1. Single molecule imaging of T cell receptor conformation dynamics

To understand the molecular mechanism of TCR ligand discrimination and conformation dynamics, we apply single molecule fluorescence resonance energy transfer (smFRET) to measure the intermolecular TCR-pMHC bond distance and intramolecular TCR-CD3 conformation with a spatiotemporal resolution of ~1 Å and ~10 ms at the membrane of a live primary T cell. This experiment uncover how TCRs discriminate structurally similar peptides by forming TCR-pMHC bonds of different conformations, which in turn control the accessibility of CD3 ITAMs for signal initiation, thus explaining the molecular mechanism of TCR ligand discrimination.

Left, A composite structural model of T Cell Receptor (TCR), IEk , scFv and CD3ζGFP to measure conformational dynamics. Ref. Sasmal et al. Cellular and Molecular Immunology 2020

Right, proposed model for TCR ligand discrimination

2. T cell receptor-pMHC interaction kinetics

We use micropipette system to measure the in situ two-dimensional (2D) TCR-pMHC binding kinetics and affinities. We are trying to find out how binding affinity (driven by the on-rate) is correlated with the pMHC potency.

2D micropipette adhesion assay

3. T cell Ca2+ signalling

To understand how the TCR-pMHC bond conformation triggers T-cell signaling, we are building a fluorescent micropipette microscope to measure the real-time T-cell calcium signaling at the single-cell level. Single T-cell calcium flux is observed upon T cell-APC contact (bond formation). Consistently, the calcium signaling amplitude and speed were dependent on the peptide potency

Single-cell Ca2+ Imaging by micropipette system, and the real-time Ca2+ flux was imaged by EMCCD camera.

4. Single molecule patch-clamp FRET anisotropy microscopy studies of ion channel activation and deactivation

We develop a new correlated technical approach- single-molecule patch-clamp FRET anisotropy imaging and demonstrate by probing the dynamics of NMDA receptor ion channel and kinetics of glycine binding with its ligand binding domain.

(Ref. Sasmal et al. J. Am. Chem Soc 2014 and Sasmal et al. J. Am. Chem Soc 2016)

Microscope setup for correlated patch-clamp and smFRET

5. Fluorescence correlation spectroscopy (FCS)

We apply fluorescence correlation spectroscopy to understand protein unfolding and refunding dynamics in solution as well as live cell.

Ref. Sasmal et al. J. Phys. Chem. B 2011, 115, 7781, Sasmal et al. J. Phys. Chem. B 2011, 115, 13075 and Sasmal et al. Langmuir 2013, 29, 2289.

Teaching:

  1. Analytical and Spectroscopic Techniques (For M.Sc.)

  2. General Chemistry Laboratory (For B.Tech.)

  3. Hands on Physical Chemistry Experiments (For M.Sc.)

  4. Quantum Chemistry and Spectroscopy (B.Tech.)

New Courses:

  1. Principles of Fluorescence Spectroscopy and Imaging (For M.Sc. and Ph.D.)

  2. Fluorescence Imaging (For Ph.D.)

  3. Single Molecule FRET and Applications (For Ph.D.)

Research Support:

SERB, DST and IIT Jodhpur