Thermoresponsive molecular interface for in-demand release of surface-immobilised proteins

In the last decades, biomedicine opened its doors to stimuli-responsive biomaterials, with applications ranging from tissue engineering to drug delivery systems. Among them, polymers are by far the most investigated, thanks to their biocompatibility and their vast range of properties, tunable to match their end-use. Protein-based thermoresponsive materials are increasingly being studied, especially for drug delivery applications, where dedicated chemistries for on-demand capture and release of biomolecules at the solid-liquid interface are essential. This is an important requirement for the synthesis of switchable surfaces used in analytical devices and for the assembly of novel smart materials with complex architectures and functions. Here the design, synthesis and characterisation of novel peptide tags for reversible protein capture and thermoresponsive release from a solid surface are reported. The peptide sequences were inspired by the selfassembling protein machinery dedicated to vesicle fusion in eukaryotes, known as the SNARE proteins. The three proteins involved, named SNAP25, syntaxin and VAMP, all present a highly preserved strand (SNARE motif) that allows them to assemble in a tight coiled-coil structure upon interaction, called SNARE complex. This four α-helix bundle has remarkable chemical and thermal stability, withstanding temperatures up to 80°C and changes of pH. The structural features of the native ternary protein complex were engineered to yield a binary self-assembling polypeptide system. The first element of the binary system is a universal protein substrate immobilised on a solid surface. This protein mimics the neuronal SNAP25, which is involved in the docking and fusion of synaptic vesicles to the synaptic membrane. The second element is a protein fusion of syntaxin and VAMP, acting as a polypeptide tag; it includes SNARE motifs from both syntaxin and VAMP, capable of self-assembly in a coiled-coil structure when coupled with SNAP25, even when immobilised on a surface. This interaction is strong but fully-reversible; therefore, this polypeptide tag can be recombinantly fused to a protein of interest to allow spontaneous assembly and stimuli-sensitive release from the surface upon heating at a predetermined temperature. Two VAMP-syntaxin protein fusions were produced, with different VAMP lengths: in the first, VAMP's SNARE motif spans for 54 amino acids, matching syntaxin's length. In the second, VAMP has been reduced to 25 amino acids, truncated before the residue involved in the ionic layer, which helps stabilise the coiled-coil structure of the SNARE complex. The protein fusion with the shortened VAMP has been designed to weaken the SNARE complex thermal stability and test its disassembly temperature. The recombinant proteins described above were characterised with pulldown assays and circular dichroism spectroscopy, testing their ability to form a stable SNARE complex. Two VAMP-syntaxin thermoresponsive tags are described: results show that the first one, with the 54-amino acid VAMP, presents remarkable thermal stability with Tm of the order of 80°C. The second tag, with the truncated VAMP, disassembles at a substantially lower temperature of about 45°C. The latter is a promising candidate for remote-controlled localised delivery of therapeutic proteins: the physiologically tolerable local increase of temperature in the 40-45°C range, also known as hyperthermia, can be achieved using magnetic fields, infra-red light or focused ultrasound. Notably, these two novel polypeptides provide an example for the engineering of future functional proteins with predictable folding and response to external stimuli.