Structural Engineering of Thermostable Fluorescent Proteins TGP-E and YTP-E and Crystal Structure of TGP-E
Date of Graduation
Master of Science in Chemistry
Chemistry and Biochemistry
Thermostable fluorescent proteins, such as thermal green protein (TGP) and yellow thermal protein (YTP), could be used as biosensors to monitor cellular activity and as a fusion tag to monitor a protein of interest. The use of fluorescent proteins can sometimes be limited in certain organelles with low pH and in thermophilic organisms. This research aims to improve the thermal stability of TGP and YTP. TGP was created from a synthetically derived eCGP123 protein to improve solubility by substituting residues on the positively charged b- barrel surface with negatively charged glutamate (E) at Los Alamos National Lab. YTP was developed by mutating a histidine residue located under the chromophore into a tyrosine (H193Y) by the DeVore lab. The properties in which we are targeting improvement are thermal stability, pH stability, overall fluorescence, and the ability to withstand extreme conditions. To accomplish this overall improvement, we altered the chromophore using sited-directed mutagenesis to mutate glutamine 66 to glutamate in both YTP and TGP proteins. The YTP-E protein improved the thermal stability and the overall pH stability compared to TGP-E and YTP. A TGP-E protein crystal structure was obtained with a resolution of 2 Å which gave us insight that there is an additional hydrogen bond formed between Glu 66 and the backbone. The conserved hydrogen bond became shorter than in TGP, making TGP-E more stable - which explains the changes to the protein’s properties.
thermostable, fluorescent, protein, GFP, TGP, X-ray diffract, stability
© Matthew Ryan Anderson
Anderson, Matthew Ryan, "Structural Engineering of Thermostable Fluorescent Proteins TGP-E and YTP-E and Crystal Structure of TGP-E" (2023). MSU Graduate Theses. 3828.