Quantitative analysis of protein stability or folding kinetics by single detection

• Main Authors: Yi-Kai Liu, 劉奕愷
• Other Authors: Pei-Fen Liu
• Format: Others
• Language: zh-TW
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/91673128531565483323

碩士 === 國立中興大學 === 食品暨應用生物科技學系所 === 105 === Part Ⅰ Abstract Study of protein folding plays important roles in several research fields and provides valuable informations for many researchers. Among these studies, protein stability and protein folding kinetics are critical properties for understanding the protein folding characteristics. New methods were then continuously developed for improving quantitative-analysis and effective-application. However, the experimental strategy used by these methods is complicated and time consuming. Trivial detection is the bottleneck that limits the application of protein folding studies on large-scale experiments pursuing high-throughput and high-precision. In order to improve the efficiency, we developed a “single detection strategy” based on the principle of integral area.This strategy was then applied to two methods including pulse proteolysis and cellular thermal shift assay (CETSA). After optimization of experimental condition, quantitative parameters represent protein folding properties were determined by “single detection strategy” with high accuracy. These results confirm that “single detection strategy” is feasible to be used for measuring protein folding properties. To our surprise, we also found that sirtinol, a known inhibitor for sirtuin 1, shows no binding effect on sirtuin 1 based on our CETSA experiment. This result indicates a possibility that inhibiting effect of sirtinol may come from other mechanisms instead of direct binding. To sum up, we believe that “single detection strategy” is applicable to many methods for studying protein folding. Especially, this strategy will benefit the applications to studies such as drug-target identification, proteomic investigation, and drug screening. Part Ⅱ Abstract Uridine phosphorylase is one of the critical enzymes in the pyrimidine salvage pathway. By incorporating uracil with ribose-1-phosphate, cells regenerate uridine for the nucleotide metabolism. Recently, it was found that Escherichia coli uridine phosphorylase is possibly destabilized in the presence of ATP. However, the mechanism of this destabilization is unclear and the influence of this interaction on the function of uridine phosphorylase is still unknown. Here, we carefully investigated the effect of ATP on the protein folding and function of Escherichia coli uridine phosphorylase. Our results demonstrate that ATP apparently decreases the stability of uridine phosphorylase, and this destabilization is amplified with increased concentration of ATP. Meanwhile, we also found that simply raising the level of ATP leads to the reduction of enzymatic activity to complete inhibition. Further studies indicate that uridine phosphorylase was accumulated as partially unfolded state in the presence of ATP. Moreover, ATP specifically accelerates the unfolding rate of uridine phosphorylase with no observable effect on the refolding regime. In summary, our finding first demonstrates that ATP changes the protein folding and function of an enzyme with apparent destabilization. This interacting mechanism may be significant to some proteins functioning in a condition with high level of ATP, like in cancer cells.