方案摘要
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参考标准 |
Severe acute respiratory syndrome (SARS) coronavirus is a novel human coronavirus and is responsible for SARS infection. SARS coronavirus 3C-like proteinase (SARS 3CLpro) plays key roles in viral replication and transcription and is an attractive target for anti-SARS drug discovery. In this report, we quantitatively characterized the dimerization features of the full-length and N-terminal residues 1–7 deleted SARS 3CLpros by using glutaraldehyde cross-linking SDS-PAGE, size-exclusion chromatography, and isothermal titration calorimeter techniques. Glutaraldehyde cross-linking SDSPAGE and size-exclusion chromatography results show that, similar to the full-length SARS 3CLpro, the N-terminal deleted SARS 3CLpro still remains a dimer/monomer mixture within a wide range of protein concentrations. Isothermal titration calorimeter determinations indicate that the equilibrium dissociation constant (Kd) of the N-terminal deleted proteinase dimer (262 M) is very similar to that of the full-length proteinase dimer (227 M). Enzymatic activity assay using the fluorescence resonance energy transfer method reveals that N-terminal deletion results in almost complete loss of enzymatic activity for SARS 3CLpro. Molecular dynamics and docking simulations demonstrate the N-terminal deleted proteinase dimer adopts a state different from that of the full-length proteinase dimer, which increases the angle between the two protomers and reduces the binding pocket that is not beneficial to the substrate binding. This conclusion is verified by the surface plasmon resonance biosensor determination, indicating that the model substrate cannot bind to the N-terminal deleted proteinase. These results suggest the N terminus is not indispensable for the proteinase dimerization but may fix the dimer at the active state and is therefore vital to enzymatic activity.
毛细流孔隙仪在均热板毛细液芯性能测试的应用
赛默飞世尔FlashSamrt 元素分析仪对于煤焦油样品中CHNS/O的分析
布鲁克3D XRM 正品飞天茅台挑选记
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