Effects of Arginine mutations on functions and thermostability of Lon protease from brevibacillus thermoruber
Date Issued
2004
Date
2004
Author(s)
Huang, Chin-Yuan
DOI
en-US
Abstract
Proteolysis in living cells serves to rid the abnormal and misfolded proteins of the cell and to limit the time and amounts of the availability of critical regulatory proteins. Most intracellular proteolysis is carried out by oligomeric ATP-dependent proteases containing both ATPase and proteolytic activities. Five ATP-dependent proteases, Lon, FtsH, ClpAP, ClpXP, and HslUV, have been discovered in Escherichia coli. Among them, Lon protease has been found in virtually all living organisms, from archaea to eubacteria to humans.
A thermostable Lon protease from thermophilic Brevibacillus thermoruber WR-249 (Bt-Lon) has been cloned and characterized in our laboratory. Previous studies showed that this protease possesses a higher ratio of arginine, and these residues may be involved in DNA-binding activity as well as responsible for thermostability of Bt-Lon under high temperature. Here, seven arginine residues of Bt-Lon, Arg98, Arg370, Arg518, Arg557, Arg566, Arg590, and Arg697, were selected for point-mutagenic analysis to investigate their functional roles.
The overall structure of each mutant determined by CD spectrum and fluorescence spectroscopy was not affected by the site-specific point mutations. Furthermore, all mutants showed a Tm value of 71.5 °C, identical to that of wild-type Bt-Lon. These results indicated that the substitution of Arg98, Arg370, Arg518, Arg557, Arg566, Arg590, or Arg697 with alanine did not affect the thermostability of the proteins.
All mutants displayed the basal peptidase and ATPase activities of the wild-type. Nevertheless, the three mutants, R518A, R557A, and R566A, showed a lower DNA-binding affinity based on the GMSA experiment. Furthermore, the two mutants, R518A and R566A, exhibited their peptidase and ATPase activities which are not stimulated by DNA. The results suggested that the DNA-binding affinity for Bt-Lon directly influences the activities of peptidase and ATPase. Based on the modeling structure of Bt-SSD, the two residues, Arg518 and Arg566, were located in the highly positively-charged surface of SSD domain that is proved to be a DNA-binding domain of Bt-Lon. In summary, our results indicated that Arg518 and Arg566, which are located in the highly positively-charged surface of SSD domain, are directly involved in DNA binding.
Subjects
嗜熱短桿菌
受質偵測識別弁鈰?聚合鍊鎖反應
AAA+
CD
PCR
GMSA
SSD domain
Bs-Lon
Bt-Lon
Type
other