X‑ray Transparent Microfluidic
Chip for Mesophase-Based
Crystallization of Membrane Proteins and On-Chip Structure Determination
- Publication date
- Publisher
Abstract
Crystallization
from lipidic mesophase matrices is a promising
route to diffraction-quality crystals and structures of membrane proteins.
The microfluidic approach reported here eliminates two bottlenecks
of the standard mesophase-based crystallization protocols: (i) manual
preparation of viscous mesophases and (ii) manual harvesting of often
small and fragile protein crystals. In the approach reported here,
protein-loaded mesophases are formulated in an X-ray transparent microfluidic
chip using only 60 nL of the protein solution per crystallization
trial. The X-ray transparency of the chip enables diffraction data
collection from multiple crystals residing in microfluidic wells,
eliminating the normally required manual harvesting and mounting of
individual crystals. We validated our approach by on-chip crystallization
of photosynthetic reaction center, a membrane protein from <i>Rhodobacter sphaeroides</i>, followed by solving its structure
to a resolution of 2.5 Å using X-ray diffraction data collected
on-chip under ambient conditions. A moderate conformational change
in hydrophilic chains of the protein was observed when comparing the
on-chip, room temperature structure with known structures for which
data were acquired under cryogenic conditions