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.

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.

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.

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.

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.

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.