X-ray transparent microfluidic chips for high-throughput screening and optimization of in meso membrane protein crystallization

Abstract

Elucidating and clarifying the function of membrane proteins ultimately requires atomic resolution structures as determined most commonly by X-ray crystallography. Many high impact membrane protein structures have resulted from advanced techniques such as in meso crystallization that present technical difficulties for the set-up and scale-out of high-throughput crystallization experiments. In prior work, we designed a novel, low-throughput X-ray transparent microfluidic device that automated the mixing of protein and lipid by diffusion for in meso crystallization trials. Here, we report X-ray transparent microfluidic devices for high-throughput crystallization screening and optimization that overcome the limitations of scale and demonstrate their application to the crystallization of several membrane proteins. Two complementary chips are presented: (1) a high-throughput screening chip to test 192 crystallization conditions in parallel using as little as 8 nl of membrane protein per well and (2) a crystallization optimization chip to rapidly optimize preliminary crystallization hits through fine-gradient re-screening. We screened three membrane proteins for new in meso crystallization conditions, identifying several preliminary hits that we tested for X-ray diffraction quality. Further, we identified and optimized the crystallization condition for a photosynthetic reaction center mutant and solved its structure to a resolution of 3.5 Å.

FIG. 1.

Architecture of a single microfluidic in meso crystallization well. (a) Optical micrograph of a single crystallization well during a crystallization experiment. Protein and precipitant meet at a mixing interface in an open-ended capillary valve for passive mixing by free interface diffusion. (b) Photograph of a microfluidic array chip for in meso crystallization, showing a chip filled with protein (red solution) and precipitants (blue, green, yellow, and clear solutions). (c) Exploded schematic of a single crystallization well. X-ray transparency is achieved by construction with <200 μm of low-scattering materials. (d) Inset of the open-ended capillary valve, highlighting key optimization parameters indicated with capped red lines or red angles. Experimental studies of width (w), expansion angle (θ), and length (L) resulted in an optimal valve geometry for high-throughput chips.

FIG. 2.

High-throughput screening chips for membrane protein crystallization. (a) Designs for 192-well (left) and 48-well (right) screening chips. Fluid introduced through protein and precipitant ports mix to generate 192 unique conditions. (b) Variable protein:lipid ratios generated in high-throughput screens. As indicated in the table, the amount of lipid for mesophase formulation remains constant while the variable size of protein compartments generates a linear gradient of protein concentrations. Each precipitant mixes with four different mesophases for extensive screening. (c) Tabulated screening results for qNOR, screened at 20 mg/ml with Cubic Screen. 96 conditions (A1–H12) were screened in high-throughput chips. Scores are indicated with colors and numbers: red (0–1) and grey (2–3) for a negative result, yellow (4–6) for optimization candidates (low quality crystals, crystallites), and green (7–9) for diffraction ready crystals (not observed in shown qNOR screen). (d) Representative crystallization screening results and scores for qNOR, cytochrome bo₃ oxidase, and the LM-dimer (3 conditions each) as visualized on-chip with light or cross-polarized microscopy.

FIG. 3.

Optimization chips for membrane protein crystallization. (a) Design of a 48-well chip (left) and fluid filling scheme (right) for a crystallization optimization experiment. The mixing array formulates fully combinatorial in meso crystallization experiments for 4 different levels of both protein and precipitant, aiding the search for diffraction-quality crystals once proper precipitant components are discovered via screening. (b) (left) Photograph of on-chip optimization experiment for L223SW observed 10d after set-up. (right) Representative optimization results from each condition formulated on a single chip. No crystallization was observed at low protein concentration, and many small crystals appeared at high protein concentration and low precipitant concentration. The best crystals were observed at higher protein concentrations (15 and 20 mg/ml) and high precipitant concentrations (14%–15%). Scale bars: 100 μm

FIG. 4.

The secondary quinone (Q_B) binding pocket in the L-subunit of R. sphaeroides reaction center. (a) The binding pocket in the wild-type structure where Q_B, a ubiquinone-8 molecule that serves as the terminal electron acceptor, is present. (b) The Q_B binding pocket in the L223SW mutant, where the mutated residue (serine to tryptophan) blocks the binding pocket, inhibiting the binding of Q_B. All 2F_O–F_c maps are contoured to ±1σ. (c) Flash-induced spectroscopy of wild-type and mutant reaction centers. In the wild-type where Q_B is present, the backward electron transfer from Q_B⁻ to the special pair of bacteriochlorophyll P⁺ has a lifetime of ∼1 s. In contrast, in the mutant where Q_B is absent, the electron transfer from Q_A⁻ to P⁺ has a lifetime of ∼0.1 s. This spectroscopic measurement indicates that a functional Q_B is absent in the L223SW mutant.