Protein structure dictates function. Despite numerous advances in the development of high-throughput and ultrahigh-throughput platforms for protein crystallization screening for structure determination, major challenges remain including: 1) generating sufficient quantities of purified protein for analysis, 2) screening of a multitude of possible crystallization conditions, 3) identifying and isolating high-quality crystals for x-ray diffraction analysis, and 4) accurate positioning of crystals less than ~ 5 ?m in dimension prior to X-ray diffraction measurements. The labor-intensive process of generating purified protein for crystallization screening often limits the number of conditions that can be assayed. Furthermore, few reliable on-site methods are currently available for rapidly and nondestructively assessing protein crystal quality and the likelihood of achieving high-resolution structures from diffraction measurements, such that the generation of high-resolution structures often requires multiple rounds of trial and error analysis on candidate crystals. For small crystals (<~5 ?m), simply identifying the locations of the crystals for diffraction measurements at synchrotron sources is nontrivial. These bottlenecks can potentially be addressed in part through the proposed development of second order nonlinear optical imaging of chiral crystals (SONICC) for highly selective detection of incipient crystal formation and initial assessment of crystal quality. Second harmonic generation is a coherent nonlinear optical technique that disappears by symmetry in randomly oriented assemblies and in most achiral materials, but is bulk-allowed for the overwhelming majority of chiral crystals, including those of proteins. We proposed the development of instrumentation and methods for SONICC detection and analysis of <5 ?m protein crystals. If successful, these proposed techniques have the potential to enable routine diffraction analysis of crystals ~1 ?m in dimension or smaller, through early detection of crystal formation, initial all-optical assessment of anticipated diffraction quality, automated looping of crystal smaller than the optical resolution, and high-fidelity positioning in the synchrotron source for diffraction analysis. Realization of these goals will require the combined efforts of a team of investigators, each with complementary expertise (Figure 1). Validation of SONICC as a general tool for protein crystal detection, characterization, and positioning for diffraction analysis will be assessed through collaborative efforts between the SONICC Team at APS, Das, and Simpson. Once the generality is confirmed, instrumentation utilizing SONICC for predicting diffraction quality from multiple-angle nonlinear optical imaging will be constructed based on a Bruker CrystalHarvester platform through collaboration between Bruker AXS, the Jonathon Amy Facility for Chemical Instrumentation (JAFCI), and Simpson. Development of ultrahigh-throughput crystallization screening platforms will also be concurrently pursued by Qi and Simpson, taking advantage of unique microfabrication resources available in the Birck Nanotechnology Center.

Public Health Relevance

Second-order nonlinear optical imaging of chiral crystals (SONICC) will be explored as a general, sensitive and highly selective detection approach for protein crystal formation. If the proposed project is successful in achieving the Specific Aims, SONICC has the potential to directly address key bottlenecks in steps common to most modern protein structure determination efforts, including: 1) rapidly assaying diverse crystallization conditions, 2) prescreening of crystal quality prior to extraction into a loop, 3) looping of crystals smaller than the resolution of the optics, and 4) reliably positioning such small crystals in tightly focused synchrotron X-ray sources for diffraction analysis. An interdisciplinary, multi-institutional team of investigators from Purdue University, Argonne National Laboratories, and Bruker will assess the general applicability of SONICC for routine protein crystal detection, for readout in high-throughput and ultrahigh throughput crystallization screenings, and for integration into larger instrument platforms.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Macromolecular Structure and Function B Study Section (MSFB)
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Wu, Mary Ann
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Purdue University
Schools of Arts and Sciences
West Lafayette
United States
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Sullivan, Shane Z; Muir, Ryan D; Newman, Justin A et al. (2014) High frame-rate multichannel beam-scanning microscopy based on Lissajous trajectories. Opt Express 22:24224-34
Snyder, Gregory R; Chowdhury, Azhad U; Simpson, Garth J (2014) Exciton coupling model for the emergence of second harmonic generation from assemblies of centrosymmetric molecules. J Phys Chem A 118:4301-8
Chowdhury, Azhad U; Dettmar, Christopher M; Sullivan, Shane Z et al. (2014) Kinetic trapping of metastable amino acid polymorphs. J Am Chem Soc 136:2404-12
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Sullivan, Shane Z; Schmitt, Paul D; Muir, Ryan D et al. (2014) Digital deconvolution filter derived from linear discriminant analysis and application for multiphoton fluorescence microscopy. Anal Chem 86:3508-16
Ghorab, Mohamed K; Toth, Scott J; Simpson, Garth J et al. (2014) Water-solid interactions in amorphous maltodextrin-crystalline sucrose binary mixtures. Pharm Dev Technol 19:247-56
Muir, Ryan D; Pogranichney, Nicholas R; Muir, J Lewis et al. (2014) Linear fitting of multi-threshold counting data with a pixel-array detector for spectral X-ray imaging. J Synchrotron Radiat 21:1180-7
Muir, Ryan D; Sullivan, Shane Z; Oglesbee, Robert A et al. (2014) Synchronous digitization for high dynamic range lock-in amplification in beam-scanning microscopy. Rev Sci Instrum 85:033703
Hsu, Hsin-Yun; Toth, Scott J; Simpson, Garth J et al. (2013) Effect of substrates on naproxen-polyvinylpyrrolidone solid dispersions formed via the drop printing technique. J Pharm Sci 102:638-48
Zhu, Qing; Toth, Scott J; Simpson, Garth J et al. (2013) Crystallization and dissolution behavior of naproxen/polyethylene glycol solid dispersions. J Phys Chem B 117:1494-500

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