Zauraiz Syeda
Mentor: Susan White
Chemistry Department, Bryn Mawr College

Stability of the L30e WT and Mutant Proteins

The Saccharomyces cerevisiae (yeast) ribosomal protein L30e can regulate its own gene splicing and translation by binding to pre-mRNA.  A kink turn motif in the structure of the RNA is needed in order for the protein to bind.  At the binding site, the protein has specific amino acids that interact with the RNA. Changing these amino acids to create mutated proteins can significantly impact the thermal stability of the protein as well as its ability to form an RNA complex. Mutants will be used in this experiment to make sure that the overall structure of the protein is not affected.

Differential scanning fluorimetry (DSF) will be used to measure the thermal stability of the mutated proteins (K28A, F85A, and F85W), which will be compared to the stability of the wild type L30e protein. Similar thermal stabilities will confirm that each mutation does not significantly change the rest of the protein structure.  Since these amino acids are involved in binding to the RNA, they are on the outside of the L30e protein, suggesting that there will be little structural adjustment. A thermal cycler instrument will use temperature to denature the protein, and, as it unfolds, a hydrophobic fluorescent dye will be able to bind to it. The increasing intensity of fluorescence will show the unfolding of the protein. This thermal denaturation experiment will determine the stability of each mutant.