We have developed a method for Enzymatic Sortase-assisted Covalent Orientation-specific Restraint Tethering (ESCORT) recombinant proteins onto surfaces directly from cell-lysate. With an improved surface passivation method, we obviate the cumbersome purification steps even for single molecule studies that demand high purity in the sample. We demonstrated high-specificity of the method, high-passivity of the surface and uncompromised functional integrity of anchored proteins using single molecule fluorescence and force-mapping. We anticipate that this method will substantially reduce the investment by way of time, money and energy in the area of single molecule studies.

Cadherin-23, a giant atypical cadherin, form homophilic interactions at the cell-cell junction of epithelial cells and heterophilic interactions with protocadherin-15 at the tip links of neuroepithelial cells. While the molecular structure of the heterodimer is solved, the homodimer structure is yet to be resolved. The homodimers play an essential role in cell-cell adhesion as the downregulation of cadherin-23 in cancers loosen the intercellular junction resulting in faster migration of cancer cells and a significant drop in patient survival. In vitro studies have measured a stronger aggregation propensity of cadherin-23 compared to typical E-cadherin. Here, we deciphered the unique trans-homodimer structure of cadherin-23 in solution and show that it consists of two electrostatic-based interfaces extended up to two terminal domains. The interface is robust, with a low off-rate of~8 9 10 À4 s À1 that supports its strong aggregation propensity. We identified a point mutation, E78K, that disrupts this binding. Interestingly, a mutation at the interface was reported in skin cancer. Overall, the structural basis of the strong cadherin-23 adhesion may have far-reaching applications in the fields of mechanobiology and cancer.

Proteins as force-sensors respond to mechanical cues and regulate signaling in physiology. Proteins commonly connect the source and response points of mechanical cues in two conformations, independent proteins in end-to-end geometry and protein complexes in handshake geometry. The force-responsive property of independent proteins in end-to-end geometry is studied extensively using single-molecule force spectroscopy (SMFS). The physiological significance of the complex conformations in force-sensing is often disregarded as mere surge protectors. However, with the potential of force-steering, protein complexes possess a distinct mechano-responsive property over individual force-sensors. To decipher, we choose a force-sensing protein, cadherin-23, from tip-link complex and perform SMFS using end-to-end geometry and handshake complex geometry. We measure higher force-resilience of cadherin-23 with preferential shorter extensions in handshake mode of pulling over the direct mode. The handshake geometry drives the force-response of cadherin-23 through different potential-energy landscapes than direct pulling. Analysis of the dynamic network structure of cadherin-23 under tension indicates narrow force-distributions among residues in cadherin-23 in direct pulling, resulting in low force-dissipation paths and low resilience to force. Overall, the distinct and superior mechanical responses of cadherin-23 in handshake geometry than single protein geometry highlight a probable evolutionary drive of protein-protein complexes as force-conveyors over independent ones.

Bacterial microcompartments (MCPs) are polyhedral organelles containing an enzyme cluster wrapped inside a protein shell and carry out specific enzyme reactions in bacteria. These organelles have been explored meticulously using genetic, structural, and biochemical tools; however, their application in material science has not been explored much. In this study, we have used the external shell surface of MCP as a scaffold for the fabrication of gold nanoparticles displayed in 3D. This resulted in the formation of a protein scaffolded gold nanoparticle shell enclosing an active enzyme cluster. The surface scaffolded gold nanoparticles demonstrated standard catalysis, while the internal enzyme cluster of the MCPs demonstrated no loss in activity due to this fabrication. Under ambient conditions for in vitro inorganic reaction, the shell proteins sturdily maintain the barrier between the luminal enzyme and the surface inorganic catalysts and preserve the functionality of the core enzyme cluster. The MCP-AuNPs hybrid catalysts inspire a pristine class of resources that can be used in a chemical reaction condition without perturbing core biological environment for enzymatic activity. This system provides insight for fabricating uniform size nanoparticles in 3D for the development of orthogonal hybrid catalytic material.

Tip-link as force-sensor in hearing conveys the mechanical force originating from sound to ion-channels while maintaining the integrity of the entire sensory assembly in the inner ear. This delicate balance between structure and function of tip-links is regulated by Ca2+-ions present in endolymph. Mutations at the Ca2+-binding sites of tip-links often lead to congenital deafness, sometimes syndromic defects impairing vision along with hearing. Although such mutations are already identified, it is still not clear how the mutants alter the structure-function properties of the force-sensors associated with diseases. With an aim to decipher the differences in force-conveying properties of the force-sensors in molecular details, we identified the conformational variability of mutant and wild-type tip-links at the single-molecule level using FRET at the endolymphatic Ca2+ concentrations and subsequently measured the force-responsive behavior using single-molecule force spectroscopy with an Atomic Force Microscope (AFM). AFM allowed us to mimic the high and wide range of force ramps (103–106 pN s−1) as experienced in the inner ear. We performed in silico network analysis to learn that alterations in the conformations of the mutants interrupt the natural force-propagation paths through the sensors and make the mutant tip-links vulnerable to input forces from sound stimuli. We also demonstrated that a Ca2+ rich environment can restore the force-response of the mutant tip-links which may eventually facilitate the designing of better therapeutic strategies to the hearing loss.