Ⅰ. Interaction Between PPARγ And Ligands Or Relative Nuclear Receptor Ⅱ. Thermodynamic And Kinetic Investigation Of SARS_CoV Spike Glycoprotein Immunological Fragment

I. Interaction between PPARy and ligands or relativenuclear receptorThe binding characteristics of a series of PPARy ligands to human PPARy ligand binding domain (LBD) have been investigated for the first time by using surface plasmon resonance biosensor (SPR) technology, circular dichroism (CD) spectroscopy and molecular docking simulation. The SPR biosensor determined equilibrium dissociation constants (Kd values) are in agreement with the results reported in the literature measured by other methods, indicating SPR biosensor can assume a direct assay method in screening new PPARy agonists or antagonists. Conformational changes of PPARy caused by the ligand binding were detected by CD determination. It is interesting that the thermal stability of the receptor, reflected by the increase of the transition temperature (Tn,), enhanced by the binding of the ligands. The increment of the transition temperature (ATm) of PPARy owing to ligand binding correlated well with the binding affinity. This finding implies that CD could be a possibly complementary technology to determine the binding affinities of ligands to PPARy. Molecular docking simulation provided reasonable and reliable binding models of the ligands to PPARr at the atomic level, which gave good explanation of the structure-binding affinity relationship for the ligands interacting to PPARy. Moreover, the predicted binding free energies for the ligands correlated well with the binding constants measured by SPR biosensor, indicating that the docking paradigm used in this study can possibly be employed in virtual screening for discovering new PPARr ligands, although the docking program cannot accurately predict the absolute ligand-PPARr binding affinity.Nuclear hormone receptors are transcription factors that require multiple protein-protein interaction to modulate target gene expression. The PPARs are believed to form heterodimers with another nuclear receptor, the 9-cis-retinoic X receptor in vivo. Although the interaction of PPAR and RXR and formation of heterodimers have been studied, the binding affinity data were never reported. Inaddition, there was no evidence to show the extent of ligands quantity dependence in the process of the heterodimerization in vitro. Here we reported that PPARr ligand binding domain (LBD) can be associated with RXRa-LBD in the absence of each ligand in vitro using native-PAGE. Although the interaction seems to be ligand-independence, the binding affinities are enhanced by receptor agonist. Both PPARr agonist and RXR ligands (9-cis-retinoic acid) can enhance the heterodimer formation. To examine the kinetic characteristics of interaction between highly purified proteins, Biacore experiment was applied to explore the binding affinities that can be increased 10-100 folds induced by difference agonist. The enhancement function of agonists was considered as the result of conformation change of receptor induced by agonists. Computation modeling also demonstrated that the ligands structurally departed from the heterodimer would decrease the interaction between PPAR and RXR. Here, the eventual purpose to detect the binding affinity between PPAR and RXR induced by ligands in vitro is to build the novel framework for the new ligands screening.II. Thermodynamic and kinetic investigation of SARS_CoVspike glycoprotein immunological fragmentSevere Acute Respiratory Syndrome (SARS) is an acute respiratory illness. SARS coronavirus (SARSCoV) has been confirmed to be responsible for SARS infection. It is found that spike glycoprotein of SARS (SARSS) plays a pivotal role in SARSCoV invasion against human cells. In this report, to supply more information on the structural features of SARSS, the immunological fragment (Ala251 to His641) of SARS_S protein (SARS_Slb) was addressed. This protein was successfully expressed in E. coli and purified by the on-column cleavage from fusion protein. The purified SARS_S1b protein was applied to studies by using circular dichroism (CD), tryptophan fluorescence and stopped-flow spectral techniques.