Mechanical forces play a central role in ubiquitous phenomena such as protein degradation, cell-adhesion, tissue organization, and muscle function in multi-cellular organisms. The key players in these phenomena are protein molecules, which act as mechanosensors and communicate the surrounding dynamic microenvironment with the cell. Hence, studying the mechanical response of these biomolecules would provide a wealth of information about their structure, function, and chemistry.
We use state-of-the-aft atomic force microscope (AFM) to probe single-molecules. By using this novel technique, we can apply stretching force to a single protein molecule, measure its mechanical response and study protein mechanics. In addition, this technique has been proven to be successful in studying protein unfolding and folding, which has been a long-standing unsolved puzzle in molecular biology.
- Mechanical properties of Ubiquitin-like proteins (ex: small ubiquitin-like modifiers SUMO1, SUMO2 etc.)
- Unfolding pathways of large multi-domain proteins (ex: Maltose binding protein (MBP))
- Investigation of mechanical behaviour and functional role of different classes of cell-adhesion biomolecules.
- Engineering novel proteins with diverse mechanical functions based on cell-adhesion proteins.
- Elucidation of mechanical unfolding/unfolding pathways of proteins and their relation with biochemical pathways.
- Mechanochemistry and kinetic characterization of chemical and biochemical reactions.
- Development of novel single-molecule assays for protein-protein, protein-DNA, and protein-RNA interactions.
Recent Research Results:
We showed Ubiquitin-like proteins (SUMO1 and SUMO2) are mechanically weaker and flexible. (Biophys. J. Vol 110, 2273-2281 (2013))
We showed that MBP follows parallel unfolding pathways (paths I and II, see the picture below) upon mechanical unfolding. The unfolding flux through path I is 62% and this is further enhanced to ~80% upon ligand (maltose or maltotriose) binding. These results are explained using an energy landscape model as shown below. (JBC 2011, Link)
Recent Relevant Publications:
- Kotamarthi et al. Single-molecule studies on polySUMO proteins reveal their mechanical flexibility. Biophys. J. 104, 2273 (2013).
- Ramanujam et. al. Iterative cloning, overexpression, purification and isotopic labeling of an engineered dimer of a Ca2+-binding protein of the bg-crystallin superfamily from Methanosarcina acetivorans. Prot. Exp. & Purif. 84 116 (2012).
- Aggarwal A et al., Ligand modulated parallel mechanical unfolding pathways of Maltose Binding Proteins (MBPs), J. Biol. Chem. 286, 28056 (2011) Link. PDF
- Sri Rama Koti A. et al., Single-Molecule Force Spectroscopy Measurements of Bond Elongation during a Bimolecular Reaction. J. Amer. Chem. Soc. 130, 6479 (2008). PDF This article has been reported in Research Highlights of Nature (2008), 453, p261. PDF
- Sri Rama Koti A. et al., A Single-Molecule Assay to Directly Identify Solvent Accessible Disulfide Bonds and Probe Their Effect on Protein Folding. J. Amer. Chem. Soc. 130, 436 (2008). PDF
- Sri Rama Koti A. et al., Contour Length and Refolding Rate of a Small Protein Controlled by Engineered Disulfide Bonds. Biophys. J. 92, 225 (2007). PDF
- Arun P. Wiita et al., Force-dependent chemical kinetics of disulfide bond reduction observed with single-molecule techniques. Proc. Natl. Acad. Sci. USA 103, 7222 (2006). PDF
- Raul Perez-Jimenez et al., Mechanical Unfolding Pathways of the Enhanced yellow Fluorescent Protein Revealed by Single Molecule Force Spectroscopy. J. Biol. Chem. 281, 40010 (2006). PDF
- Sri Rama Koti A. et al., Ligand binding modulates the mechanical stability of dihydrofolate reductase (DHFR). Biophys. J. 89, 3337 (2005). PDF