Rational engineering of self-assembling thermosensitive protein complexes and their applications

Key aims of this research are to understand fundamental molecular mechanisms underlying the unusual stability of soluble n-ethylmaleimide-sensitive factor attachment-protein receptor (SNARE) proteins and to apply any discoveries to engineer novel self-assembling proteins where their assembly of disassembly could be controlled with externally applied chemical or physical stimuli. SNAREs are a family of membrane proteins responsible for driving membrane fusion in eukaryotic cell. Neuronal SNAREs comprise three proteins, which are referred to as syntaxin, SNAP-25 (which contributes two α-helices), and Vesicle Associated Membrane Protein (VAMP), also referred to interchangeably as synaptobrevin. These proteins contain SNARE domains, which drive the self- assembly of the three proteins into stable heterotetrameric coiled coil complexes exhibiting extraordinary chemical tolerance to proteases and detergents, and thermoresistance to temperatures of ~90°C. We compiled a database of all SNAREs with elucidated structures obtained from Protein Data Bank, and built a SNARE protein structure compactness analysis tool to analyse their sequences and draw our initial hypotheses about the mechanisms of SNARE assembly. We subsequently used the tool to predict in silico, how modifications to the secondary structures of a full-length neuronal SNARE complex (sp. Rattus norvegicus) would affect the assembly, and hence the stability of the complex. By virtue of its molecular structure, the individual native SNARE domains exist as fully unfolded polypeptides, which adopt alpha-helical coiled-coil following the assembly. Therefore, circular dichroism (CD) spectroscopy was used as the main method for the experimental evaluation of assembly and stability of the engineered proteins. Where possible other methods were applied, including using native polyacrylamide gel electrophoresis (PAGE), pull down assays and thermal shift assay screen.

A number of peptides were rationally engineered to systematically check key residues predicted to strongly influence complex assembly, stability, and disassembly. Special emphasis was on the development of proteins capable of disassembly in response to small temperature changes within the physiological range and in response to the change in pH. Both of these challenges have been addressed in the course of this research. The newly engineered proteins showed disassembly over an extensive range of 34 – 83°C, displaying the wide potential available for modifying complexes with bespoke stability. New pH sensitive complexes were also engineered with increased sensitivity to high pH (disassembly at pH 8). Furthermore, we explored strategies for nanoparticle-protein conjugation using full-length SNARE complexes, to investigate the applicability of SNARE proteins as potential linkers in delivery devices.