title: 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.
authors.affiliation: Department of Chemistry and Biochemistry, Arizona State University , Tempe, Arizona 85287, United States.
authors.forename: Bahige G
authors.initials: BG
authors.lastname: Abdallah
authors.forename: Christopher
authors.initials: C
authors.lastname: Kupitz
authors.forename: Petra
authors.initials: P
authors.lastname: Fromme
authors.forename: Alexandra
authors.initials: A
authors.lastname: Ros
mesh_terms: D002021:Buffers; D003198:Computer Simulation; D003460:Crystallization; D018360:Crystallography, X-Ray; D000458:Cyanobacteria / Q000737:chemistry; D004058:Diffusion; D057927:Hydrophobic and Hydrophilic Interactions; D007477:Ions; D008274:Magnesium / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry*; D046210:Microfluidic Analytical Techniques; D044085:Microfluidics; D008962:Models, Theoretical; D009113:Muramidase / Q000737:chemistry; D036103:Nanotechnology; D045331:Photosystem I Protein Complex / Q000737:chemistry*; D010798:Phycocyanin / Q000737:chemistry; D012492:Salts / Q000737:chemistry; D012997:Solvents / Q000737:chemistry; D013431:Sulfates / Q000737:chemistry; D020524:Thylakoids / Q000737:chemistry*
chemical_list: D002021:Buffers; D007477:Ions; D008565:Membrane Proteins; D045331:Photosystem I Protein Complex; D012492:Salts; D012997:Solvents; D013431:Sulfates; D010798:Phycocyanin; D009113:Muramidase; D008274:Magnesium