Substrate-induced phase transition within liquid condensates reverses the catalytic activity of nanoparticles.

Liquid-liquid phase separation is reported to enhance the catalytic reaction rates severalfold. Herein, we explored the interactions between a catalyst and a range of substrate concentrations to understand the impact on the droplet phase and catalytic reaction kinetics. We observed that the substrate above a critical concentration induces phase transitions within liquid condensates and restricts the free movement of both the substrate and products, resulting in an overall reduction of the reaction rate, an observation not reported earlier.

Emergence of slip-ideal-slip behavior in tip-links serve as force filters of sound in hearing.

Tip-links in the inner ear convey force from sound and trigger mechanotransduction. Here, we present evidence that tip-links (collectively as heterotetrameric complexes of cadherins) function as force filters during mechanotransduction. Our force-clamp experiments reveal that the tip-link complexes show slip-ideal-slip bond dynamics. At low forces, the lifetime of the tip-link complex drops monotonically, indicating slip-bond dynamics. The ideal bond, rare in nature, is seen in an intermediate force regime where the survival of the complex remains constant over a wide range. At large forces, tip-links follow a slip bond and dissociate entirely to cut-off force transmission. In contrast, the individual tip-links (heterodimers) display slip-catch-slip bonds to the applied forces. While with a phenotypic mutant, we showed the importance of the slip-catch-slip bonds in uninterrupted hearing, our coarse-grained Langevin dynamics simulations demonstrated that the slip-ideal-slip bonds emerge as a collective feature from the slip-catch-slip bonds of individual tip-links.

Redefining the Structure of Tip Links in Hair Cells.

Tip links are seen under microscopes as double-helical tetrameric complexes of long nonclassical cadherins, cadherin-23 and protocadherin-15. The twisted filamentous structure enables tip links to regulate mechanotransduction in hearing and balance. While the molecular details of the double-helical protocadherin-15 cis dimers have been deciphered, a similar conformation of cadherin-23 is still elusive. In a search of cadherin-23 cis dimers, we performed photoinduced cross-linking of unmodified proteins in solution and on lipid membranes and observed no trace of cadherin-23 cis dimers. Reportedly, tip links are dynamic connections, assembling and disassembling in seconds. Using lipid vesicles, we measured significantly slower aggregations between cis dimers of tip link cadherins than via dimer-monomer interactions, indicating that the trans interactions between two cis dimers may possess steric restraints and defer reassociations. Reconnections of tip links are thus kinetically most desired between protocadherin-15 cis dimers and cadherin-23 monomers. Here we propose that the helical geometry of tip links is induced by protocadherin-15 cis dimers, while cadherin-23 remains single before tip linking.

Polyprotein synthesis: a journey from the traditional pre-translational method to modern post-translational approaches for single-molecule force spectroscopy.

Polyproteins, an array of protein units of similar or differential functions in tandem, have been extensively utilized by organisms, unicellular or multicellular, as concentrators of the myriad of molecular activities. Most eukaryotic proteins, two-thirds in unicellular organisms, and more than 80% in metazoans, are polyproteins. Although the use of polyproteins continues to evolve in nature, our understanding of the structure-function-property of polyproteins is still limited. Cumbersome recombinant strategies and the lack of convenient in vitro synthetic routes of polyproteins have been rate-determining factors in the progress. However, in this review we have discussed the revolutionary journey of polyprotein synthesis with a major focus on surface-based structure-function-property studies, especially using force spectroscopy at the single-molecule level.

Transient interactions drive the lateral clustering of cadherin-23 on membrane.

Cis and trans-interactions among cadherins secure multicellularity. While the molecular structure of trans-interactions of cadherins is well understood, work to identify the molecular cues that spread the cis-interactions two-dimensionally is still ongoing. Here, we report that transient, weak, yet multivalent, and spatially distributed hydrophobic interactions that are involved in liquid-liquid phase separations of biomolecules in solution, alone can drive the lateral-clustering of cadherin-23 on a membrane. No specific cis-dimer interactions are required for the lateral clustering. In cells, the cis-clustering accelerates cell-cell adhesion and, thus, contributes to cell-adhesion kinetics along with strengthening the junction. Although the physiological connection of cis-clustering with rapid adhesion is yet to be explored, we speculate that the over-expression of cadherin-23 in M2-macrophages may facilitate faster attachments to circulatory tumor cells during metastasis.

Aminoindole and naphthalimide based charge transfer fluorescent probes for pH sensing and live cell imaging.

Fluorescent probes are essential for imaging of cancer cells and for tracking organelles inside cells. We have synthesized three molecular rotors AIN, AINP and F-AINP based on 1-aminoindole (AI) as an electron donor and naphthalimide as an electron acceptor. All compounds showed charge transfer (CT) character, aggregation induced emission (AIE) and emission responsiveness towards temperature variation and solvent viscosity. AINP was most sensitive towards viscosity among all molecules with a viscosity sensitivity of ∼0.37. AIN, AINP and F-AINP showed negative temperature coefficients in chloroform with internal sensitivities of -0.04% °C-1, -0.08% °C-1 and -0.1% °C-1, respectively. Furthermore, all the rotors were sensitive towards the pH of the solvent environment as revealed by acid titration and base back-titration and served as colorimetric pH sensors with intriguing photophysical characteristics. Additionally, AINP and F-AINP were used to image the live cancer cell line A549 and the fibroblast cell line L929, and the imaging studies revealed the incorporation of dyes in the cytoplasmic space of the cells except for the nuclei.

Barrier-free liquid condensates of nanocatalysts as effective concentrators of catalysis.

Traditional methods of molecular confinement have physicochemical barriers that restrict the free passage of substrates/products. Here, we explored liquid-liquid phase separation as a method to restrain protein-metal nanocomposites within barrier-free condensates. Confinement within liquid droplets was independent of the protein's native conformation and amplified the catalytic efficiency of metal nanocatalysts by one order of magnitude.

Instantaneous splicing and excision of inteins to synthesize polyproteins on a substrate with tunable linkers.

Nature has adapted chimeric polyproteins to achieve superior and multiplexed functionality in a single protein. However, the hurdles in in vitro synthesis have restricted the biomimicry of and subsequent fundamental studies on the structure-function relationship of polyproteins. Recombinant expression of polyproteins and the synthesis of polyproteins via the enzyme-mediated repetitive digestion and ligation of individual protein domains have been widely practiced. However, recombinant expression often suffers from an in vitro refolding process, whereas enzyme-assisted peptide conjugation results in heterogeneous products, primarily due to enzymatic re-digestion, and prolonged and multistep reactions. Moreover, both methods incorporate enzyme-recognition residues of varying lengths as artifacts at interdomain linkers. The linkers, although tiny, regulate the spatiotemporal conformations of the polyproteins differentially and tune the folding dynamics, stability, and functions of the constituent protein. In an attempt to leave no string behind at the interdomain junctions, here, we develop a 'splice and excise' synthetic route for polyproteins on a substrate using two orthogonal split inteins. Inteins self-excise and conjugate the protein units covalently and instantaneously, without any cofactors, and incorporate a single cysteine or serine residue at the interdomain junctions.

Anisotropy in mechanical unfolding of protein upon partner-assisted pulling and handle-assisted pulling.

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.

Interdomain linkers tailor the stability of immunoglobulin repeats in polyproteins.

Linkers in polyproteins are considered as mere spacers between two adjacent domains. However, a series of studies using single-molecule force spectroscopy have recently reported distinct thermodynamic stability of I27 in polyproteins with varying linkers and indicated the vital role of linkers in domain stability. A flexible glycine rich linker (-(GGG)n, n ≥ 3) featured unfolding at lower forces than the regularly used arg-ser (RS) based linker. Interdomain interactions among I27 domains in Gly-rich linkers were suggested to lead to reduced domain stability. However, the negative impact of inter domain interactions on domain stability is thermodynamically counter-intuitive and demanded thorough investigations. Here, using an array of ensemble equilibrium experiments and in-silico measurements with I27 singlet and doublets with two aforementioned linkers, we delineate that the inter-domain interactions in fact raise the stability of the polyprotein with RS linker. More surprisingly, a highly flexible Gly-rich linker has no interference on the stability of polyprotein. Overall, we conclude that flexible linkers are preferred in a polyprotein for maintaining domain's independence.

Weakening of interaction networks with aging in tip-link protein induces hearing loss.

Age-related hearing loss (ARHL) is a common condition in humans marking the gradual decrease in hearing with age. Perturbations in the tip-link protein cadherin-23 that absorbs the mechanical tension from sound and maintains the integrity of hearing is associated with ARHL. Here, in search of molecular origins for ARHL, we dissect the conformational behavior of cadherin-23 along with the mutant S47P that progresses the hearing loss drastically. Using an array of experimental and computational approaches, we highlight a lower thermodynamic stability, significant weakening in the hydrogen-bond network and inter-residue correlations among ß-strands, due to the S47P mutation. The loss in correlated motions translates to not only a remarkable two orders of magnitude slower folding in the mutant but also to a proportionately complex unfolding mechanism. We thus propose that loss in correlated motions within cadherin-23 with aging may trigger ARHL, a molecular feature that likely holds true for other disease-mutations in ß-strand-rich proteins.

Superior Proton-Transfer Catalytic Promiscuity of Cytochrome†c in Self-Organized Media.

Evolutionarily elderly proteins commonly feature greater catalytic promiscuity. Cytochrome c is among the first set of proteins in evolution to have known prospects in electron transport and peroxidative properties. Here, we report that cyt c is also a proficient proton-transfer catalyst and enhances the Kemp elimination (KE; model reaction to show proton transfer catalytic property) by ∼750-fold on self-organized systems like micelles and vesicles. The self-organized systems mimic the mitochondrial environment in vitro for cyt c. Using an array of biophysical and biochemical mutational assays, both acid-base and redox mechanistic pathways have been explored. The histidine moiety close to hemin group (His18) is mainly responsible for proton abstraction to promote the concerted E2 pathway for KE catalysis when cyt c is in its oxidized form; this has also been confirmed by a H18A mutant of cyt c. However, the redox pathway is predominant under reducing conditions in the presence of dithiothreitol over the pH range 6-7.4. Interestingly, we found almost 750-fold enhanced KE catalysis by cyt c compared to aqueous buffer. Overall, in addition to providing mechanistic insights, the data reveal an unprecedented catalytic property of cyt c that could be of high importance in an evolutionary perspective considering its role in delineating the phylogenic tree and also towards generating programmable designer biocatalysts.

Variable Mutations at the p53-R273 Oncogenic Hotspot Position Leads to Altered Properties.

Mutations in p53 protein, especially in the DNA-binding domain, is one of the major hallmarks of cancer. The R273 position is a DNA-contact position and has several oncogenic variants. Surprisingly, cancer patients carrying different mutant variants of R273 in p53 have different survival rates, indicating that the DNA-contact inhibition may not be the sole reason for reduced survival with R273 variants. Here, we probed the properties of three major oncogenic variants of the wild-type (WT) p53: [R273H]p53, [R273C]p53, and [R273L]p53. Using a series of biophysical, biochemical, and theoretical simulation studies, we observe that these oncogenic variants of the p53 not only suffer a loss in DNA binding, but they also show distinct structural stability, aggregation, and toxicity profiles. The WTp53 and the [R273H]p53 show the least destabilization and aggregation propensity. [R273C]p53 aggregation is disulfide mediated, leading to cross-ß, thioflavin-T-positive aggregates, whereas hydrophobic interactions dominate self-assembly in [R273L]p53, leading to a mixture of amyloid and amorphous aggregates. Molecular dynamics simulations indicate different contact maps and secondary structures for the different variants along the course of the simulations. Our study indicates that each of the R273 variants has its own distinct property of stability and self-assembly, the molecular basis of which may lead to different types of cancer pathogenesis in vivo. These studies will aid the design of therapeutic strategies for cancer using residue-specific or process-specific protein aggregation as a target.

Broken force dispersal network in tip-links by the mutations at the Ca2+-binding residues induces hearing-loss.

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.

The strong propensity of Cadherin-23 for aggregation inhibits cell migration.

Cadherin-23 (Cdh23), a long-chain non-classical cadherin, exhibits strong homophilic and heterophilic binding. The physiological relevance of strong heterophilic binding with protocadherin-15 at neuroepithelial tip links is well-studied. However, the role of Cdh23 homodimers in physiology is less understood, despite its widespread expression at the cell boundaries of various human and mouse tissues, including kidney, muscle, testes, and heart. Here, we performed immunofluorescence studies that revealed that Cdh23 is present as distinct puncta at the cell-cell boundaries of cancer cells. Analysis of patient data and quantitative estimation of Cdh23 in human tissues (normal and tumor) also indicated that Cdh23 is down-regulated via promoter methylation in lung adenocarcinoma (AD) and esophageal squamous cell carcinoma (SCC) cells; we also observed a clear inverse correlation between Cdh23 expression and cancer metastasis. Using HEK293T cells and four types of cancer cells differentially expressing Cdh23, we observed that cell migration was faster in cells with reduced levels of Cdh23 expression. The cell migration rate in cancer cells is further accelerated by the presence of excretory isoforms of Cdh23, which loosen its cell-adhesion ability by competitive binding. Overall, our data indicate the role of Cdh23 as a suppressor of cell migration.

Force-activated catalytic pathway accelerates bacterial adhesion against flow.

Mechanical cues often influence the factors affecting the transition states of catalytic reactions and alter the activation pathway. However, tracking the real-time dynamics of such activation pathways is limited. Using single-molecule trapping of reaction intermediates, we developed a method that enabled us to perform one reaction at one site and simultaneously study the real-time dynamics of the catalytic pathway. Using this, we showed single-molecule calligraphy at nanometer resolution and deciphered the mechanism of the sortase A enzymatic reaction that, counter-intuitively, accelerates bacterial adhesion under shear tension. Our method captured a force-induced dissociation of the enzyme-substrate bond that accelerates the forward reaction 100×, proposing a new mechano-activated catalytic pathway. In corroboration, our molecular dynamics simulations in the presence of force identified a force-induced conformational switch in the enzyme that accelerates proton transfer between CYS184 (acceptor) and HIS120 (donor) catalytic dyads by reducing the inter-residue distances. Overall, the present study opens up the possibility of studying the influence of factors affecting transition states in real time and paves the way for the rational design of enzymes with enhanced efficiency.

Tailored Polyproteins Using Sequential Staple and Cut.

Polyproteins, individual protein units joined covalently in tandem, have evolved as a promising tool for measuring the dynamic folding of biomacromolecules in single-molecule force spectroscopy. However, the synthetic routes to prepare polyproteins have been a bottleneck, and urge development of in vitro methods to knit individual protein units covalently into polyprotein. Employing two enzymes of orthogonal functionalities periodically in sequence, we synthesized monodispersed polyproteins on a solid surface. We used Sortase A (SrtA), the enzyme known for sequence specific transpeptidation, to staple protein units covalently through peptide bonds. Exploiting the sequence-specific peptide cleaving ability of TEV protease, we controlled the progress of the reaction to one attachment at a time. Finally, with unique design of the unit proteins we control the orientation of proteins in polyprotein. This simple conjugation has the potential to staple proteins with different functionalities and from different expression systems, in any number in the polyprotein and, above all, via irreversible peptide bonds. Multiple chimeric constructs can also be synthesized with interchangeable protein units.

The Prospects of Cadherin-23 as a Mediator of Homophilic Cell-Cell Adhesion.

Cadherins (calcium-dependent adhesion proteins) constitute a family of cell surface proteins that mediate cell-cell adhesion and actively participate in tissue morphogenesis and in mediating tissue integrity. The ecto-domains of cadherins from opposing cell surfaces interact with each other to form the load-bearing trans-dimers and mechanically hold cells together. The "classical" cadherins and desmosomes that form separate groups in cadherin superfamily are mostly explored for their roles in cell-cell adhesion. However, majority of cadherins in cells belong to "nonclassical" group which is poorly explored in the context of their cell-binding properties. This review focuses on the role of "nonclassical" cadherin, cadherin-23, in cell-cell adhesion. Overall, this review highlights the need for further investigations on the role of "nonclassical" cadherin-23 in cell-cell adhesion.

ESCORTing proteins directly from whole cell-lysate for single-molecule studies.

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.

Resolving the molecular mechanism of cadherin catch bond formation.

Classical cadherin Ca(2+)-dependent cell-cell adhesion proteins play key roles in embryogenesis and in maintaining tissue integrity. Cadherins mediate robust adhesion by binding in multiple conformations. One of these adhesive states, called an X-dimer, forms catch bonds that strengthen and become longer lived in the presence of mechanical force. Here we use single-molecule force-clamp spectroscopy with an atomic force microscope along with molecular dynamics and steered molecular dynamics simulations to resolve the molecular mechanisms underlying catch bond formation and the role of Ca(2+) ions in this process. Our data suggest that tensile force bends the cadherin extracellular region such that they form long-lived, force-induced hydrogen bonds that lock X-dimers into tighter contact. When Ca(2+) concentration is decreased, fewer de novo hydrogen bonds are formed and catch bond formation is eliminated.