Thermodynamic Characterization of DNA-binding Activity of Human Lon Protease
Date Issued
2006
Date
2006
Author(s)
Chen, Si-Han
DOI
en-US
Abstract
Lon is a class of ATP-dependent protease, which is committed to several crucial functions including proteolytic, ATPase, chaperone, and DNA-binding activities. Previously, human Lon protease (huLon) has been reported that it binds to an element of antisense light strand promoter (5’-AATAATGTGTTAGTTGGGGGGTGA-3’, designated as 24-mer LSPas) within the displacement loop (D-loop) region of mitochondrial genome. In this study, the potential specific binding site for huLon containing six consecutive guanine bases, 24-mer LSPas, was found to form G-quartet in various ionic conditions with electrophoretic and circular dichroism (CD) spectroscopic analysis. We have investigated the thermodynamics of the binding reactions of huLon to 24-mer LSPas and the G-quartet-forming unit (5’-TGGGGGGT-3’, or simply called 8-mer TG6T) respectively by using isothermal titration calorimetry (ITC). The results revealed that the interactions between huLon and 24-mer LSPas are primarily driven by enthalpy change, which exhibits strong dependence on temperature, rendering a large negative heat capacity, -607.82±31.39 cal?mol-1?K-1. Though, the binding of 8-mer TG6T to huLon is quite comparable in binding constant and free energy change with that of 24-mer LSPas. In contrast to what was observed in the binding reaction of 24-mer LSPas, the binding of huLon to 8-mer TG6T is mainly entropically driven, and associated with a small change in the heat capacity of 67.29±4.52 cal?mol-1?K-1, which reflects a lack of strong enthalpy-entropy compensation.
The thermal property of each binding component as well as complex was further examined with differential scanning calorimetry (DSC). We observed a DNA-dependence of excess molar heat capacity of huLon when it is in the presence of 24-mer LSPas. The results are consistent with what were observed in the ITC experiments. We concluded that the polyanionic G-quadruplex with high charge density interacts with huLon with appreciable affinity but in a nonspecific manner. Our thermodynamic analysis has revealed that huLon specifically binds 24-mer LSPas, and accompanies structural rearrangement, which is also indicated in the CD experiments. By evaluating the entropic cost, we found that about 32 residues of huLon are coupled with folding to the site-specific binding. The energetic interpretations may shed new light on the DNA-binding mechanism of human Lon protease.
Subjects
粒線體DNA
蛋白水解酵素
生物熱力學
DNA四重螺旋
端粒
端粒結合蛋白
DNA結合蛋白
熱容
焓
亂度
熵
恆溫滴定熱卡計
差異性掃描熱卡計
圓二色光譜
Lon protease
DNA-binding protein
thermodynamic driving force
G-quartet
G-quadruplex
G-quartet-binding protein
mitochondrial DNA
D-loop
hydrophobic interactions
heat capacity
enthalpy
entropy
isothermal titration calorimetry
differential scanning calorimetry
circular dichroism
Type
other
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