-based drug design.