What do we work on? Like any other macromolecular crystallography laboratory, we also work primarily on proteins. If you ask what proteins do we work on, our answer would be that we work on proteins from viruses, bacteria, yeast, plants and animals (including human). Yeah! All of it actually and DNA as well. However, in fact, it is not the source that we are particularly interested in, it is their function that we try to address through structural studies. In our pursuit to ‘solve’ structural problems on a particular theme, we take up proteins from varying sources.
What kind of experiments we often do? Routine experiments in our laboratory include PCR, cloning, protein over-expression, protein purification, biochemical, biophysical and functional characterization, crystallization and structure solution. As a structural biology group, our major tool is crystallography. We are getting poised to initiate some cryo-EM work as well.
How & where do we get the proteins from? We over-express most of it in bacteria, in recombinant form. We are also looking at the possibility of expanding our horizon by expressing proteins in systems like yeast, mammalian and insect cells.
Do we have a specific area of interest? Over the initial years, we have gained a certain sense of directionality with respect to our science. Details of our current focus areas can be seen below:
(1) Chromatin Structural Biology: Histone proteins package eukaryotic DNA into chromatin. The dynamic DNA-protein complex called nucleosomes forms the basic element of chromatin. All eukaryotic genomic processes, both natural and pathological, including transcription, replication, recombination, repair, chromosome segregation, carcinogenesis and viral infection can be fully understood only in the context of nucleosomes. The core of a nucleosome, called the nucleosome core particle (NCP) consists of fourteen turns of B-form DNA around an octamer of histone proteins, as can be seen in the image here.
A nucleosome is known to be the binding platform for several protein factors. In order for a protein to gain access to chromosomal DNA/histone proteins, it needs to interact and/or compete with the histone proteins/DNA of the nucleosome. Studies involving nucleosomes are of greater significance as compared to isolated studies focusing on the interaction of protein factors with individual histone(s) or DNA. In this context, we work on some nucleosome-interacting proteins.
Around the theme of chromatin structural biology, another of our interest is histone chaperones. Histone chaperones are a group of proteins that bind histones and regulate nucleosome assembly. Mode of action of several histone chaperones is not well-understood. Also, it is not completely clear regarding what decides the specificity of certain histone chaperones for H3/H4, H2A/H2B or histone variants. In order to better understand these molecular machines, we work on a few projects dealing with the structural characterization of some important, but poorly characterized histone chaperones. Our endeavours with histone chaperones have gotten us to initiate structural characterization work on certain histone deacetylases (HDACs) as well!
(2) Structural Biology of Clp Machinery Proteins: The caseinolytic protease (Clp) machinery is present in bacteria, plants and other eukaryotes. These proteases usually associate with Clp chaperone proteins that help in ATP-dependent unfolding of substrates, which need to be taken in for proteolysis by the proteases. ClpD is a Clp-associated chaperone unique to plants. Plant ClpC and ClpD proteins both get localized into chloroplast stroma. However, our work from ILS has shown that the structure of ClpD is quite different from that of ClpC in its N-terminus. Our interest in plant Clp machinery proteins is to understand their distinct roles in protein homeostasis and stress response. In addition, we also work on mycobacterial Clp chaperones due to their importance as a target in anti-mycobacterial research.
(3) Infectious Disease Structural Biology: A couple of our chromatin structural biology and Clp machinery projects have infectious disease flavour. In addition, we also work on proteins from bacteria, fungi, viruses and parasites that are infectious to human. More recently, we have started working on candidate proteins from the pathogens that infect cultured shrimps.
Over and above these, additional crystallographic projects are being taken up from time to time, on a collaborative basis.
Whom do we collaborate with? We do have several collaborators.
Our current & active in-house collaborators are:
- Dr Balachandran Ravindran (immunology)
- Dr Narottam Acharya (yeast genome instability)
- Dr Soma Chattopadhyay (virology)
- Dr Tushar Kant Beuria (bacterial cell division)
- Dr V. Arun Nagaraj (plasmodium biology)
In addition, we have ongoing collaborative projects with:
- Prof. Kim Lewis, North Eastern University, Boston, USA (antibacterials)
- Prof. Sheng Luan, University of California at Berkeley, USA (plant FKBPs)
- Prof. Klaas van Wijk, Cornell University, USA (plant protein homeostasis)
- Prof. Claudia Jonak, Austrian Inst. of Technology, Austria (plant stress factors)
- Prof. Aline V. Probst, CNRS, France (histone variants and chaperones)
- Prof. Anil Grover, University of Delhi-South Campus, India (plant stress tolerance)
- Prof. Ranjith Padinhateeri, IIT, Bombay, India (chromatin biophysics)
- Prof. Jagneshwar Dandapat, Utkal University, Bhubaneswar, India (immunology)
- Prof. Rabindra Behera, NIT, Rourkela, India (ferritin nanocages)
- Prof. Meenu Kapoor, Indraprastha University, New Delhi, India (plant DNMTs)
- Dr Devinder Sehgal, National Institute of Immunology, New Delhi, India (bacterial virulence)
- Dr Bhavani Shankar Sahu, National Brain Research Centre, Manesar, India (vesicle biogenesis)
- Dr Umasankar PK, Rajiv Gandhi Centre for Biotechnology, Trivandrum, India (intracellular trafficking)
- Dr Rahul Modak, KIIT University, Bhubaneswar, India (bacterial deacetylases)
- Dr Subhendu K. Otta, CIBA, Chennai, India (shrimp pathogens)
- Dr Insaf Ahmed Qureshi, University of Hyderabad, Hyderabad, India (Leishmania proteins)
Are we keen on new collaborations? Being a crystallography-based laboratory, we believe in evolving ourselves by adding on newer projects and starting new collaborations. Each project, in fact, comes with a good amount of learning experience for our lab members. However, with the limited space, manpower and resources that we have now, our hands are relatively full. Due to the inherent risk involved in crystallography, each one of us juggle with 2-4 projects at any given point of time – 1-2 projects of lab’s main theme and others mostly collaborative! Having said this, you could still persuade us to pursue a bit of your science. We don’t say ‘NO’ that easily. 🙂