Studies of Protein Conformation and Folding Using Biochemical and Biophysical Methods

ow a protein spontaneously folds into its biologically active folded structure is an extremely interesting and still an open question. In order to attain its final active structure, a protein needs to search an enormous number of possible conformations. This process may lead to some conformational defects and formation of partially folded intermediate states.These partially folded intermediates often contain exposed hydrophobic surface through which they can interact leading to aggregation. Protein aggregation has been implicated in several physiological disorders. In addition, aggregated proteins are often immunogenic and hence un –desirable in the formulation development of bio - therapeutics. An understanding of the conformational changes during protein folding is important to elucidate the mechanism of protein aggregation. It is also important to look for an appropriate agent or small molecule, which can prevent the protein aggregation. In this thesis, we have studied different aspects of protein folding, aggregation and explored the possible use of a chemical osmolyte to combat protein aggregation. We begin by investigating the thermodynamics of folding of a small, globular mycobacterial secretory protein, MPT63 using chemical and thermal denaturation. We observe significant similarities in the thermodynamic parameters obtained using different methods. We have also observed the presence of residual structures in the unfolded states. Our study demonstrates that key processes leading to protein folding can be understood through detailed structural studies using conventional biophysical methods (like fluorescence, circular dichroism etc) along with less conventional phosphorescence spectroscopy. Phosphorescence spectroscopy has been used to unravel valuable insights into the surroundings of individual tryptophan residues of this multi – tryptophan protein (containing four tryptophan residues). We have studied the conformational changes of MPT63 in different solution pH conditions and observed spatial rearrangement in two tryptophan environments. The other two tryptophan local environments are found unperturbed at low pH leading to protein aggregation. The study highlights the promise of phosphorescence spectroscopy for increasing our understanding of the link between partially folded intermediates and protein aggregation. Finally, we use arginine, as a small molecule stabilizer to inhibit protein aggregation and to increase refolding yield of the thermally unfolded proteins. We indicate that arginine inhibits the formation of partially folded intermediates during unfolding, which would otherwise lead to protein aggregation

pH-induced structural change of a multitryptophan protein MPT63 with immunoglobulinlike fold: identification of perturbed tryptophan residue/residues

The structural change of M. tuberculosis MPT63, which is predominantly a β-sheet protein having an immunoglobulin like fold, has been investigated in the pH range 7.5–1.5 using various biophysical techniques along with low-temperature phosphorescence (LTP) spectroscopy. MPT63 contains four Tryptophan (Trp) residues at 26, 48, 82, and 129. Although circular dichroism, steady-state and time-resolved fluorescence, time-resolved anisotropy, 1-aniline-8-naphthalene sulfonic(ANS) acid binding, and analytical ultracentrifuge depict more open largely unfolded structure of MPT63 at pH 1.5 and also more accessible nature of Trp residues to neutral quencher at pH 1.5, it is, however, not possible to assign the specific Trp residue/residues being perturbed. This problem has been resolved using LTP of MPT63, which shows optically resolved four distinct (0, 0) bands corresponding to four Trp residues in the pH range 7.5–3.0. LTP at pH 1.5 clearly reveals that the solvent-exposed Trp 82 and the almost buried Trp 129 are specifically affected compared with Trp 48 and Trp 26. Lys 8 and Lys 27 are predicted to affect Trp 129. Tyrosine residues are found to be silent even at pH 1.5.This type of specific perturbation in a multi-Trp protein has not been addressed before. LTP further indicates that partially exposed Trp 48 is preferentially quenched by acrylamide compared with other Trp residues at both pH 7.5 and 1.5. The solvent-exposed Trp 82 is surprisingly found to be not quenched by acrylamide at pH 7.5. All the results are obtained using micromolar concentration of protein and without using any Trp-substituted mutan

SOIL FERTILITY EVALUATION FOR FERTILISER RECOMMENDATION USING HYPERION DATA

Soil fertility characterised by nitrogen, phosphorus, potassium, calcium, magnesium and sulphur is traditionally measured from soil samples collected from the field. The process is very cumbersome and time intensive. Hyperspectral data available from Hyperion payload of EO 1 was used for facilitating preparation of soil fertility map of Udaipur district of Rajasthan state, India. Hyperion data was pre-processed for band and area sub setting, atmospheric correction and reflectance data preparation. Spectral analysis in the form of SFF and PPI were carried out for selecting the ground truth sites for soil sample collection. Soil samples collected from forty one sites were analysed for analysis of nutrient composition. Generation of correlogram followed by multiple regressions was done for identifying the most important bands and spectral parameters that can be used for nutrient map generation