Much of life’s complexity depends upon contacts between proteins with precise affinity and specificity. The successful application of engineered proteins often depends on high-stability binding to their target. In recent years, various approaches have enabled proteins to form irreversible covalent interactions with protein targets. However, the rate of such reactions is a major limitation to their use. Infinite affinity refers to the ideal where such covalent interaction occurs at the diffusion limit. Prototypes of infinite affinity pairs have been achieved using nonnatural reactive groups. After library-based evolution and rational design, here we establish a peptide–protein pair composed of the regular 20 amino acids that link together through an amide bond at a rate approaching the diffusion limit. Reaction occurs in a few minutes with both partners at low nanomolar concentration. Stopped flow fluorimetry illuminated the conformational dynamics involved in docking and reaction. Hydrogen–deuterium exchange mass spectrometry gave insight into the conformational flexibility of this split protein and the process of enhancing its reaction rate. We applied this reactive pair for specific labeling of a plasma membrane target in 1 min on live mammalian cells. Sensitive and specific detection was also confirmed by Western blot in a range of model organisms. The peptide–protein pair allowed reconstitution of a critical mechanotransmitter in the cytosol of mammalian cells, restoring cell adhesion and migration. This simple genetic encoding for rapid irreversible reaction should provide diverse opportunities to enhance protein function by rapid detection, stable anchoring, and multiplexing of protein functionality.

There is need for effective and affordable vaccines against SARS-CoV-2 to tackle the ongoing pandemic. In this study, we describe a protein nanoparticle vaccine against SARS-CoV-2. The vaccine is based on the display of coronavirus spike glycoprotein receptor-binding domain (RBD) on a synthetic virus-like particle (VLP) platform, SpyCatcher003-mi3, using SpyTag/SpyCatcher technology. Low doses of RBD-SpyVLP in a prime-boost regimen induce a strong neutralising antibody response in mice and pigs that is superior to convalescent human sera. We evaluate antibody quality using ACE2 blocking and neutralisation of cell infection by pseudovirus or wild-type SARS-CoV-2. Using competition assays with a monoclonal antibody panel, we show that RBD-SpyVLP induces a polyclonal antibody response that recognises key epitopes on the RBD, reducing the likelihood of selecting neutralisation-escape mutants. Moreover, RBD-SpyVLP is thermostable and can be lyophilised without losing immunogenicity, to facilitate global distribution and reduce cold-chain dependence. The data suggests that RBD-SpyVLP provides strong potential to address clinical and logistic challenges of the COVID-19 pandemic.

β3 integrin residue Y747 is required for cell spreading and paxillin adapter recruitment to substrate-bound integrins in response to substrate stiffness.

Serological detection of antibodies to SARS-CoV-2 is essential for establishing rates of seroconversion in populations, and for seeking evidence for a level of antibody that may be protective against COVID-19 disease. Several high-performance commercial tests have been described, but these require centralised laboratory facilities that are comparatively expensive, and therefore not available universally. Red cell agglutination tests do not require special equipment, are read by eye, have short development times, low cost and can be applied at the Point of Care. Here we describe a quantitative Haemagglutination test (HAT) for the detection of antibodies to the receptor binding domain of the SARS-CoV-2 spike protein. The HAT has a sensitivity of 90% and specificity of 99% for detection of antibodies after a PCR diagnosed infection. We will supply aliquots of the test reagent sufficient for ten thousand test wells free of charge to qualified research groups anywhere in the world.

Poly(vinylidene fluoride) (PVDF) is a biocompatible material with excellent electroactive properties. Nonelectroactive α-PVDF and electroactive β-PVDF were used to investigate the substrate polarization and polarity influence on the focal adhesion (FA) size and number as well as on human adipose stem cells (hASCs) differentiation. hASCs were cultured on different PVDF surfaces adsorbed with fibronectin and FA size and number, total adhesion area, cell size, cell aspect ratio and FA density were estimated using cells expressing vinculin fused to enhanced green fluorescent protein. Osteogenic differentiation was also determined using a quantitative alkaline phosphatase assay. The surface charge of the poled PVDF films (positive or negative) influenced the hydrophobicity of the samples, leading to variations in the conformation of adsorbed extracellular matrix proteins, which ultimately modulated the stem cell adhesion on the films and induced their osteogenic differentiation.

The mechanical unfolding of proteins is a cellular mechanism for force transduction with potentially broad implications in cell fate. Despite this, the mechanism by which protein unfolding elicits differential downstream signalling pathways remains poorly understood. Here, we used protein engineering, atomic force microscopy, and biophysical tools to delineate how protein unfolding controls cell mechanics. Deleted in liver cancer 1 (DLC1) is a negative regulator of Ras homolog family member A (RhoA) and cell contractility that regulates cell behaviour when localised to focal adhesions bound to folded talin. Using a talin mutant resistant to force-induced unfolding of R8 domain, we show that talin unfolding determines DLC1 downstream signalling and, consequently, cell mechanics. We propose that this new mechanism of mechanotransduction may have implications for a wide variety of associated cellular processes.

Cells adhere to the surrounding tissue and probe its mechanical properties by forming cell-matrix adhesions. Talin is a critical adhesion protein and participates in the transmission of mechanical signals between extracellular matrix and cell cytoskeleton. Force induced unfolding of talin rod subdomains has been proposed to act as a cellular mechanosensor, but so far evidence linking their mechanical stability and cellular response has been lacking. Here, by utilizing computationally designed mutations, we demonstrate that stepwise destabilization of the talin rod R3 subdomain decreases cellular traction force generation, which affects talin and vinculin dynamics in cell-matrix adhesions and results in the formation of talin-rich but unstable adhesions. We observed a connection between talin stability and the rate of cell migration and also found that talin destabilization affects the usage of different integrin subtypes and sensing of extracellular matrix proteins. Experiments with truncated forms of talin confirm the mechanosensory role of the talin R3 subdomain and exclude the possibility that the observed effects are caused by the release of talin head-rod autoinhibition. In conclusion, this study provides evidence into how the controlled talin rod domain unfolding acts as a key regulator of adhesion structure and function and consequently controls central cellular processes such as cell migration and substrate sensing.

Talin protein is one of the key components in integrin-mediated adhesion complexes. Talins transmit mechanical forces between β-integrin and actin, and regulate adhesion complex composition and signaling through the force-regulated unfolding of talin rod domain. Using modified talin proteins, we demonstrate that these functions contribute to different cellular processes and can be dissected. The transmission of mechanical forces regulates adhesion complex composition and phosphotyrosine signaling even in the absence of the mechanically regulated talin rod subdomains. However, the presence of the rod subdomains and their mechanical activation are required for the reinforcement of the adhesion complex, cell polarization and migration. Talin rod domain unfolding was also found to be essential for the generation of cellular signaling anisotropy, since both insufficient and excess activity of the rod domain severely inhibited cell polarization. Utilizing proteomics tools, we identified adhesome components that are recruited and activated either in a talin rod-dependent manner or independently of the rod subdomains. This study clarifies the division of roles between the force-regulated unfolding of a talin protein (talin 1) and its function as a physical linker between integrins and the cytoskeleton.