Entanglements Present in Protein Sub-Populations Impact the Protein's Structure and Function

Open Access
- Author:
- Berek, Taylor
- Area of Honors:
- Chemistry
- Degree:
- Bachelor of Science
- Document Type:
- Thesis
- Thesis Supervisors:
- Edward Patrick O'Brien, Jr., Thesis Supervisor
David D Boehr, Thesis Honors Advisor
C Denise Okafor, Faculty Reader - Keywords:
- Proteins
entanglements
synonymous mutations - Abstract:
- This thesis investigates how the existence of near-native like misfolded states influence the overall specific activity and structural stability of a protein population. The rate of co-translational folding is influenced by synonymous mutations, which can make certain proteins more prone to co-translational entanglements. In the protein D-Alannine D-Alannine Ligase B and Hexokinase, computational analysis has identified synonymous mutations with high probabilities of entanglements. This was proven experimentally, by growing genetically engineered cultures, isolating the protein of interest, and measuring the activity. For each case, a wildtype protein is compared to mutants with both faster and slower rates of co-translational folding. The fast mutant repeatedly showed lower enzymatic function than the slower mutant, proving that co-translational folding rate can impact a proteins function. The third protein, pyruvate kinase, was exposed to a denaturing environment where it was forced to unfold and then later refold. When compared to the native state, the refolded protein had similar enzyme substrate binding affinity with a maximal reaction rate less than half than that of the native state. The results of the pyruvate kinase refolding assay, along with the D-alannine D-alannine ligase B and hexokinase study, prove that a sub-population of misfolded proteins exists in the cell, and is similar enough to the native state to function, but not as efficiently as the native state.