X-ray transparent microfluidic platforms for membrane protein crystallization with microseeds.

Crystallization of membrane proteins is a critical step for uncovering atomic resolution 3-D structures and elucidating structure-function relationships. Microseeding, the process of transferring sub-microscopic crystal nuclei from initial screens into new crystallization experiments, is an effective, yet underutilized approach to grow crystals suitable for X-ray crystallography. Here, we report simplified methods for crystallization of membrane proteins that utilize microseeding in X-ray transparent microfluidic chips. First, a microfluidic method for introduction of microseed dilutions into metastable crystallization experiments is demonstrated for photoactive yellow protein and cytochrome bo3 oxidase. As microseed concentration decreased, the number of crystals decreased while the average size increased. Second, we demonstrate a microfluidic chip for microseed screening, where many crystallization conditions were formulated on-chip prior to mixing with microseeds. Crystallization composition, crystal size, and diffraction data were collected and mapped on phase diagrams, which revealed that crystals of similar diffraction quality and size typically grow in distinct regions of the phase diagram.

[1]  Yanwei Jia,et al.  Control and measurement of the phase behavior of aqueous solutions using microfluidics. , 2007, Journal of the American Chemical Society.

[2]  Naomi E Chayen,et al.  Methods for separating nucleation and growth in protein crystallisation. , 2005, Progress in Biophysics and Molecular Biology.

[3]  R. Allen Waggoner,et al.  Diffusion in Aqueous Solutions of Poly(ethylene glycol) at Low Concentrations , 1995 .

[4]  Alan Van Heuvelen,et al.  Intermediate physics for medicine and biology , 1989 .

[5]  N. Sträter,et al.  New crystal forms of NTPDase1 from the bacterium Legionella pneumophila. , 2013, Acta crystallographica. Section F, Structural biology and crystallization communications.

[6]  Sudipto Guha,et al.  Fabrication of X-ray compatible microfluidic platforms for protein crystallization. , 2012, Sensors and actuators. B, Chemical.

[7]  G. Borgstahl,et al.  1.4 A structure of photoactive yellow protein, a cytosolic photoreceptor: unusual fold, active site, and chromophore. , 1995, Biochemistry.

[8]  J. A. Gavira,et al.  Combining Counter-Diffusion and Microseeding to Increase the Success Rate in Protein Crystallization , 2011 .

[9]  J. Luft,et al.  A method to produce microseed stock for use in the crystallization of biological macromolecules. , 1999, Acta crystallographica. Section D, Biological crystallography.

[10]  Jessica D. Schiffman,et al.  Graphene-based microfluidics for serial crystallography. , 2016, Lab on a chip.

[11]  Wilhelm Pfleging,et al.  Microfluidic chips for the crystallization of biomacromolecules by counter-diffusion and on-chip crystal X-ray analysis. , 2009, Lab on a chip.

[12]  N. Shimizu,et al.  Preparation of Large Crystals of Photoactive Yellow Protein for Neutron Diffraction and High Resolution Crystal Structure Analysis † , 2007, Photochemistry and photobiology.

[13]  D. Hekmat.  Large-scale crystallization of proteins for purification and formulation , 2015, Bioprocess and Biosystems Engineering.

[14]  N. Chayen,et al.  Random Microseeding: A Theoretical and Practical Exploration of Seed Stability and Seeding Techniques for Successful Protein Crystallization , 2011 .

[15]  Garth J. Williams,et al.  From Macrocrystals to Microcrystals: A Strategy for Membrane Protein Serial Crystallography. , 2017, Structure.

[16]  N. Schormann,et al.  Crystallization and preliminary X-ray diffraction analysis of three recombinant mutants of Vaccinia virus uracil DNA glycosylase. , 2013, Acta crystallographica. Section F, Structural biology and crystallization communications.

[17]  A. Puustinen,et al.  Purification, crystallization and preliminary crystallographic studies of an integral membrane protein, cytochrome bo3 ubiquinol oxidase from Escherichia coli. , 2000, Acta crystallographica. Section D, Biological crystallography.

[18]  R. Gennis,et al.  The quinone-binding sites of the cytochrome bo3 ubiquinol oxidase from Escherichia coli. , 2010, Biochimica et biophysica acta.

[19]  Neer Asherie,et al.  Protein crystallization and phase diagrams. , 2004, Methods.

[20]  Bernhard Rupp,et al.  Approaches to automated protein crystal harvesting. , 2014, Acta crystallographica. Section F, Structural biology communications.

[21]  A. Margolin,et al.  Protein crystals for the delivery of biopharmaceuticals , 2004, Expert opinion on biological therapy.

[22]  J. A. Gavira,et al.  Heterogeneous Crystallization of Proteins: Is it a Prenucleation Clusters Mediated Process? , 2013 .

[23]  R. Gennis,et al.  X-ray transparent microfluidic chips for high-throughput screening and optimization of in meso membrane protein crystallization. , 2017, Biomicrofluidics.

[24]  P. Kenis,et al.  X-ray Transparent Microfluidic Chip for Mesophase-Based Crystallization of Membrane Proteins and On-Chip Structure Determination , 2014, Crystal growth & design.

[25]  J. Drenth,et al.  Nucleation in protein crystallization. , 1998, Acta crystallographica. Section D, Biological crystallography.

[26]  Claude Sauter,et al.  ChipX: A Novel Microfluidic Chip for Counter-Diffusion Crystallization of Biomolecules and in Situ Crystal Analysis at Room Temperature , 2013 .

[27]  G. Gilliland,et al.  Promoting crystallization of antibody–antigen complexes via microseed matrix screening , 2010, Acta crystallographica. Section D, Biological crystallography.

[28]  S. Quake,et al.  Monolithic microfabricated valves and pumps by multilayer soft lithography. , 2000, Science.

[29]  Zhongqiang Yang,et al.  Heterogeneous nucleants for crystallogenesis and bioseparation , 2015 .

[30]  Elizabeth M. Horstman,et al.  Crystallization Optimization of Pharmaceutical Solid Forms with X-ray Compatible Microfluidic Platforms , 2015 .

[31]  A. Kuglstatter,et al.  Acoustic matrix microseeding: improving protein crystal growth with minimal chemical bias. , 2010, Acta crystallographica. Section D, Biological crystallography.

[32]  S. Sainsbury,et al.  Semi-automated microseeding of nanolitre crystallization experiments. , 2008, Acta crystallographica. Section F, Structural biology and crystallization communications.

[33]  Naomi E Chayen,et al.  Turning protein crystallisation from an art into a science. , 2004, Current opinion in structural biology.

[34]  A. D'arcy,et al.  An automated microseed matrix-screening method for protein crystallization. , 2007, Acta crystallographica. Section D, Biological crystallography.

[35]  P. Nollert,et al.  A plug-based microfluidic system for dispensing lipidic cubic phase (LCP) material validated by crystallizing membrane proteins in lipidic mesophases , 2010, Microfluidics and nanofluidics.

[36]  Microseeding – A Powerful Tool for Crystallizing Proteins Complexed with Hydrolyzable Substrates , 2008, International journal of molecular sciences.

[37]  G. Gilliland,et al.  Protein crystallization with microseed matrix screening: application to human germline antibody Fabs , 2014, Acta crystallographica. Section F, Structural biology communications.

[38]  Rustem F Ismagilov,et al.  A droplet-based, composite PDMS/glass capillary microfluidic system for evaluating protein crystallization conditions by microbatch and vapor-diffusion methods with on-chip X-ray diffraction. , 2004, Angewandte Chemie.

[39]  C. Oloman,et al.  Development of a continuous reactor for the electro-reduction of carbon dioxide to formate – Part 1: Process variables , 2006 .

[40]  Naomi E Chayen,et al.  Separating nucleation and growth in protein crystallization using dynamic light scattering. , 2002, Acta crystallographica. Section D, Biological crystallography.

[41]  S. Eom,et al.  Crystallization and preliminary X-ray crystallographic analysis of sterol transcription factor Upc2 from Saccharomyces cerevisiae. , 2013, Acta crystallographica. Section F, Structural biology and crystallization communications.

[42]  K. Volz,et al.  Crystallization of wild-type and mutant ferricytochromes c at low ionic strength: seeding technique and X-ray diffraction analysis. , 1994, Acta crystallographica. Section D, Biological crystallography.

[43]  Jan Drenth,et al.  Understanding protein crystallization on the basis of the phase diagram , 1999 .

[44]  E. Pozharski,et al.  The role of bias in crystallization conditions in automated microseeding. , 2008, Acta crystallographica. Section D, Biological crystallography.

[45]  T. Boggon,et al.  Structure-guided studies of the SHP-1/JAK1 interaction provide new insights into phosphatase catalytic domain substrate recognition. , 2013, Journal of structural biology.

[46]  Wei Liu,et al.  Preparation of microcrystals in lipidic cubic phase for serial femtosecond crystallography , 2014, Nature Protocols.

[47]  S. Quake,et al.  A robust and scalable microfluidic metering method that allows protein crystal growth by free interface diffusion , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[48]  Garth J. Williams,et al.  Low-Z polymer sample supports for fixed-target serial femtosecond X-ray crystallography , 2015 .

[49]  A. D'arcy,et al.  Microseed matrix screening for optimization in protein crystallization: what have we learned? , 2014, Acta crystallographica. Section F, Structural biology communications.

[50]  Methods : A Companion to Methods in Enzymology , 2022 .

[51]  E. Garman,et al.  Structure of arylamine N-acetyltransferase from Mycobacterium tuberculosis determined by cross-seeding with the homologous protein from M. marinum: triumph over adversity. , 2013, Acta crystallographica. Section D, Biological crystallography.

[52]  D. Yin,et al.  Sensitivity of lysozyme crystallization to minute variations in concentration. , 2012, Acta crystallographica. Section D, Biological crystallography.

[53]  K. Takano,et al.  Spatially Precise, Soft Microseeding of Single Protein Crystals by Femtosecond Laser Ablation , 2012 .

[54]  D. Yin,et al.  Uncertainties in crystallization of hen‐egg white lysozyme: reproducibility issue , 2008 .

[55]  Jane M. Shaw,et al.  Micromachining applications of a high resolution ultrathick photoresist , 1995 .

[56]  G. Whitesides,et al.  Rapid Prototyping of Microfluidic Systems in Poly(dimethylsiloxane). , 1998, Analytical chemistry.

[57]  Rustem F Ismagilov,et al.  Time-controlled microfluidic seeding in nL-volume droplets to separate nucleation and growth stages of protein crystallization. , 2006, Angewandte Chemie.

[58]  B. Stoddard,et al.  Microseed matrix screening to improve crystals of yeast cytosine deaminase. , 2004, Acta crystallographica. Section D, Biological crystallography.

[59]  K. Hellingwerf,et al.  The xanthopsins: a new family of eubacterial blue‐light photoreceptors. , 1996, The EMBO journal.

[60]  Beyond the International Year of Crystallography , 2015 .

[61]  E. Pardon,et al.  Combining in-situ proteolysis and microseed matrix screening to promote crystallization of PrPc-nanobody complexes. , 2011, Protein engineering, design & selection : PEDS.