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.
- Mechanical stability, stiffness of Ubiquitin and Ubiquitin-like proteins (SUMO1 and SUMO2)
- Unfolding pathways of large multi-domain proteins (Maltose binding protein (MBP), Leucine binding protein (LBP) and Ribose binding protein (RBP))
- Mechanical properties proteins on the malarial parasite, Plasmodium falciparum sporozoites (circumsporozoite protein, CSP)
- 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
Recent Research Results:
Small ubiquitin-related modifiers (SUMO1 and SUMO2) are ubiquitin family proteins which are structurally similar to ubiquitin, but differ in terms of their amino acid sequence and function. Therefore, they provide a great platform for investigating sequence-structure-stability-function relationship. We observed that ubiquitin is thermodynamically more stable compared to SUMOs. SUMOs mainly exhibit 2-state folding pathway, while ubiquitin has a kinetic intermediate. The spontaneous unfolding rate of ubiquitin is ~40 times lower than SUMOs. Unique energy landscape properties of ubiquitin-like proteins that we find might have implications in their function (Proteins: Structure, Function, Bioinformatics 2019, Link).
Protein conformational flexibility is important for stability and function. Here we investigated the temperature dependent protein stiffness of Small Ubiquitin-related MOdifier 2 (SUMO2) using single-molecule force spectroscopy and ensemble fluorescence spectroscopy and circular dichroism. We find that the protein tertiary structure at the core gradually melts with temperature making the overall protein malleable (J Phys Chem B 2018, Link).
Appeared on Cover of JPCB (Link)
We have shown that the mechanical unfolding pathways of azurin (apo- and copper-bound holo-forms are complex and involve intermediates and collective (diffuse) and localized transition states. (Sci. Rep. 2018, Link). We have also shown unambiguously that copper-binding changes the unfolding contour length and transition states.
We have shown that the Plasmodium falciparum vaccine candidate circumsporozoite protein (CSP) of sporozoite surface has mechanically compliant structure. (J. Biol. Chem. 2017, Link). The 43-NANP repeat region is conformationally heterogeneous while near full-length CSP has collapsed and open conformations. Most importantly, these heterogeneous conformations could be modulated with small forces <70pN, suggesting entropic elasticity could be an important factor in providing lubricating (or mechanical buffering) capacity required during the navigation of sporozoites through the mosquito and vertebrate host tissues.
We have shown that small peptide ligands stiffen the ubiquitin-like protein SUMO1 (Biophys J 2015, Link). The stiffness of SUMO1 (measured as a spring constant for the deformation response along the line joining the N- and C-termini) increased from ∼1 N/m to ∼3.5 N/m upon binding to peptides derived from SUMO-binding motifs (SBMs). The relatively higher flexibility of ligand-free SUMO1 might play a role in accessing various conformations before binding to a target.
We have shown that Ribose Binding Protein (RBP) also follows multiple pathways during mechanical unfolding and explained the detailed unfolding pathways of periplasmic binding proteins (PBPs: MBP, LBP and RBP) using topology diagrams (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
This paper is highlighted in ‘JACS Select Issue 31, December 2014: ‘Protein Dynamics in Simulation and Experiment’. Link
It is also is highlighted in ‘JACS Editorial on ‘Protein Dynamics in Simulation and Experiment’. 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:
- Nandi T, Yadav A, and Ainavarapu SRK, Experimental comparison of energy landscape features of ubiquitin family proteins. Proteins: Structure, Function, and Bioinformatics (2020), 88, p449-461. Link
- Bhattacharya S and Ainavarapu SRK, Mechanical Softening of a Small Ubiquitin-related Modifier Protein (SUMO2) Due to Temperature Induced Flexibility at the Core. J Phys Chem B (2018), 122, p9128-9136. Link – This article appeared on JPCB ‘Cover‘. Link – This article is recommended by ‘Faculty of 1000‘. Link
- Yadav A, Paul S, Venkatramani R, and Ainavarapu SRK, Differences in the mechanical unfolding pathways of apo- and copper-bound azurins. Sci Rep (2018), 8:1989, p1-13. Link
- Patra AP, Sharma S, and Ainavarapu SRK, Force spectroscopy of the Plasmodium falciparum vaccine candidate circumsporozoite protein suggests a mechanically pliable repeat region. J Biol Chem (2017), 292, 6, 2110. Link
- Kotamarthi HC, Yadav A, and Ainavarapu SRK, Small peptide binding stiffens the ubiquitin-like protein SUMO1. Biophysical Journal (2015), 108, 360. Link
- 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 – This paper is highlighted in ‘JACS Select Issue 31, December 2014: ‘Protein Dynamics in Simulation and Experiment’. Link - It is also is highlighted in ‘JACS Editorial on ‘Protein Dynamics in Simulation and Experiment’ 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)