This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. While efforts in microfluidics for protein crystallization have developed dramatically in the past ten years[1-9] the idea of coupling microfluidic technology with advanced crystallographic techniques for in situ analysis is an area where significant advances can be made. Thus far efforts have focused mainly on either simple devices where monochromatic X-rays can be used to interrogate the sample.[2 4 5 10-26] However this approach has been of limited utility because of the need to rotate samples in order to fully sample phase space. An alternative to the more traditional monochromatic X-ray analysis is a polychromatic method termed Laue crystallography.[27] In this method the use of a range of X-ray wavelengths allows for significantly faster data collection enabling the analysis of tiny or fragile crystals that are susceptible to radiation damage[28 29] as well as kinetic experiments.[30] Because of fast data collection it is possible to perform time-resolved experiments by matching the X-ray exposure time to the timescale of chemical and structural changes in the protein. Thus Laue crystallography provides a very elegant platform for performing extremely meaningful kinetic experiments that directly probe changes that occur during enzymatic function. Microfluidic platforms have the benefit of not only enabling experiments at very small volumes but also by creating an environment free of inertial or convective effects and allowing for exquisite control over local conditions and gradients. Here we propose coupling microfluidic platforms for protein crystallization with in situ Laue crystallographic analysis. By combining the control and throughput of microfluidic platforms with this powerful structural analysis method we hope to further enable the field of dynamic crystallographic analysis. Experimentally we need to first validate our platforms for use with Laue analyses in terms of signal to noise rotational requirements visualization and sample handling. A second proof-of-concept effort would involve the structure determination of a model protein either from a single crystal or from an array of microcrystals. Subsequent efforts will involve creating an array of crystals that have been exposed to a range of conditions. For instance the array of crystals could be exposed to a range of ligand molecule concentrations or pH values. More advanced studies will involve coupling real-time fluid handling with in situ analysis for dynamic crystallization experiments. For example a chamber containing a crystal could be controllably exposed to a second chamber containing a chemical of interest. Time resolved structural data could be collected as the chemical of interest diffuses into the crystal.
| Weingarten, Adam S; Dannenhoffer, Adam J; Kazantsev, Roman V et al. (2018) Chromophore Dipole Directs Morphology and Photocatalytic Hydrogen Generation. J Am Chem Soc 140:4965-4968 |
| Yang, Cheolhee; Choi, Minseo; Kim, Jong Goo et al. (2018) Protein Structural Dynamics of Wild-Type and Mutant Homodimeric Hemoglobin Studied by Time-Resolved X-Ray Solution Scattering. Int J Mol Sci 19: |
| Kazantsev, Roman V; Dannenhoffer, Adam J; Weingarten, Adam S et al. (2017) Crystal-Phase Transitions and Photocatalysis in Supramolecular Scaffolds. J Am Chem Soc 139:6120-6127 |
| Fournier, Bertrand; Sokolow, Jesse; Coppens, Philip (2016) Analysis of multicrystal pump-probe data sets. II. Scaling of ratio data sets. Acta Crystallogr A Found Adv 72:250-60 |
| Cho, Hyun Sun; Schotte, Friedrich; Dashdorj, Naranbaatar et al. (2016) Picosecond Photobiology: Watching a Signaling Protein Function in Real Time via Time-Resolved Small- and Wide-Angle X-ray Scattering. J Am Chem Soc 138:8815-23 |
| Pande, Kanupriya; Hutchison, Christopher D M; Groenhof, Gerrit et al. (2016) Femtosecond structural dynamics drives the trans/cis isomerization in photoactive yellow protein. Science 352:725-9 |
| Weingarten, Adam S; Kazantsev, Roman V; Palmer, Liam C et al. (2015) Supramolecular Packing Controls H? Photocatalysis in Chromophore Amphiphile Hydrogels. J Am Chem Soc 137:15241-6 |
| Pfoh, Roland; Pai, Emil F; Saridakis, Vivian (2015) Nicotinamide mononucleotide adenylyltransferase displays alternate binding modes for nicotinamide nucleotides. Acta Crystallogr D Biol Crystallogr 71:2032-9 |
| Mariette, Céline; Guérin, Laurent; Rabiller, Philippe et al. (2015) The creation of modulated monoclinic aperiodic composites in n-alkane/urea compounds. Z Kristallogr Cryst Mater 230:5-11 |
| Yang, Xiaojing; Stojkovi?, Emina A; Ozarowski, Wesley B et al. (2015) Light Signaling Mechanism of Two Tandem Bacteriophytochromes. Structure 23:1179-89 |
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