Function-Structural Analysis of Lon Protease from hyperthermophilic archaeon Thermococcus kodakaraensis KOD1
Date Issued
2011
Date
2011
Author(s)
Chou, Hui-Jou
Abstract
ATP-dependent Lon proteases degrade specific short-lived regulatory proteins and are key components of the protein quality control systems in the cell, which are universally distributed in all kingdoms of life. Lon protease can be divided into two subfamilies, LonA and LonB. LonA is well-studied as compared with LonB. Previous studies have shown that Lon protease from Thermococcus kodakaraensis KOD1 (TK-Lon) which belong to LonB is composed of an N-terminal ATPase domain and a C-terminal protease domain and is a membrane-bound protein in its native host. In this study, we designed a TK-Lon mutant protein (TK-LonΔTM) with a deletion of the membrane-anchoring region and characterized its function and structure. TK-LonΔTM was overexpressed in E.coli and purified from soluble fraction displaying ATPase and proteolytic activity. Electrophoresis mobility shift assay showed that TK-LonΔTM has DNA-binding activity. Chaperone activity assay indicated that TK-LonΔTM can prevent aggregation of denature proteins under thermal stress or chemical stress. The melting temperature of TK-LonΔTM was observed at 98.9 ℃ by differential scanning calorimetry, suggesting the extreme thermostable of TK-LonΔTM. Far-UV CD and near-UV CD measurements revealed that TK-LonΔTM consists of α-helices as the major secondary structure and possesses well-defined three-dimensional structure, respectively. Our gel-filtration chromatography assay, analytical ultracentrifugation and transmission electron microscopy all displayed that TK-LonΔTM assembles into hexameric rings that likely mimic the oligomerization state of the holoenzyme. These findings showed that function and structure of Lon protease are conserved in the LonA and LonB subfamilies.
Subjects
Lon protease
Thermococcus kodakaraensis KOD1
Chaperone
Hexamer
Type
thesis
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