Chaperone Assisted Protein Folding, Protein Engineering and Molecular Biophysics Group
Biological systems in itself are not less than miracle. They carry out millions of processes to sustain what we know as life processes. Protein molecules are the real working horse behind it and play directly or indirectly role in every process going on inside a cell; viz-structural, functional as well as regulatory roles. Every second, thousands of proteins self-pack into the unique shapes that hold the key to their function.
It is known from the time of C.B. Anfinsen, the final design of a protein is cumulative effect of the information inscribed into its amino acid sequence allowing it to cause bends, form loops, twists, coils and collapses on itself to achieive the final functional conformation. A substantial fraction of that information needed for the folding process comes from the specific environment in which the protein is located. In contrast to our knowledge about nucleic acids, available information about protein conformation and folding is enigmatic till now. The chemical forces that direct folding act in an incompletely understood, nebulous way and remains one of science's holiest grails.
Often, neither of these information inputs is, or can be, optimized for the efficient folding of a protein. As a result, a great many proteins are very prone to mis-folding and aggregation inside a cell. Molecular chaperones appear to have evolved to facilitate protein folding by somehow preventing these deleterious side reactions.
In the case of the bacterial chaperonin GroEL, protein folding is facilitated by the entrapment of folding intermediates on the interior of a large cavity formed between GroEL and its co-chaperonin GroES. ATP binding and hydrolysis controls the formation of this chamber in a cooperative and dynamic process that leads to oscillating rounds of protein capture and release.
This type of mechanism of GroEL assisted protein folding, in which bound polypeptide and GroES share the same GroEL ring is known as cis mechanism, and it has been observed that polypeptides within the molecular weight range of 52 kDa follow the cis- mechanism. However, for larger proteins, GroES cannot encapsulate the bound one and it has been found that GroES and polypeptide share different GroEL rings during the process of chaperone assisted protein folding. The later mechanism has been termed as trans- folding mechanism. Our group is pursuing cutting edge research in the area of GroEL-GroES assisted in vivo and in vitro folding of large substrate proteins in Escherichia coli with a view for the better understanding of the long lasting issue of how larger and aggregation prone proteins fold inside the cell.
We are also carrying out equilibrium protein folding studies and kinetics of fast folding reactions using CD, fluorescence, IR spectroscopy and stopped-flow technique. Through these studies we determine thermodynamic parameters associated with the folding-unfolding process which in turn provide information on protein stability. We try to demonstrate the role of intermediate species in the folding and unfolding processes of smaller and larger multi-domain proteins.
Our group is also actively involved in the preparation and characterisation of recombinant therapeutic proteins and proteins of commercial interests in Escherichia coli system. We are trying to develop chaperone assisted method to enhance the production of functional recombinant proteins for therapeutic and commercial purposes. A very new direction of our reseach is to use GroEL chaperonin as a tool on Biolayer interferometry platform to monitor protein folding process as well as to discriminate between properly folded and misfolded proteins.