| dc.description.abstract | In the process of recombinant protein production, we often get inactive and insoluble inclusion bodies instead of target proteins due to high peptide concentration. In industrial application, denaturants such as urea or guanidine hydrochloride are used to solve these inclusion bodies to renature and purify/separate proteins and then denaturants are removed to get the protein we want.
Factors to stabilize protein structures include hydrophobic interactions, hydrogen bonding, electrostatic interaction and covalent disulfide bonding. Urea is mainly used to break hydrophobic interactions and guanidine hydrochloride is to destroy hydrophobic and electrostatic interactions. Sometimes, salts such as sodium chloride/lithium chloride are added in urea as electrostatic interaction destroyer to enhance the denaturant power. Another commonly used reduced reagent (dithiothreitol, DTT) is to reduce disulfide bonding within protein. All these different mechanisms bring various influences to protein structures.
In our study, we examine secondary and tertiary structure changes of lysozyme in various conditions (different concentrations of urea / guanidine hydrochloride / salts (sodium chloride), experimental temperature, with/without reduced reagent) with the aid of circular dichroism and fluorescence spectrophotometer to investigate the relationship between above factors and spectrum structure.
When reduced reagent exists, guanidine hydrochloride causes greater changes in protein structure than urea and sodium chloride. Without reduced reagent, only high concentration of guanidine chloride causes significant protein structure changes. The results of fluorescence experiments indicates that tryptophan inside protein didn’’t expose easily when disulfide bond remained unbroken.
Besides, from temperature manipulation experiments, we see the denature reaction with denaturants and reducing agents added is an exothermic reaction. At the mean time, the entropy of system decreases and melting temperature (Tm) is lower than that in buffer solution.
From the above experiments, we know that if the disulfide bond is not reduced, we have to use denaturant with the capacity to break hydrophobic and electrostatic interactions in high concentration to induce significant changes in lysozyme structure. When disulfide bond is reduced, denaturants causes changes of lysozyme structure easier that indicate lysozyme is a hard protein itself.
It’s possible to investigate the relationship between activity and structure of enzymes with the aid of ultraviolet spectrometer. In further studies, comparing reaction enthalpy in various conditions measured by isothermal titration calorimeter with thermodynamic data from spectrometer, we can learn the pathway of lysozyme denature process to see it’s two state or multi-state phase transformation reaction. | en_US |