Biennial Report 2005-2007 - Saha Institute of Nuclear Physics

Biophysical Sciences 203containing glycophorins and other glycoconjugates. We propose that in thalassemia, the globinchain aggregation is resulted under oxidative stressed conditions, could then induce loss of PSasymmetry and release of silylated glycoconjugates through membrane vesiculations.Sumanta Basu, Debashis Banerjee†, Sharmila Chandra†, Abhijit ChakrabartiSG6.1.1.14 Structural and conformational studies on the DNA binding domain of RFX5RFX5, RFXANK and RFXAP are the subunits of RFX complex involved in transcription of majorhistocompatibility complex II (MHC II) molecules. RFX complex binds to the X and S box regionof the promoter of MHC II. In bare lymphocyte syndrome (BLS) the promoter of MHC II isneither occupied nor transcribed and that causes severe immune deficiency in individuals. So acomprehensive structural analysis of the protein complex will play a vital role in interpretationof their functionality. In this project the DNA binding domain (DBD) of RFX5 (12kD) and thefull length RFXANK (35kD) were subcloned in a GST tag plasmid, expressed in bacterial hostcells and purified by two-step chromatographic process: - i) Affinity Chromatography and ii) SizeExclusion Chromatography. The biophysical studies of these two proteins revealed the formationof dityrosine derivatives upon UV irradiation. It is an intense fluorophore having emission maxima∼400nm when excited at 315nm. Its formation is favoured under alkaline condition and the reactionis also catalysed by H 2 O 2 /peroxidase system. Reducing agents such as DTT and glutathione inhibitdityrosine formation. RFX5 DBD is more susceptible to dityrosine formation than RFXANK. NMRspectroscopic studies have been initiated for structure elucidation of RFX5 DBD. Preliminaryproton 1D spectrum and 2D spectrum (TOCSY and NOESY) were taken in 500MHz and cryoprobe 600MHz NMR spectrometer. Analysis of the spectral data and preparation of 15 N and 13 Cisotope enriched proteins are underway. RFX DBD has been isotopically enriched with 15 N nucleiand 15 N- 1 H HSQC experiments were performed. The analysis of these data is in progress.Madhumita Chakraborty, Amitabha Sengupta, Subrata Banerjee, Chaitali Mukhopadhyay†, AbhijitChakrabartiSG6.1.1.15 Red Cell Proteomics with and without HemoglobinRed blood cells have been an obvious target of proteomics study for the past few years. However,even after the emergence of sophisticated and high throughput mass spectrometry instrumentation,only a handful number of proteomics work have been published till date identifying proteins fromthe cytosolic fractions of the red cells. The major difficulty of identification of other proteins hasbeen the large abundance of hemoglobin in the cytosol (∼95%), masking s the detection of most ofthe lower abundant proteins on 2D gel electrophoretic separation. Depletion of hemoglobin exclusivelyand effectively has remained largely unsuccessful till date. We have succeeded in depletinghemoglobin from the hemolysate using ion-exchange chromatography on SP-Sephadex matrix. Thehemoglobin-depleted red cell cytosolic fractions showed larger number of protein spots in the 2Dgels compared to the undepleted hemolysate. Using tandem MALDI ToF/ToF mass spectrometry(AB 4700 from Applied Biosystems), we have been able to identify more than 30 proteins including20 known proteins and about 10 proteins, so far not detected in the hemolysate by the earlier