The Rational Design of a Functional Solvent-free Liquid Protease

Abstract

Recent research into the rational design and synthesis of solvent-free liquid proteins has yielded remarkable results. For example, these novel anhydrous biofluids maintain secondary structure at temperatures in excess of 150 °C, are able to dynamically unfold and refold, and retain enzymatic activity towards small molecules at extreme temperature. Accordingly, the global aim of this thesis was to demonstrate proteolytic activity in the absence of solvent via the synthesis of a solvent-free liquid protease and an analogous protein-based substrate. This involved increasing the positive surface charge density of the enzyme Proteinase K (PK) and the substrate protein bovine serum albumin (BSA) through chemical cationization, followed by the electrostatic surface grafting of anionic polymer-surfactant molecules, and subsequent lyophilization and melting. Significantly, this methodology was used to successfully synthesise solvent-free liquids of both PK and BSA for the first time, both exhibiting high concentrations (over 250 mg/ml) and lower than solvation levels of water (fewer than 30 water molecules per protein construct).

Biophysical analysis, including circular dichroism spectroscopy, was undertaken on the modified proteins after each stage of the synthesis, assessing how the surface modifications had altered the conformation and structural thermal stability. High levels of secondary structure were present throughout the synthesis for both proteins, although deviation away from the native-like structure was seen after cationization, which was to some extent recovered after surfactant conjugation and solvent-free liquid formation. This indicated that the polymer-surfactant corona created during the synthesis could replace structurally significant water molecules and replicate the majority of the associated water-protein interactions, resulting in melting points in the vicinity of 40 °C.

Enzymatic kinetic assessments of solvent-free liquid PK and its aqueous precursors were conducted via the development of a novel circular dichroism spectroscopy-based enzymatic assay; a spectroscopic technique often utilised to examine the secondary structure of a protein. By tracking the reduction in substrate secondary structure, protease activity towards solvent-free liquid BSA, and its aqueous precursors, was evaluated. In general, cationization of either the PK or BSA was shown to reduce proteolytic activity due to both structural alterations and an increase in electrostatic repulsion between the protease and the substrate. Surfactant conjugation also reduced the proteolytic activity, in this case because of the surfactant molecules increasing steric hinderance of the protease active site. Formation of the solvent-free liquid state again reduced the proteolytic activity, due in part to the large increase in viscosity hampering diffusion of the macromolecules. However, the retention of even low levels of proteolytic activity in the absence of solvent indicated that the dynamical freedom given by the water hydration shell to the protein had also been replicated by the polymer corona. This retention of similar surface interactions enabled the protease to perform the dynamic conformational fluctuations required for proteolytic activity.

In light of the persistent structure, activity, and extremely high protein concentrations in the solvent-free liquids (ca. 250 mg mL-1), a novel protocell encapsulation platform was developed. Here, the successful encapsulation of highly concentrated protein-polymer conjugate within silica colloidosomes was achieved through the use of solvent-free liquid myoglobin. The resulting hybrid protein-containing colloidosomes were structurally stable, shown to maintain spherical geometry under the vacuum of scanning electron microscopy, and did not exhibit signs of protein construct leakage.

The outcomes of the work presented in this thesis challenge the perceived necessity of water in roles such as stabilization of protein structure and proteolytic function, and also expands the gallery of macromolecular liquids. Moreover, the ability to encapsulate extremely high concentrations of structurally stable protein nano-conjugates paves the way for a whole new class of protocells with complex internal chemistry.

Date of Award	19 Mar 2019
Original language	English
Awarding Institution	University of Bristol
Sponsors	GlaxoSmithKline
Supervisor	Stephen Mann (Supervisor), Adam W Perriman (Supervisor) & Christabel Fowler (Supervisor)