Nonlinear optical imaging of membrane protein crystals
Abstract
Protein structure determination is a key step in developing molecular-level understandings of the role of proteins in cell signaling pathways, which in turn can guide understanding of diseases and the rational design of potential drugs for treatment. High-resolution structures of relatively large proteins are generally obtained by X-ray diffraction from protein crystals. Consequently, identification of the conditions amenable to the formation of diffraction-quality protein crystals remains a major bottleneck in the process of determining protein structures. In this thesis work, two novel methodologies based on second harmonic generation and intrinsic visible luminescence were developed for detecting protein crystals in traditional ex-vivo crystallization platforms and in more complex matrices such as in lipidic mesophase and in living cells. In order to facilitate the screening of numerous crystallization trials, a high speed laser scanning nonlinear optical instrument was designed and built with five independent channels of detection (brightfield, two-photon excited fluorescence, epi generated SHG, and crossed and coparallel polarized transmission generated SHG) for protein crystal imaging. Fast (MHz) polarization capabilities were also built into the instrument to allow for the assessment of crystal orientations, polymorphs and quality through the polarization dependent SHG response.
Degree
Ph.D.
Advisors
Simpson, Purdue University.
Subject Area
Biochemistry
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