Dr. Trevor Douglas

Our research is aimed at understanding mineral formation in biological systems and applying what we learn to the synthesis of novel materials. Of particular interest is the iron storage protein, ferritin, which sequesters and stores iron as a small particle (~6 nm diam.) of iron oxide (rust). Ferritin is a 24 subunit protein which assembles into a cage-like structure with hollow central cavity. We are interested in how the electrostatics of the protein affect the uptake of Fe and subsequent mineralization reactions. Specifically, we are investigating how altering regions of charge on the protein (interior and exterior) affects the oxidative hydrolysis and mineralization reactions transition metals such as Fe, Co, and Mn. This involves modification of the protein using site directed mutagenesis as well as direct chemical modification. We have begun to use this biomimetic approach to inorganic materials synthesis in conjunction with carboxyl terminated poly(amido)amine dendrimers and shown that we can affect a high degree of control over the nanoparticle phase and morphology. The oxidative hydrolysis and mineralization reactions are monitored by dynamic titration of H⁺ released during the reaction, spectroscopically in uv-visible through the development of charge transfer bands, and by dynamic light scattering which allows us to monitor spatially selective mineralization within the protein cage of ferritin. Analysis of the resulting mineral-protein composite is undertaken using transmission electron microscopy which allows us to collect electron diffraction and elemental composition of a single (6 nm) mineral core.

Our work on ferritin has also inspired our investigation of a wide range of protein cage structures with the potential to act as nano-containers for encapsulation of both inorganic and organic materials. These include the use of virus capsid proteins which assemble into protein cage-like structures based on an icosahedral symmetry. We are applying a biomimetic approach to the synthesis of size and shape constrained materials in these virus-derived protein cages. In collaboration with Prof. Mark Young (Plant Sciences and Microbiology) we have used the empty virus coat protein from Cowpea Chlorotic Mottle Virus (CCMV) as a model system to demonstrate the feasibility of this approach. These protein cages self-assemble into 180-subunit assemblies with a well defined central cavity which we have shown can be used as a constrained reaction environment. These proteins are remarkable in that they are pleomorphic (i.e. can adopt many sizes and shapes). Also, through extensive site directed mutagenesis, we have mutants available which are extremely stable to pH (3-9) and temperature (70^oC). Changing basic residues on the interior surface of the viral cage to acidic results in a protein which acts, much like L-chain ferritin, to selectively induce oxidative hydrolysis and mineralization of an iron oxide nano-particle within the protein cage. We believe this to be the result of a interfacial aggregation of ions at the highly anionic interior interface of the protein.

Perhaps one of the most remarkable features of this viral capsid assembly is the potential for switchable gating. That is, the virion undergoes a dramatic conformational change in response to a change in pH and/or metal ion concentration. As shown in the cryo image reconstruction below the virion switches between a non-swollen and a swollen form, which results in the opening of 60 separate pores (~20 Å in diam.) in the protein cage. In the open (swollen) state the virion allows free access to the interior, while in the closed state the pores in the virion are sealed to large molecules. This allows us to switch between two conformations, thus we have a self-assembled and gated host with the capacity to hold and deliver a large number of molecules trapped within the 180 Å diameter cavity. We are exploring the nature of the materials which can be encapsulated (and released) via this gating mechanism and also designing new chemical switches which can be employed to initiate the response. Also, we are quantifying the pH and metal ion dependence of the conformational response using a number of biophysical techniques including fluorescence and dynamic light scattering. By using fluorescence resonance energy transfer from Trp to Tb³⁺, we can probe the metal binding affinity of the viral cage. Competition experiments allow us to measure the affinity of the virus for the native Ca²⁺ ion as well as other metal ions of interest.