• RAJARAM SWAMINATHAN

      Articles written in Journal of Chemical Sciences

    • Employing the fluorescence anisotropy and quenching kinetics of tryptophan to hunt for residual structures in denatured proteins

      Satish Kumar Rajaram Swaminathan

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      Residual structures in denatured proteins have acquired importance in recent years owing to their role as protein-folding initiation sites. Locating these structures in proteins has proved quite formidable, requiring techniques like NMR. Here in this report, we take advantage of the ubiquitous presence of tryptophan residues in residual structures to hunt for their presence using steady-state fluorescence spectroscopy. The surface accessibility and rotational dynamics of tryptophan in putative residual structures among ten different proteins, namely glucagon, melittin, subtilisin carlsberg, myelin basic protein, ribonuclease T1, human serum albumin, barstar mutant, bovine serum albumin, lysozyme and Trp-Met-Asp-Phe-NH2 peptide, was studied using steady state fluorescence quenching and anisotropy, respectively. Five proteins, namely ribonuclease T1, bovine serum albumin, melittin, barstar and hen egg white lysozyme appear likely to possess tryptophan(s) in hydrophobic clusters based on their reduced bimolecular quenching rates and higher steady-state anisotropy in proportion to their chain length. We also show that the fluorescence emission maximum of tryptophan is insensitive to the presence of residual structures.

    • Ordered structure induced in human c-Myc PEST region upon forming a disulphide bonded dimer

      MOHD ZIAUDDIN ANSARI SHAH EKRAMUL ALOM RAJARAM SWAMINATHAN

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      PEST rich protein sequences are intrinsically disordered and serve as degradation hotspots in eukaryotic cells. The structure of PEST degrons in proteins and their mechanism of action remain poorly understood. Deregulation or overexpression of human transcription factor c-Myc causes cancer. The PEST region in c-Myc is implicated for its rapid degradation. Here, we investigate how the structure of 77-residue PEST fragment of c-Myc and its mutant (M1, Trp inserted) are affected by the formation of a covalent dimer. DLS and size exclusion data indicated a ~30% increase in Stokes radius upon the formation of the dimer. CD showed an increase in helix and strand content with dimer formation in both Wt and mutant. However,counterintuitively, Trp fluorescence anisotropy decay was marginally faster in the dimer compared to monomer. This anomaly was traced to Trp-Trp Fo¨rster resonance energy homotransfer in M1 dimer, suggesting close interaction between two chains in the dimer. Our data suggest that indole rings in the PEST M1 dimer are no further than 24 A ° apart. These results may hold the key to higher stability of c-Myc in tumours and the poorly understood physiological role of c-Myc oligomer in cells.

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