Rajagopal Chattopadhyaya
Bose Institute, India
Title: Thermal and chemical denaturation of Colocasia esculenta tuber agglutinin from α2β2 to unfolded state, its 1.74Å crystal structure and a complex crystal structure at 1.85Å with mannose
Biography
Biography: Rajagopal Chattopadhyaya
Abstract
The major tuber storage protein of Colocasia esculenta, is a monocot mannose-binding, widely used dietary lectin. This tuber agglutinin contains two polypeptides of 12.0 and 12.4 kDa by MALDI-TOF analysis. By both gel filtration and dynamic light scattering at pH 7.2 show the lectin has a α2β2 form; however, at pH 3, it converts to αβ form. Our circular dichroism spectroscopy studies show that the lectin retains approximately 100% of its secondary structure between pH 2-8. The fluorescence emission maxima of 346 to 350 nm for pH 4 to 10 show that the tryptophan residues are relatively exposed. The unfolding is a simple two-state process, N4 ↔ 4U, as seen in our denaturation scan profiles, when monitored by fluorescence, far-UV CD, and near-UV CD, are completely super imposable. Analyses of these profiles provide an estimate of several thermodynamic parameters at each guanidinium chloride concentration, including the melting temperature Tg, which is 346.9 K in 0 M, but lowers to 321.8 K in 3.6M. Mannose-free mannose-bound lectin crystals were obtained by hanging-drop, vapor-diffusion method at room temperature and high-resolution X-ray diffraction data were collected using a home X-ray source. The mannose-free structure (5D5G) and mannose-bound structure (5D9Z) are both available in the PDB, along with the X-ray data. Some highlights of both structures will be presented. Such high resolution structures obtained from a home X-ray source is rare among this class of lectins, and it has not been possible to crystallize simple mannose with such lectins before.