Prof. Tapan K. Chaudhuri Laboratory

It was a long time when C. B. Anfinsen came with the conclusion the information needed by a nascent polypeptide chain to achieve its correct conformation is already inscribed within its own amino acid sequence. The surrounding environment of nascent polypeptide is also substantially responsible for its folding process. This folding process involves polypeptide chains to bend, form loops, twists, coils and collapses on itself to produce the final finished functional design. The process looks straight forward but our knowledge is not as precise and complete as for nucleic acids. More than fifty years passed but there is no concrete predictive model to ascertain protein folding and conformation and remains the holy grail of protein research.

Our lab is interested in studying and understanding the folding processes; i.e. spontaneous and chaperone mediated using small and large multi-domain protein molecules utilizing biomolecular, biophysical tools and techniques. We also try to utilize the obtained information’s from these experiments as well as already existing knowledge pool for production of recombinant protein molecules of commercial and therapeutical importance. We employ protein engineering and designing approaches to learn more about folding processes and understanding the stability and structure-function relationship. Research interests of our lab include, but are not limited to:

Research works going on in lab:

A- Chaperone assisted protein folding.

Often, neither the information present in nascent polypeptide chain nor the environmental contribution for correct folding is optimum enough to allow the correct and efficient folding of a protein. As a result, a great number of proteins are susceptible to misfolding and aggregation inside the cell. Molecular chaperones appear to have evolved to facilitate protein folding by somehow preventing these adverse side reactions. Mechanism of chaperone assisted protein folding in Escherichia coli cells as well as in vitro. Our specific aim is to understand how the relatively larger and multi-domain aggregation prone polypeptides folds by the help of chaperonin GroEL and GroES. Our group is also involved in developing chaperone assisted process for the large scale preparation of functional recombinant proteins having commercial as well as therapeutic importance. Our long term goal is to develop chaperone based therapies for protein misfolding disorders.


B- Equilibrium, Kinetics and Thermodynamics of protein folding reactions:

Understanding the mechanism and pathways of protein folding and unfolding is an important aspect of protein biosynthesis. Intermediate species formed during the folding and unfolding pathway may give rise to the identification of drug targets, and characterization of those intermediates leads to the discovery of molecular aspects of diseases and physiological processes. To have definite idea about stability and function of recombinant proteins, thermodynamic and kinetic parameters are of quite importance. We carry out equilibrium unfolding and refolding experiments to know the stability of the proteins. The stability of the molecules is further be verified from fast kinetics experiment like stopped-flow CD, or fluorescence as well as measuring activity of the protein molecules. Characterization of the intermediate species formed during the unfolding and refolding pathways is done through various biochemical and biophysical methods involving Circular dichroism, fluorescence spectroscopy, FT-IR spectroscopy, HPLC based methods and other methods whenever required. A very new direction of our research 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.

Folding funnel traps in protein folding

C- Understanding the mechanism of inhibition of eukaryotic chaperone system(s) by certain putative anti-cancer compounds:

We are working on characterizing the behavior of certain putative anti-cancer compounds against the eukaryotic chaperone Heat Shock Protein 90. Hsp90 has been targeted extensively to design or screen anti-cancer compounds, as it has over 200 client proteins inside the cell, including several kinases and steroid hormone receptors that have been found to be mutated in certain types of melanomas and carcinomas. This chaperone, along with a host of other co-chaperones that tightly regulate the role of Hsp90 in binding and folding nascent polypeptides, play a major role in the progression of tumors by facilitating mutant oncogenic proteins to perform their function. This work deals with characterizing inhibitors that bind to Hsp90, and includes probing all the chaperone modalities of Hsp90 both inside and outside the cell.

Mechanism of hsp90 inhibition by putative anticancer drugs

D- Understanding the mechanism of different pH stress chaperones in protecting periplasmic E.coli substrate proteins:

Enterohaemorrhagic Escherichia coli(EHEC) is a most common and emerging pathogen that produces a toxin known as Shiga toxin which causes severe damage to the lining of the intestinal tract. This extremely virulent pathogen can cause hemorrhagic colitis and hemolytic uremic syndrome in humans. These bacteria survive in extreme acid conditions during its passage through the host’s stomach. Since the bacterial periplasm quickly equilibrates with the external acidic environment, it is the most prominent site for the acid attack which may cause many proteins to denature /aggregate at low pH. Therefore, an efficient acid tolerance mechanism is required for bacterial cell viability. How these pathogens deal with such an extremely acidic environment has been a question of central importance in understanding host-pathogen interactions during physiological and pathological processes.

We are trying to understand the mechanism of two periplasmic chaperones, Hde A and Hde B which helps in protecting these pathogens from harsh acidic conditions. Hde A facilitates the refolding as well as the resolubilization of acid denatured substrate proteins and prevents aggregation of various periplasmic proteins at very low pH. Hde A functions in concert with another protein, Hde B, which is transcribed from the same operon. This peculiar molecular chaperone system doesn’t require co-chaperone and ATP for its activity. The study of these chaperones may give close insight on the virulence factors of pathogens, given that chaperones have been associated with the invasion, infection, and survival of virulent bacteria like Enterohaemorrhagic, Shiga-toxin producing Escherichia coli, which can easily survive in the low pH of the mammalian stomach.

We are also attempting to understand the pathways and mechanism of chaperone folding and unfolding using various biochemical and biophysical studies. Detail characterization of intermediate species formed during the folding-unfolding phenomenon may give us opportunity to explore the molecular aspect of the disease and discovery of novel drug targets against Enterohaemorrhagic bacteria.

Periplasmic chaperones hdeA and hdeB

E- Chaperone Stability:

Molecular chaperone GroEL is a tetra-decameric protein which assists folding of many non-native substrate proteins. Along with folding activity in normal cell, GroEL also helps in the protection of denaturing substrate proteins under stress conditions like heat and pH by its holding activity. The motivation for undertaking this work is an unanswered question as to why GroEL is stable under stress conditions. To answer this question we have chosen to compare the thermodynamic stability of wild type monomeric GroEL and its mutants to see the effect of specific amino acid residue on the stability of GroEL protein.

Monomeric GroEL theoritical curve showing different populations and free energy extrapolated at zero urea concentration

F- Recombinant production and characterization of therapeutically important proteins:

This work focusses on using recombinant DNA technology to enhance the production of therapeutically important proteins like serratiopeptidase and Human Serum Albumin (HSA).

1- Currently, HSA is primarily obtained from the fractionation of collected human blood, which is a limited and unsafe source possessing the risk of contamination by blood derived pathogens. Thus there exists an indispensable need to promote non animal derived HSA production. In this work we aim to produce functional recombinant HSA (rHSA) in the well-studied, scalable, fast growth and convenient expression host system Escherichia coli through recombinant DNA technology as it is well established fact that production of various therapeutic proteins in Escherichia coli is free from above mentioned risk and 30% of the FDA approved recombinant pharmaceuticals are derived from Escherichia coli which has been neglected as a host for the production of rHSA. Hence, developing such a system to prepare immensely important, multi- application oriented recombinant therapeutic protein like HSA is truly important and immensely demand driven.

recombinant HSA production in <em>E.coli</em> improvement

2- Serratiopeptidase, a major protease produced from Serratia marcescens; is reported for having various therapeutical properties and marketed as an integral compound in various generic drugs with combination or an individual component. It is routinely used in medical treatment as potent anti-inflammatory and analgesic. It is also used as a fibrinolytic compound. Isolation and production of serratiopeptidase is solely performed using wild type Serratia marcescens strains. The bacteria is major cause behind nosocomial infections and also reported to causing pneumonia, septicemia and associated to cystic fibrosis. Hence development of a recombinant production strategy for the production of active and efficient serratiopeptidase is a major requisite among scientific community. We are also working on elucidating the folding pathway of serratiopeptidase by understanding the role of domains and it’s propeptide. We are using protein engineering and directed evolution to enhance its yield and develop highly efficient version of serratiopeptidase.

Serratiopeptidase and protein engineering

G- Studying the Mycobacterial chaperonin system(s):

1- The 3-D structure of proteins is determined by the amino acid sequence as well as the environment in which they fold. Protein folding within the cellular environment is facilitated by a class of proteins known as Molecular Chaperones. These Chaperones, also known as Heat Shock Proteins (HSPs), enable proteins to overcome the problems caused by crowding of macromolecules in the cell. They also protect and refold proteins during stress, by protecting the exposed hydrophobic regions, and preventing them from forming non-productive interactions that eventually lead to protein misfolding. The best characterized of them all are referred to as Chaperonins. Most bacteria possess a larger 60kDa chaperonin and a smaller 10kDa co-chaperonin. Mycobacteria were the first bacteria shown to have multiple 60kDa chaperonins. The goals of this work are to understand the reason behind the existence of multiple 60kDa chaperonins in Mycobacteria, and to understand the mechanism behind the function of the multiple chaperonins.

mycobacterial chaperone and small Hsps

2- The other work carried out is studying and characterizing the chaperone like properties of MIP_05962 obtained from Mycobacterium indicus pranii (MIP). MIP, a saprophytic, non-pathogenic organism, is emerging as a promising intervention against a number of diseases by virtue of its strong immunomodulatory functions and continues to be extensively used as an intervention against leprosy. Comparative genome sequences analyses of MIP, BCG and M. leprae, revealed that MIP and M. leprae contain 29 novel genes having significant antigenicity index revealed by in-silico analysis and absent in BCG. Most of these gene products are potentially highly immunogenic proteins. One of these putatively immunogenic proteins, MIP_05962 belongs to Hsp20 protein family due to the presence of α-crystallin domain and also has a very high protein identity with Hsp18 of M. leprae. The chaperone like properties have been investigated in-vitro with non-native substrates and also in-vivo refolding studies have been carried out. This protein has substantial potential for being used as a subunit vaccine or as a booster with BCG against tuberculosis.

H- Amyloidogenesis:

A proper conformation is essential for proteins to be functional inside the cells. Problems such as misfolding & mutations that are related to improper folding of proteins may result in the development of toxic protein aggregates. Such protein aggregates have a tendency to form amyloid fibrils, which interact with other cellular components, and adversely affect the structure and function of tissues and organs in contact, causing several degenerative pathologies such as Alzheimer’s disease, Parkinson’s disease, and many others. In our effort towards understanding such events of protein misfolding and amyloidogenesis, our interest is to find and study potential novel inhibitors against a rare and serious disease of Dialysis-related amyloidosis (DRA). Patients of DRA suffer from amyloid deposits of misfolded beta-2 microglobulin protein, specifically in the osteoarticular joints and visceral organs of the human body. These amyloid deposits cause chronic arthropathies affecting bones and joint tissues.

Human beta-2 microglobulin crystal structure