Folding and unfolding of a non-fluorescent mutant of green fluorescent protein.

Journal of Physics. Condensed Matter : an Institute of Physics Journal
Beata Wielgus KutrowskaPatricia L Clark

Abstract

Green fluorescent protein (GFP), from the Pacific jellyfish A. victoria, has numerous uses in biotechnology and cell and molecular biology as a protein marker because of its specific chromophore, which is spontaneously created after proper protein folding. After formation, the chromophore is very stable and remains intact during protein unfolding, meaning that the GFP unfolding process is not the reverse of the original folding reaction; i.e., the principles of microscopic reversibility do not apply. We have generated the mutant S65T/G67A-GFP, which is unable to form the cyclic chromophore, with the goal of investigating the folding, unfolding and competing aggregation of GFP under fully reversible conditions. Our studies have been performed in the presence of GdnHCl. The GFP conformation was monitored using intrinsic tryptophan fluorescence, and fluorescence of bis-ANS. Light scattering was used to follow GFP aggregation. We conclude from these fluorescence measurements, that S65T/G67A-GFP folding is largely reversible. During equilibrium folding, the first step is formation of molten globule, prone to aggregation.

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Citations

Nov 7, 2009·Nature Nanotechnology·Thomas M HermansE W Meijer
Aug 21, 2008·Proceedings of the National Academy of Sciences of the United States of America·Benjamin T AndrewsPatricia A Jennings
Nov 13, 2012·PloS One·Olesya V StepanenkoKonstantin K Turoverov
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Mar 27, 2020·International Journal of Biological Macromolecules·Gargi BiswasRahul Banerjee

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