Membrane proteins represent among the most challenging protein targets for structure determination and yet also the most important for human health. Major emerging advances in X-ray sources, including the use of X-ray free electron laser (XFEL) diffraction, tightly focused synchrotron XRD, and improvements in electron diffraction (ED) instrumentation have inexorably reduced the crystal sizes required for structure determination into the protein nanocrystal regime. These trends are increasingly leading to a major bottleneck in protein nanocrystal screening, the importance of which will only increase as these new diffraction capabilities continue to advance. The primary objective of the proposed effort will be to develop measurement tools based on nonlinear optical interactions from ultrafast laser sources for selectively screening protein nanocrystal formation. Three key novel approaches will be developed. In the first, polarization-dependent TPE-UVF will be developed and validated as a complement to conventional SONICC for the detection of immobile protein crystals, such as those encountered in lipidic mesophases and under cryogenic conditions. Ordered crystalline domains produce polarization-dependent TPE-UVF patterns distinct from aggregates and solvated proteins. In the second, SHG and TPE-UVF autocorrelation methods will be developed for improving the detection limits of diffusing nanocrystals, targeting 2D and 3D nanocrystals generated from aqueous solutions. Autocorrelation relies on analysis of the combined weak responses from many nanocrystals for signal to noise enhancement, while simultaneously providing information on translational diffusion. Finally, nonlinear optical cross-correlation spectroscopy methods will be advanced using polarization-dependent SHG and TPE-UVF for the selective detection of protein nanocrystal rotational dynamics. The correlations within the polarization-dependence of TPE-UVF will preserve crystal-specific detection by both TPE-UVF and SONICC for freely diffusing nanocrystals. Validation of the proposed approaches will be performed using a combination of focused XRD, XFEL diffraction, and ED, with diverse and representative membrane protein nanocrystal samples provided through collaboration with Vadim Cherezov (Scripps) for lipidic mesophase crystallizations, Petra Fromme (U. Arizona) for crystals amenable to XFEL analysis, and David Stokes (NYU) for 2D nanocrystals for structure determination by ED. Combined SONICC and synchrotron XRD measurements will be performed at the APS using a prototype capable of combined SHG imaging and focused """"""""minibeam"""""""" XRD. Although the primary purposes of the present studies are to fundamentally advance membrane protein nanocrystal detection, close collaboration with Formulatrix will help lower the barriers to rapidly transition he findings of these inquiries to high-throughput commercial platforms for routine protein nanocrystal detection should the proposed efforts prove successful.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Edmonds, Charles G
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Purdue University
Schools of Arts and Sciences
West Lafayette
United States
Zip Code
Zhang, Shijie; Song, Zhengtian; Godaliyadda, G M Dilshan P et al. (2018) Dynamic Sparse Sampling for Confocal Raman Microscopy. Anal Chem 90:4461-4469
Scarborough, Nicole M; Godaliyadda, G M Dilshan P; Ye, Dong Hye et al. (2017) Dynamic X-ray diffraction sampling for protein crystal positioning. J Synchrotron Radiat 24:188-195
Ding, Changqin; Ulcickas, James R W; Deng, Fengyuan et al. (2017) Second Harmonic Generation of Unpolarized Light. Phys Rev Lett 119:193901
Scarborough, Nicole M; Godaliyadda, G M Dilshan P; Ye, Dong Hye et al. (2017) Synchrotron X-Ray Diffraction Dynamic Sampling for Protein Crystal Centering. IS&T Int Symp Electron Imaging 2017:6-9
Dow, Ximeng Y; Dettmar, Christopher M; DeWalt, Emma L et al. (2016) Second harmonic generation correlation spectroscopy for characterizing translationally diffusing protein nanocrystals. Acta Crystallogr D Struct Biol 72:849-59
Newman, Justin A; Zhang, Shijie; Sullivan, Shane Z et al. (2016) Guiding synchrotron X-ray diffraction by multimodal video-rate protein crystal imaging. J Synchrotron Radiat 23:959-65
Newman, Justin A; Simpson, Garth J (2016) Nonlinear Optical Characterization of Membrane Protein Microcrystals and Nanocrystals. Adv Exp Med Biol 922:91-103
Dow, Ximeng Y; DeWalt, Emma L; Newman, Justin A et al. (2016) Unified Theory for Polarization Analysis in Second Harmonic and Sum Frequency Microscopy. Biophys J 111:1553-1568
Simpson, Garth J (2016) Connection of Jones and Mueller Tensors in Second Harmonic Generation and Multi-Photon Fluorescence Measurements. J Phys Chem B 120:3281-302
Deng, Fengyuan; Ulcickas, James R W; Simpson, Garth J (2016) Theoretical Foundation for Electric-Dipole-Allowed Chiral-Specific Fluorescence Optical Rotary Dispersion (F-ORD) from Interfacial Assemblies. J Phys Chem Lett 7:4248-4252

Showing the most recent 10 out of 23 publications