The project will be executed through two main experimental pathways. Both experimental setups utilize FRET to visualize the conformational change that is the result of the activation of the SNF1 protein complex. The first approach consists of creating a fusion protein consisting of the SNF1 protein and two fluorescent proteins, namely EYFP and ECFP. The idea is that when the protein is activated it undergoes a conformational change and a FRET-signal will be visible. The second approach utilizes a SAMS-peptide with fluorescent proteins fused to each end. The SAMS-peptide will be phosphorylated by the active SNF1-complex and will undergo a conformational change that will be visible due to the fluorescent tags.
To reach the project goal several procedures need to be performed. Firstly the most suitable positions of the fluorescent proteins need to be determined. This will be done by studying parts of the crystal structure of the protein complex. The distances between the fluorophores are measured to secure that they are optimal with respect to the intrinsic properties of the flurophore pair. After this the fusion protein genome will be synthesized through fusion PCR. The fusion protein genome is then inserted into the yeast plasmid pSGM1 with the aid of several restriction endonucelases. The plasmids will be amplified in E. coli after which they are sent to be sequenced to make sure that the correct insert has been created. The last step is to transform a yeast strain with the plasmids, expose the cells to different stress factors and study the FRET signal. The FRET-signal will be used to quantify the expression level of the SNF1 complex.
A secondary task is to measure the expression levels of the SNF1 complex through western blot with probes that only bind to the phosphorylated (active) protein. These levels will be compared to the expression levels derived through the FRET-analysis. Our expectation is to find a linear correlation between both measurements.
The long term ambition of this project it is to ultimately use the results in the pharmaceutical industry when performing high-throughput screening for new substances or finding the correct drug concentrations to use. The yeast cells with the modified SNF-complex can be moved through a micro-fluidic system, gradually exposing them to an array of substances or a concentration gradient and easily finding out at which concentration or substance that the cells are stressed. |
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