Crystallization of the large membrane protein complex photosystem I in a microfluidic channel

Abstract

Traditional macroscale protein crystallization is accomplished nontrivially by exploring a range of protein concentrations and buffers in solution until a suitable combination is attained. This methodology is time-consuming and resource-intensive, hindering protein structure determination. Even more difficulties arise when crystallizing large membrane protein complexes such as photosystem I (PSI) due to their large unit cells dominated by solvent and complex characteristics that call for even stricter buffer requirements. Structure determination techniques tailored for these "difficult to crystallize" proteins such as femtosecond nanocrystallography are being developed yet still need specific crystal characteristics. Here, we demonstrate a simple and robust method to screen protein crystallization conditions at low ionic strength in a microfluidic device. This is realized in one microfluidic experiment using low sample amounts, unlike traditional methods where each solution condition is set up separately. Second harmonic generation microscopy via second-order nonlinear imaging of chiral crystals (SONICC) was applied for the detection of nanometer- and micrometer-sized PSI crystals within microchannels. To develop a crystallization phase diagram, crystals imaged with SONICC at specific channel locations were correlated to protein and salt concentrations determined by numerical simulations of the time-dependent diffusion process along the channel. Our method demonstrated that a portion of the PSI crystallization phase diagram could be reconstructed in excellent agreement with crystallization conditions determined by traditional methods. We postulate that this approach could be utilized to efficiently study and optimize crystallization conditions for a wide range of proteins that are poorly understood to date.

Figure 1

a) Cross section of the microfluidic channel used for crystallization. Two reservoirs are located at channel ends where solutions can be introduced. The saline protein solution is injected into the channel from the reservoir on the right and fills via capillary action. The hydrophobic valve is placed at the left channel end to stop protein flow, effectively setting up a discrete crystallization zone. Hydrophobic surfaces are indicated in red and blue designates hydrophilic regions. On the left side of the image, the reservoir containing the Buffer A gel plug is shown. b) Photograph of the Buffer A reservoir/channel interface without the hydrophobic valve illustrating leakage of protein out of the channel and into the reservoir. c) Photograph of the same interface with the hydrophobic valve showing impeded protein flow and no leakage out of the channel.

Figure 2

Top-down view of the channel structure, laid out similarly to that in Figure 1. PSI crystal characteristics are drawn within the channel as hexagons, indicating expected changes along the channel. At the gel/channel interface, salt concentration is the lowest; therefore, protein rapidly precipitates out of solution and forms amorphous precipitate (small dots). Moving toward the protein solution reservoir, crystal size increases and abundance decreases. In terms of the phase diagram, each phase can be mapped out along the channel beginning with supersaturated near the gel/channel interface and transitioning towards non-saturated at the opposite end of the channel.

Figure 3

Simulated relative concentration profiles of protein and both salt ions along the microfluidic channel. Distance on the x-axis is the location relative to the gel/channel interface (0 cm). The simulations consider spatial and time dependent diffusion of Mg²⁺ (a), SO₄²⁻ (b), and PSI (c). Each plot shows the concentration profiles at four different time points ranging from 3 to 14 days. At 3 days, the ions exhibit a slightly curved trend that becomes linear at later time points. The PSI curves show a hyperbolic trend due to its lower diffusion coefficient compared to the ions. The 10 day profile given by the solid bold line corresponds to the experimental data shown in Figure 4.

Figure 4

Microfluidic device dimensions with a zoomed in region showing SONICC and bright field images of the microchannel: The capability of SONICC for detection is illustrated as a high contrast image compared to the bright field image where crystals are not detectable in the dark protein solution. Only a small portion of the channel (3 mm) is shown where crystals were detected as further downstream conditions did not favor crystallization. Six positions are marked (P1–P6) corresponding to the labels shown on the phase diagram in Figure 5. The region depicted beyond P6 is representative of the remainder of the microchannel, which did not exhibit PSI crystals within the channel.

Figure 5

Possible phase diagram for PSI based on experimental and simulation data. Positions along the microchannel were correlated with simulation data to determine salt and protein concentration assignments for each phase. For the 10 day experiment, positions are labeled according to Figure 4 (P1–P6). At position P1, protein concentration was lowest and crystals were not observed, likely indicating the metastable region. P2–P6 are positions where crystals were observed, indicating conditions of the nucleation zone. Additionally, 3 (▲) and 6 (■) day experiments were performed in a similar way and corresponding salt and protein concentrations were extracted from the simulations. For those experiments, crystals were observed at each corresponding data point indicating nucleation zone conditions. Negligible variation (~ 0.15 mM MgSO₄) was observed based on duplication of the 10 day trial, thus error bars are encompassed by the marker size.