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Gautam Krishnan

Bits Pilani Kk Birla Goa Campus, India India

Title: A New Functional Model for Prediction of Chaperone Activity of the Recombinant M. tb Acr (α-Crystallin) Using Insulin as Substrate

Biography

Biography: Gautam Krishnan

Abstract

Mycobacterium tuberculosis Acr is an important protein expressed in latent tuberculosis which is active as an oligomer in preventing misfolding of cellular proteins. In this study, Mycobacterium alpha crystallin (acr) gene was cloned and expressed in E. coli. The recombinant Acr protein was purified by Nickel-NTA resin. The oligomeric state of Acr was confirmed by gel filtration chromatography using Sephacryl S-200 and Native-PAGE. The activity of recombinant Acr was checked by preventing thermal aggregation of citrate synthase at 45°C and the chaperone activity against insulin B chain aggregation at 60ºC and 37°C. Chaperone activity studies were performed with insulin at different mole ratios of Acr with 2 types of samples, His tag elutes (H) and His tag elutes with gel filtration (G). Polynomial graphs were plotted which could be used to predict activity. It was observed that ratio of different sizes of oligomers (9 to 24 mers) had a significant effect on chaperone activity. Using mole ratio of Acr for both (H) and (G) samples to insulin substrate and ratio of oligomers, we determined number of Acr molecules binding to insulin as a model substrate. We found that if 1.54% of the insulin chain is covered completely by the (G) samples, aggregation is completely inhibited as compared to 6% with (H) samples. Pre-heat treatment studies were carried out at 37ºC, 60ºC and 70ºC. Far-Ultraviolet circular dichroism (UV-CD) analysis provided fresh insights into the role of β sheets and α helices in activity, especially in (H) samples suggesting a reversible transition fromhelices to  sheets. This enabled us to formulate a functional model for binding of Acr to insulin B chain which incorporated 4 types of secondary structure molecules. This is a useful tool to analyse in vitro preparations of recombinant Acr and build more consensus on the structure activity relationship; especially in terms of oligomeric ratios.