Single-Molecule Protein Mechanics, Mechanobiology and Mechanochemistry

 

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.

Our laboratory uses a combination of experimental techniques and theoretical tools (Protein engineering, polymer synthesis, fluorescence spectroscopy, state-of-the-aft custom-built single-molecule atomic force microscope (SM-AFM), theoretical methods and steered molecular dynamics (SMD)) to probe the stability and dynamics of proteins and polymers. Using SM-AFM and SMD, we probe the mechanical response of proteins and synthetic polymers.

We use custom-built atomic force microscope (AFM) to probe single-molecules. By using this novel technique, we can apply stretching forces to single protein molecules, measure their mechanical response and study protein mechanics. This technique has proven to be very useful in studying protein (forced) unfolding and folding processes, which have implications in protein translocation, degradation, muscular function and cell adhesion.  

 

 

Research Interests:

  • Mechanical properties of Ubiquitin-like proteins (eg: small ubiquitin-like modifiers SUMO1, SUMO2 etc.)
  • Unfolding pathways of large multi-domain proteins (eg: Maltose binding protein (MBP), Leucine binding protein (LBP) and Ribose binding protein (RBP))
  • Ligand binding effects on the (mechanical) stability of Calcium binding proteins (Ca2+ binding crystallins)
  • Structural topology versus mechanical stability relationship through single-molecule studies
  • 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 have shown that Leucine binding protein (LBP) follows multiple pathways during mechanical unfolding. (JACS 2013, Link)

This paper is highlighted in ‘Spotlights on Recent JACS Publications’: Pulling on a Protein To Map Its Unfolding Pathways. Link

 

 

We have shown that Ubiquitin-like proteins (SUMO1 and SUMO2) are mechanically weaker and flexible. (Biophys J 2013, Link)

We have shown 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. (J Biol Chem 2011, Link)

Selected Recent Publications:                                                                                                                             

  • Ramanujam et. al., Ca2+ binding enhanced mechanical stability of an archaeal crystallin, PLOS ONE, 2014, 9, e94513. Link
  • Kotamarthi et. al., Multiple unfolding pathways of leucine binding protein (LBP) probed by single-molecule force spectroscopy (SMFS). J. Am. Chem. Soc. 2013, 135, 14768-14774. Link

This paper is highlighted in ‘Spotlights on Recent JACS Publications’: Pulling on a Protein To Map Its Unfolding Pathways. Link

  • Kotamarthi et. al., Single-molecule studies on polySUMO proteins reveal their mechanical flexibility. Biophys. J. 2013, 104, 2273. Link
  • Ramanujam et. al., Iterative cloning, overexpression, purification and isotopic labeling of an engineered dimer of a Ca2+-binding protein of the βγ-crystallin superfamily from Methanosarcina acetivorans Prot. Exp. & Purif. 2012, 84, 116. Link
  • Aggarwal A et. al., Ligand modulated parallel mechanical unfolding pathways of Maltose Binding Proteins (MBPs), J. Biol. Chem. 2011, 286, 28056. PDF
  • Sri Rama Koti A. et. al., Single-Molecule Force Spectroscopy Measurements of Bond Elongation during a Bimolecular Reaction. J. Am. Chem. Soc. 2008, 130, 6479. 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. Am. Chem. Soc. 2008, 130, 436.  PDF 
  • Sri Rama Koti A. et al., Contour Length and Refolding Rate of a Small Protein Controlled by Engineered Disulfide Bonds. Biophys. J. 2007, 92, 225. PDF
  • Arun P. Wiita et al., Force-dependent chemical kinetics of disulfide bond reduction observed with single molecule techniques. Proc. Natl. Acad. Sci. USA, 2006, 103, 7222. PDF
  • Raul Perez-Jimenez et al., Mechanical Unfolding Pathways of the Enhanced yellow Fluorescent Protein Revealed by Single Molecule Force Spectroscopy. J. Biol. Chem. 2006, 281, 40010. PDF
  • Sri Rama Koti A. et. al., Ligand binding modulates the mechanical stability of dihydrofolate reductase (DHFR). Biophys. J. 2005, 89, 3337. PDF