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 Ribose binding protein (RBP) also follows multiple pathways during mechanical unfolding. (J Phys Chem B 2014, Link)

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:                                                                                                                            

  • Kotamarthi HC, Yadav A, and Ainavarapu SRK, Small peptide binding stiffens the ubiquitin-like protein – SUMO1. Biophysical Journal (Accepted, November 24, 2014).
  • Bera et al, Characterization of unfolding mechanism of human lamin A Ig fold by single-molecule force spectroscopy – implications in EDMD. Biochemistry (2014), 53, 7247. Link
  • Kotamarthi et al, Mechanical Unfolding of Ribose Binding Protein (RBP) and its Comparison with other Periplasmic Binding Proteins (PBPs). J Phys Chem B (2014), 118, 11449. Link
  • 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). JACS (2013)135, 14768. 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
  • Aggarwal et al, Ligand modulated parallel mechanical unfolding pathways of Maltose Binding Proteins (MBPs). J Biol Chem (2011)286, 28056. PDF
  • Ainavarapu et al, Single-Molecule Force Spectroscopy Measurements of Bond Elongation during a Bimolecular Reaction. JACS (2008)130, 6479. PDF This article has been featured in Research Highlights of Nature (2008), 453, p261. PDF
  • Ainavarapu et al, A Single-Molecule Assay to Directly Identify Solvent Accessible Disulfide Bonds and Probe Their Effect on Protein Folding. JACS (2008)130, 436. PDF
  • Ainavarapu et al, Contour Length and Refolding Rate of a Small Protein Controlled by Engineered Disulfide Bonds. Biophys J (2007)92, 225. PDF
  • Wiita et al, Force-dependent chemical kinetics of disulfide bond reduction observed with single molecule techniques. PNAS (2006)103, 7222. PDF
  • 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
  • Ainavarapu et al, Ligand binding modulates the mechanical stability of dihydrofolate reductase (DHFR). Biophys J (2005)89, 3337. PDF

(For complete list of publications click here)