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Expression and molecular characterization of the Mycobacterium tuberculosis PII protein

IR@IGIB: CSIR-Institute of Genomics & Integrative Biology, New Delhi

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Title Expression and molecular characterization of the Mycobacterium tuberculosis PII protein
Creator Bandyopadhyay, Anannya
Arora, Amit
Jain, Sriyans
Laskar, Aparna
Mandal, Chhabinath
Ivanisenko, Vladimir A.
Fomin, Eduard S.
Pintus, Sergey S.
Kolchanov, Nikolai A.
Maiti, Souvik
Ramachandran, Srinivasan
Subject Q Science (General)
Description The signal transduction protein PII plays an important role in cellular nitrogen assimilation and regulation. The molecular characteristics of the M. tuberculosis PII (Mtb PII) were investigated using biophysical experiments. The Mtb PII coding ORF Rv2919c was cloned and expressed in Escherichia coli. The binding characteristics of the purified protein with ATP and ADP were investigated using Surface Plasmon Resonance (SPR) and Isothermal Titration Calorimetry (ITC). Mtb PII binds to ATP strongly with Kd in the range 1.93-6.44 µM. This binding strength was not significantly affected by the presence of 2-ketoglutarate even in molar concentrations of 66 (ITC) or 636 (SPR) fold excess of protein concentration. However, an additional enthalpy of 0.3 kcal/mol was released in presence of 2-ketoglutarate. Binding of Mtb PII to ADP was weaker by an order of magnitude. Binding of ATP and 2-ketoglutarate were analysed by docking studies on the Mtb PII crystal structure (PDB id 3BZQ). We observed that hydrogen bonds involving the γ-phosphate of ATP contribute to enhanced binding of ATP compared with ADP. Glutaraldehyde Crosslinking showed that Mtb PII exists in homotrimeric state which is consistent with other PII proteins. Phylogenetic analysis showed that Mtb PII consistently grouped with other actinobacterial PII proteins.
Date 2009-11-02
Type Article
Format application/pdf
Identifier http://openaccess.igib.res.in/14/1/jb.mvp174.full.pdf
Bandyopadhyay, Anannya and Arora, Amit and Jain, Sriyans and Laskar, Aparna and Mandal, Chhabinath and Ivanisenko, Vladimir A. and Fomin, Eduard S. and Pintus, Sergey S. and Kolchanov, Nikolai A. and Maiti, Souvik and Ramachandran, Srinivasan (2009) Expression and molecular characterization of the Mycobacterium tuberculosis PII protein. Journal of Biochemistry.
Relation http://jb.oxfordjournals.org/content/early/2009/11/02/jb.mvp174.abstract