Protein characterization and crystal structure of yellow thermostable protein (YTP) Q66E E148D
Discipline: Chemistry and Chemical Sciences
Subcategory: Biochemistry (not Cell and Molecular Biology and Genetics)
Session: 4
Room: Woodley Park
Victoria Ogbeifun - Missouri State University
Co-Author(s): Caitlin M. Padgett, Missouri State University, Springfield, MO; Natasha DeVore, Missouri State University, Springfield, MO
Fluorescent proteins have become popular tools in many research areas. They are mostly used for the visualization of structures and processes in living cells. Green fluorescent protein (GFP) was isolated from the Aequorea victoria jellyfish by scientists in the 1960’s. To further the stability and range of usefulness of GFP, several different mutations have been carried out. Some mutations have been implemented to produce different colors of fluorescence such as blue, yellow, and cyan. The various colors are needed to visualize varying processes at different wavelengths. Thermo-green protein (TGP) was engineered to withstand harsher temperature conditions compared to GFP. Further mutations are constantly being performed to create better versions of these proteins. This poses the question of whether a better fluorescing protein that can withstand harsh temperature conditions can be synthesized by the introduction of certain mutations. The goal of this project is to examine the stability of the thermostable fluorescent protein YTP-E-D. The protein is a mutated version of the yellow thermostable protein (YTP). The color change from green to yellow was accomplished by altering a histidine that is located near the chromophore at the 197 location into a tyrosine (H197Y). Site-directed mutagenesis was then utilized to conduct the following mutations Q66E and E148D. YTP-E-D did express at a higher level than YTP and YTP-E and had less of a lower molecular weight contaminant during purification. Protein crystals of YTP-E-D were superior to those obtained for YTP and YTP-E and led to two protein crystal structures solved with x-ray diffraction to 2.6 Å and the other to 3.0 Å resolution. The structure was solved with molecular replacement using monomeric Azami Green as a search model. The model of YTP-E-D was built with iterative steps of refinement and model building. These structures give insight into how to further improve yellow thermostable protein. Despite crystallizing better, YTP-E-D had a significantly lower quantum yield than its counterpart YTP-E. This study focused on evaluating the stability of the YTP-E-D by performing several assays. The pH assay revealed a curve that showed about 50% fluorescence at pH 7, with a maximum fluorescence at pH 10. The chemical stability of YTP-E-D had a Cm of 1.9 M Guanidium Hydrochloride, which was not as stable as YTP-E. Likewise, the thermal stability of YTP-E-D is not as good as that of YTP-E or YTP, but better than the corresponding green TGP and TGP-E. Future research involves utilizing the information from the structure to make a better yellow thermostable protein with site-directed mutagenesis.
Funder Acknowledgement(s): I would like to thank Dr. Tayo Obafemi-Ajayi for her work in the LSAMP program and her dedication to helping students succeed in STEM. I would also like to thank Dr. DeVore for all her continuous help, support, and guidance not only in my research but throughout my entire undergraduate career.
Faculty Advisor: Natasha DeVore, NDeVore@MissouriState.edu
Role: For this project, I grew the protein crystals and used the crystals for X-ray diffraction. I was also able to obtain the crystal structures for the specific protein. I also completed all of the assays that evaluate the stability of the protein. The pH assay, thermo-stability assay, and the chemical stability assay. All of these assays were able to help determine the characteristics that the protein has. In addition, I will be solving the second structure using molecular replacement to build an online model of the protein.