Changes Of Microenvironment Around The Center Of Protein Activity Induced By Chemical Small Molecules

Activity and function of proteins in life depends on its structure and conformation. The conformational structures of proteins are affected by a number of noncovalent interactions such as hydrogen bonds, van der Waals interactions, electrostatic interactions, hydrophilic/hydrophibic effects, and steric interactions of the protein with itself and with solvents, etc. The stability can be perturbed by changing the temperature, pressure, and solvent conditons of the system. One group of components that are significantly important for the stability and function of a proteinare chemical moleculars, including chiemical denaturants and drug moleculars. Therefore, studying on the rules and influence factors of protein folding/unfolding can help to further understand the dependence on conformation and function of proteins, which have significance in relation to further development of biomedicines and the field of safety-engineered drug delivery.In this dissertation, we used glucose oxidase (GOx), human serum albumin (HSA), and sperm whale myoglobin (Mb) as protein models, using electrochemical method, spectroscopic techniques and molecular docking method study the conformational structures and activities of proteins induced by chemical denaturants; the interaction of the anticancer drug (5-fluorouracil (FU)) with the human serum albumin (HSA), and the FU-binding-induced microenvironment alterations in subdomain ⅡA of HSA; the interaction of the photosensitizer (znic phthalocyanines derivatives, ZnPcs) with the human serum albumin (HSA), and the ZnPcs-binding-induced microenvironment alterations in subdomain ⅡA of HSA; the structural alterations around the heme group in sperm whale myoglobin (Mb) induced by solution pH. The main results are as follows:1. The glucose oxidase (GOx) and the guanidinium ion (Gdm+) was used as a protein-small molecular interaction model for studying the conformational structures and activities of proteins induced by chemical denaturants with the purpose of revealing the mechanism of chemical unfolding of proteins. The results demonstrate that Gdm+ions could enter the hydrophobic pocket of GOx molecule and interact with FAD group, leading to significant alteration in the electronic characteristics and hydrogen bond networks formed between FAD and the amino acid residues around the cofactor. These interactions could significantly alter the conformational structure of the enzyme both at secondary and tertiary structure level, resulting to a decrease in the catalytic activity of the enzyme to glucose oxidation.2. We studied the interaction of the anticancer drug,5-fluorouracil (FU), with the human serum albumin (HSA), and the FU-binding-induced microenvironment alterations in subdomain ⅡA of HSA molecule using spectroscopic techniques and molecular docking method with the purpose of understanding the drug-binding-induced microenvironment alterations of proteins. The results demonstrated that FU molecule could enter the inside a hydrophobic cavity of subdomain ⅡA (Sudlow’s site Ⅰ) near Trp 214 amino acid residue with the formation of specific hydrogen bonding with Trp 214 and Lys 199 residues, causing the fluorescence quenching of Trp 214 through a static quenching mechanism. The nature of forces derived binding interaction between HSA and FU molecule were mainly van der Waal’s forces and hydrogen bonding interactions, which were concluded by docking study and analysis of the thermodynamic parameters of the binding process. These interactions resulted in the formation of the FU-HSA complex, making the local microenvironment of the protein more hydrophobic than its native state. Moreover, the interaction of HSA with low concentration of FU (less than 150 μmol/L) led to the increase in the amount of the compact α-helix structures, whereas interaction of HSA with high concentration of FU (higher than 150 μmol/L) made the compact a-helix structure decreasing, probably due to the protein undergoing some sort of distortion.3. We studied the interaction of the znic phthalocyanines derivatives ZnPc1, ZnPc2, and ZnPc3 with the human serum albumin (HSA), respectively, and the ZnPcs-binding-induced microenvironment alterations in binding-site of HSA molecule using spectroscopic techniques and molecular docking method. The results demonstrated that three ZnPc derivatives molecules could enter the inside a hydrophobic cavity of subdomain ⅡA (Sudlow’s siteⅠ) near Trp 214 amino acid residue, causing the fluorescence quenching of Trp 214 through a static quenching mechanism. The nature of forces derived binding interaction between HSA and ZnPcs molecules were mainly electrostatic interactions and hydrophobic effects, which were concluded by docking study and analysis of the thermodynamic parameters of the binding process. These interactions resulted in the formation of the HSA-ZnPcs complex, making the local microenvironment around Trp 214 in the protein more hydropholic than its native state. Moreover, comparing to the ZnPc1 and ZnPc2, the interaction of HSA with ZnPc3 was the strongest, probably due to the strongest elecgtrostatic potential of ZnPc3.4. We used the facile electrochemical voltammetry for probing the structural alterations around the heme group in sperm whale myoglobin (Mb) induced by solution pH. The results demonstrated that the subtle structural changes around the heme group in Mb induced by the solution pH can be sensitively probed based on the ET signals recorded using voltammetry. In the acidic and basic conditions, the heme group dissociates from the hydrophobic pocket and becomes exposed to the solution because of the breakage of the Fe-His93 bond, causing a significant enhancement in the ET signals. We also verify such a breakage of the Fe-His93 bond by using UV-vis spectroscopic measurements and molecular dynamics (MD) simulations. The good consistency of the results obtained with the electrochemical method with those of spectroscopic and MD simulations substantially validates our proposed electrochemical method based on the ET signal of the redox process of the heme group as a facile and effective way for probing structural changes around the heme group and the conformation alterations of heme-containing proteins. Compared with the existing methods used for the study of structural changes of protein, our electrochemical method demonstrated here could be advantageous in terms of its sensitivity, ease of operation, cost-effective feature, simple use of bare electrodes, and availability of abundant dynamic information. This study essentially offers a facile and effective electrochemical route to probe the structural changes around the heme group in heme-containing proteins.