Efficient preparation and metal specificity of the regulatory protein TroR from the human pathogen Treponema pallidum.
TroR is a putative metal-dependent regulatory protein that has been linked to the virulence of the human pathogen Treponema pallidum. It shares high homology with the well-known iron-dependent regulatory protein DtxR from Corynebacterium diphtheriae, as well as the manganese-dependent MntR from Bacillus subtilis. However, it has been uncertain whether manganese or zinc is the natural cofactor of TroR to date. Herein, we established an efficient method named "double-fusion tagging" to obtain soluble TroR for the first time. A series of studies, including ICP, CD, fluorescence, ITC, and electrophoresis mobility shift assay (EMSA), were performed to resolve the discrepancies in its metal-binding specificity. In addition, bioinformatic analysis as well as mutation studies were carried out to find the genetic relationships of TroR with its homology proteins. In conclusion, our findings indicate that TroR is a manganese-dependent rather than a zinc-dependent regulatory protein.
Institutes of Biomedical Science, Fudan University, Shanghai 200433, China. xstan@fudan.edu.cn.
Yi
Y
Liu
Wei
W
Li
Yaozhu
Y
Wei
Yindi
Y
Jiang
Xiangshi
X
Tan
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002151:Calorimetry; D002942:Circular Dichroism; D019295:Computational Biology; D024202:Electrophoretic Mobility Shift Assay; D005822:Genetic Vectors; D006801:Humans; D008670:Metals / Q000378:metabolism*; D008969:Molecular Sequence Data; D055550:Protein Stability; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014210:Treponema pallidum / Q000378:metabolism*
D001426:Bacterial Proteins; D008670:Metals; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C121664:TroR protein, Treponema pallidum