Biotechnological applications of a surfactant protein, ranaspumin-2

Surfactant activity is generally associated with small molecules rather than biological macromolecules like proteins. Only a few proteins have good intrinsic surfactant activity, an example being the natural surfactant ranaspumin 2 (Rsn2) from the foam nests of the túngara frog. In solution, Rsn2 has a hydrophobic core and hydrophilic exterior, but when Rsn2 comes in contact with an air-water interface, it changes conformation to expose its hydrophobic core to interact with the air and present a hydrophilic face to the water. The unique combination of biocompatibility along with surface activity offers the possibility of developing biomedical applications based on Rsn2. Some of the possible applications, including cell patterning, functionalising scaffolds and stabilising droplets, have been explored in the work described in this thesis. The ability of Rsn2 to coat hydrophobic surfaces persistently, rendering them wettable and the nature of coating and interaction with the surfaces were investigated. This formed the basis for the development of a method to coat a range of hydrophobic polymers, which are commonly used for biomedical applications. These Rsn2 coated surfaces were tested for their capability to control cell adhesion on surfaces which usually do not support cell adhesion. Rsn2 coating was demonstrated to promote, and thus allowed the spatial control over, cell adhesion on otherwise non-cell compatible surfaces. The potential of Rsn2 to be used as a protein fusion partner for the production of further functionalised cell engineering substrates was explored by constructing five different integrin binding sequence (IBS)-Rsn2 conjugates. Specific IBS-Rsn2 proteins proved successful in increasing the adhesion and biomineralising potential of osteoblasts isolated from neonatal rats. In addition, Rsn2's ability to stabilise microscopic oil droplets were investigated. Rsn2 stabilised oil droplets were stable for more than six months. Thus, the surfactant properties of Rsn2 can be used for many potential biomedical applications

Aqueous solubilization of C60 fullerene by natural protein surfactants, latherin and ranaspumin-2

C60 fullerene is not soluble in water and dispersion usually requires organic solvents, sonication or vigorous mechanical mixing. However, we show here that mixing of pristine C60 in water with natural surfactant proteins latherin and ranaspumin-2 (Rsn-2) at low concentrations yields stable aqueous dispersions with spectroscopic properties similar to those previously obtained by more vigorous methods. Particle sizes are significantly smaller than those achieved by mechanical dispersion alone, and concentrations are compatible with clusters approximating 1:1 protein:C60 stoichiometry. These proteins can also be adsorbed onto more intractable carbon nanotubes. This promises to be a convenient way to interface a range of hydrophobic nanoparticles and related materials with biological macromolecules, with potential to exploit the versatility of recombinant protein engineering in the development of nano-bio interface devices. It also has potential consequences for toxicological aspects of these and similar nanoparticles

The Conformation of Interfacially Adsorbed Ranaspumin-2 Is an Arrested State on the Unfolding Pathway

Ranaspumin-2 (Rsn-2) is a surfactant protein found in the foam nests of the t\'{u}ngara frog. Previous experimental work has led to a proposed model of adsorption which involves an unusual clam shell-like `unhinging' of the protein at an interface. Interestingly, there is no concomitant denaturation of the secondary structural elements of Rsn-2 with the large scale transformation of its tertiary structure. In this work we use both experiment and simulation to better understand the driving forces underpinning this unusual process. We develop a modified G\={o}-model approach where we have included explicit representation of the side-chains in order to realistically model the interaction between the secondary structure elements of the protein and the interface. Doing so allows for the study of the underlying energy landscape which governs the mechanism of Rsn-2 interfacial adsorption. Experimentally, we study targeted mutants of Rsn-2, using the Langmuir trough, pendant drop tensiometry and circular dichroism, to demonstrate that the clam-shell model is correct. We find that Rsn-2 adsorption is in fact a two-step process: the hydrophobic N-terminal tail recruits the protein to the interface after which Rsn-2 undergoes an unfolding transition which maintains its secondary structure. Intriguingly, our simulations show that the conformation Rsn-2 adopts at an interface is an arrested state along the denaturation pathway. More generally, our computational model should prove a useful, and computationally efficient, tool in studying the dynamics and energetics of protein-interface interactions.Comment: 8 figure