In Silico structural & functional study of BoNT/A LC

We have explored the folding dynamics of the BoNT endopeptidase using BoNT serotype A Light Chain (BoNT/A LC) as a model system. We have utilized urea denaturation assays and Molecular Dynamics (MD) simulations to unmask the folding mechanism. A highly unusual folding pattern has emerged, which does not show a typical folding co-operativity but rather, it follows a unique three-step denaturation process with two intermediate states, i.e. N <=>I1 <=>I2<=>U. This finding agreed with results from MD simulations which enabled us to resolve the structural underpinning of BoNT in the native state (N), intermediate state (I1 and I2), and unfolded state (U). Except for the intermediate state I2, all the states of BoNT/A retained full enzymatic activity for the substrate SNAP-25, including the unfolded state U stable in 7 M urea solution. Direct observation of the functionally active intermediate states of BoNT defines a unique folding pattern and specifies the crucial role played by partially unfolded intermediate conformations. Our results stress the importance of the structural heterogeneity and conformational flexibility in the toxin's mechanism of intracellular survival and action. We are also examining the characteristics of native and PRIME (Pre Imminent Molten Globule Enzyme) conformations and mechanism of enzyme-substrate binding, utilizing MD simulation.

   Molten Globule Conformation : Implication on the function of a Protein

The MG state of proteins is believed to be part of the cell engineering which forms important intermediate conformations required for protein folding. The non-native conformations not only involve transmembrane translocation but also become a part of processes raking place within the cells. The MG conformations are required for various biological processes including translocation of proteins across the organelles or cell membranes, assisting molecular chaperones, binding to proteins to prevent then from aggregating until the quaternary structure is formed, ligand transfer, binding to multiple ligands, protein degradation at the acidic pH, channel or pore formation under low pH conditions, and for enzymatic activity. The MG state definitely plays some role in such genetic diseases owing to their existence in the denatured state under physiological conditions. However, the diverse nature of non-native conformations can also not be ignored which involves conformations other than a MG. More native MG structures need to be unraveled to better understand the unfolding process of proteins. The growing database of bacterial as well as human genomes will help in the further understanding of native MG structures.

Various members of toxin family have demonstrated to acquire the functional state by transforming their native conformation to molten globule conformation. The vital role of the MG structure is significant in translocation of various toxins in view of their exquisite specificity of their mechanism of actions. The remarkable utility of unravelling of MG state in the steps of intoxication will help in designing of effective diagnostics and specific antidotes against their effects. This family of proteins provides an integrated model to study various aspects of protein chemistry including factor affecting structural conformations of a protein, specifically, binding, translocation, longevity and various cellular functions.

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