Nonlinear Optical Imaging Methods for Protein Crystal Detection and Characterization
Abstract
Rational drug design is largely dependent on the successful generation of high-resolution protein structures. Synchrotron X-ray diffraction is the most widely used approach for generating these high resolution structures of macromolecules. These facilities are increasingly moving towards fully automated crystal analysis and the target protein crystals are becoming smaller and the demand for user time is also increasing. As such there is a need for techniques to decrease time, cost, and crystal size necessary along all steps of the crystallography pipeline, from crystallization to diffraction. Nonlinear optical (NLO) methods such as second harmonic generation (SHG) and two-photon excited ultraviolet excited fluorescence (TPE-UVF) have demonstrated the ability to provide rapid, non-destructive detection and characterization of protein crystals. From identifying crystals in vivo to selectively and automatically assessing crystal quality, SHG in combination with SHG active dyes or covalently labeled proteins, has emerged as a rapid and reliable way for finding protein crystals. Furthering the capabilities of NLO methods is the ability to quickly and accurately characterize multicomponent samples and differentiate between crystalline or amorphous material through the use of hyperspectral imaging with SHG and two-photon excited fluorescence While NLO imaging methods are in play for the initial detection and characterization of protein crystals and allow for increasingly smaller crystals to be collected, another key step in the pipeline is the accurate positioning of the sample prior to diffraction analysis. An adaptation of X-ray raster scanning, in which dynamic sampling greatly reduces the X-ray exposure of the crystal prior to data collection has been implemented at a beamline further increasing the odds of successful diffraction and protein structure determination.
Degree
Ph.D.
Advisors
Simpson, Purdue University.
Subject Area
Analytical chemistry
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