High resolution transmission electron microscopy of green fluorescent protein


High resolution transmission electron microscopy of green fluorescent protein

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Title: High resolution transmission electron microscopy of green fluorescent protein
Author: Knobles, Micah
Abstract: Using fluorescence and transmission electron microscopy (TEM), this study investigated the structure of a novel recombinant protein used in tagging molecules. This molecule, green fluorescent protein (GFP) exhibited unexpected aggregation phenomena. The fluorescence, an indicator of protein integrity, could be used to test exposure to conditions of the electron microscope. Negative staining with uranyl acetate was explored to demonstrate how TEM might be employed to observe biological samples with high resolution. Extensive circumstantial evidence suggests that discrete GFP molecules were resolved. This study demonstrated that protein structure can be observed with relatively simple negative staining techniques using uranyl acetate. It was determined that the electron beam was primarily responsible for the degradation of protein structure. Crystal formations with uranyl acetate and GFP produced highly ordered monolayers and complex aggregates. These aided in the analysis of the TEM images. Low dose electron beam exposure and the uranyl acetate stain likely protected the GFP sample. His tag labeling of the recombinant GFP sample with nickel-nitrilotriacetic acid Nanogold (Ni-NTA-Nanogold) was used to definitively identify GFP molecules with inconclusive results. Our findings indicate a newfound use of TEM and negative staining in high resolution transmission electron microcopy studies of globular proteins, biological polymers, and small molecules. With further refinements, these techniques would offer a valuable addition to the contributions of cryo-electron microscopy and X-ray crystallography. The work may help us to interpret complex aggregation phenomena and to increase our understanding of amyloid and prion diseases whose insoluble protein aggregates consisting of tightly packed beta sheets are not amenable to conventional structural biology techniques.
Department: Biological Sciences, School of
Subject: College of Natural Sciences transmission electron microscopy proteins green fluorescent protein uranyl acetate
URI: http://hdl.handle.net/2152/13391
Date: 2009

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