| When nanoparticles enter into organism, it will encounter and interact with thousands of protein, leading to a diverse biological effects and even cytotoxicity. The main reason is that nanomaterials can interact with cells or proteins in the body, with some undesirable consequences on protein in terms of configuration, activity and stability. Due to their unique three-dimensional conjugate structure and physical and chemical properties, fullerenes have a wide range of potential applications in biomedical science, including photodriven DNA cleavage, enzyme inhibition, anti-tumor avtivity, drug delivery, antibacterial activity and cell protection.The study has shown that fullerene C60 nanocrystals can cause the same biological effect with receptor subunit NR2 B, by binding seclectively and stably to CaMKⅡα. The results include the ability to lock CaMKⅡα in an active conformation, leading to the cellular signaling process and enhancement of learing and memory. Therefore, to clarify the interaction of nanomaterials with proteins is helpful to investigate the mechanism. Furthermore, it will probably provide a theoretical basis for the bio-safety designs and the potential biomedical applications of the engineered nanomaterials.The research consisits of the following two parts:(1) In this part, to improve the expression level, the optimization of expression conditions for the gene CaMKⅡα cloned into different vectors, were studied in the E.coli BL21(DE3) Rosetta. The optimal effect of vector pET-22 b was found: 0.1% inoculation quantity and 0.2 mM IPTG under the induced condition of 16℃ for 16 h. Subsequently, the soluble proteins were purified by affinity chromatography and gel filtration chromatography, to get enough samples for subsequent experiments.(2) The Nano-C60 solutions were prepared by solvent substitution, and then were characterized by UV and DLS. According to the Langmiur equilibrium adsorption isotherm model, the saturated protein adsorption to C60 were 5.435 μg/μg. DLS showed that nanocrystals existed in the form of single dimer, with a small amount of aggregate after adsorption protein. In simulated physiological consitions, the interaction between C60 and CaMKⅡα was investigated by spectrocopy methods and molecular simulation to explore the binding parameters. The results are shown as follows:The fluorescence quenching of CaMKⅡα by addition of C60 is due to static quenching, and the binding constants were 4.87×105, 6.47×105, 7.65×105 L·mol-1 at 298, 310 and 318 K, respectively. The thermodynamic parameters were calculated as follows: enthalpy change ΔH is a positive value(209.805 kJ/mol) and entropy change ΔS is a positive value(778.364J/mol/K). The corresponding results indicated that C60 can bind with CaMKⅡα mainly via hydrophobic force spontaneously, which were agree with that from the molecular modeling. Furthermore, the study of molecular modeling showed that there were mainlyπ-πinteractions between the aromatic or charged residues of CaMKⅡα and C60, which is a good complement of thermodynamics experiment. The spectra of UV absorption and synchronous fluorescence indicated the polarity increased and the hydrophobicity decreased around the tryptophan residues. In addition, circular dichroism was employed to calculate quantitatively the change of the secondary structure. The results indicated that C60 had not significantly effect on the secondary structure of CaMKⅡα. The content of alpha helix was reduced by 1.8%, β-fold increased by 1.1%. |
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