Brendan Orner research group, King's College London

Research Interests:

The overall research undertaken in our lab has two fundamental goals, both focused on the chemistry/biology interface. One goal is to develop new methods and technologies, while the second goal is to investigate fundamental questions. The philosophy of the lab is to mesh each of the goals so that they can feed into the other in a smooth and dynamic manner. The new technologies will be utilized to help answer fundamental questions and this basic research will necessitate the development of additional methods. The novel techniques will be developed with an eye on generality and additional applications. They will initially focus on combinatorial techniques that not merely employ vast collections of molecules, but unleash the power and intellectual satisfaction of focused design. The fundamental biological systems of interest will be focused on understanding the role protein-protein interactions play in the key cellular process of apoptosis (controlled cell death), how primary, secondary, and tertiary structure of proteins affect their quaternary structure and self assembly processes, and whether it is possible to convert overly reactive, non-specific compounds into specific ones that target key HIV and Alzheimer’s Disease proteins.

Technology developmen<-->fundamental research
 

Understanding and Exploiting Protein-Protein Interactions in Ferritin Nano-Assembly
Protein-protein interactions are essential to most biological functions resulting in increasing medicinal interest. Furthermore, they are used cellularly as the foundation to assemble large protein structures. Therefore understanding the fundamentals of protein-protein interactions and how they control protein oligomer self-assembly could shed light on rudimentary biological processes. In addition they could be the key to creating novel protein-based structures with unique functions.


This project focuses on establishing the fundamentals of the protein-protein interactions that govern the assembly of the ferritin nano-cage family of proteins and setting up tools for downstream applications. These proteins form oligomers that are highly symmetric and have a hollow central cavity. One goal of this proposal is to probe two hypotheses that have been proposed to describe the mechanism of self-assembly of the ferritins by using computational design, mutagenesis, various biophysical techniques and crystallography.

The second goal is to establish a new fluorescence-based technique to further understand the oligomerization of these proteins. This technique would allow us to rapidly screen multiple conditions and to screen libraries of proteins that have been randomized at the protein-protein interfaces to discover proteins with enhanced stability. The third goal is to push these proteins in new directions and lay the ground work for new technologies based on them. Ferritin-based systems will be established that utilize small-molecule switches to control cage formation with the intention of using these systems to explore the cellular role of cage protein self-assembly and control release of cargo store inside the cage.
This project bridges a) hypothesis-focused, fundamental research to understand elemental properties of a ubiquitous class of proteins, with b) the design of a flexible and powerful system to further understand them and serendipitously discover variants with enhanced properties, and c) the foundational development of a technique to advance these proteins into novel, synthetic applications.
 

Protein Cages to Generate and Supra-Assemble Inorganic Nanoparticles
Protein templates in material science can provide precisely defined reactors to control both particle shape and size, often on the nanoscale. In addition they can enhance particle solubility and protect particles from aggregation. Because of advances in bioconjugation, molecular biology, and protein engineering techniques, they can also assist in the supra-assembly of particles in highly controlled and possibly symmetric ways.
Recently we have developed a method for the generation of gold nanoparticles inside nanocage proteins. Because, unlike other techniques, this method does not require the modification of the cage protein, it is general and could be used with any nanocage therefore potentially producing encapsulated nanoparticles of any shape and size. This technique employs a two step reduction strategy where we first rapidly form a nanocluster of gold which is used as a seed to assist in slow growth of the full nanoparticle. The first half of this proposal will be to use the cluster to seed the formation of particles using other metals thereby generating core-shell nanoparticles.
 

The second part of this project will be to assemble an organized array of nanocages in a way that would allow alternating cages (small/big; filled/unfilled etc) in highly organized and symmetric, three-dimensional arrays. This technique will then be applied to a colorimetric and tunable high throughput assay for the discovery of inhibitors of protein-protein interactions.


The Discovery of New Bio-Orthogonal Reactions Through Genetically Encoded Organo-Catalyst Screening

Many ferritin proteins self-assemble into protein capsules. This project will focus on formation of protein capsules with novel sizes and shapes by domain swapping of two four helix bundle ferritin proteins which naturally self-assemble into different size protein capsules. Whole helicies or parts of the helicies will be swapped to form a small library of proteins.
These libraries will be screened for the formation of assemblies that differ in size and shape from those formed by the native proteins. Along with investigating the fundamental question of protein folding, this strategy will be applied to the templating of inorganic nano-particles of novel size, shape, and materials properties.
 
The Cell Surface Specific Ligands and Small Molecule Mimicry through Phage Display
 
The overall objective of this project is to develop flexible phage display-based strategies to discover first generation ligands that are either specific to cancer and stem cell types, and can therefore be used for a myriad of applications, or specific as universal mimics of natural ligands involved in essential cellular pathways and could therefore function as tool compounds or leads toward inhibitory or agonistic drugs. As a proof of concept, these ligands will be applied to characterize various types of stem cells, to target cytotoxic agents to specific types of cancer cells and in phenotypic/high content screens in both classes of cells.
Stem cells have great clinical potential, however, the fundamentals of their properties are only just beginning to be understood. Many pluripotent stem cell lines are available including those whose pluripotency has been induced in previously differentiated cells. However the commonalities and differences of these lines need to be characterized for them to be safely utilized in the clinic.
Carbohydrates are involved in many cell/cell and cell/pathogen interactions and phosphoinositides are ubiquitous mediators of signal transduction. Therefore both classes of molecules, when administered exogenously, should be able to manipulate medicinally important biological processes. However the chemical synthesis and drug advancement of these compounds can be difficult due to their polarity and to the subtle substitution and stereochemical differences between molecules of the same class. Peptides can often be rationally advanced to peptidomimetic, small molecule drugs, however understanding how a peptide can functionally mimic carbohydrates and phosphoinositides cannot be rationally deduced.
The first goal of this research is to establish a technique to aid in the characterization of cells and to compare and contrast different cell lines, even those that are thought to be quite similar. Phage display libraries will be screened against human embryonic stem cells and induced pluripotent stem cells to discover ligands that bind to cell surface receptors on one cell line and not the other or on both. These ligands will be used to characterize a descriptive phenotype of these cells. This technique will also be used to discover ligands that are specific for cancer cell types.
The second goal of this research is to develop a phage display method, involving the screening of sets of protein receptors with known selectivity for carbohydrates and phosphoinositides, to discover peptides that can mimic, with specificity, different members of these two classes of molecules.
The third goal of this research is to determine if the ligands derived from these screens have activity in the directed differentiation or maintenance of pluripotency, can alter the phenotype in high-content screens, or can be used to direct cytotoxic agents toward specific cancer cells. In addition thei cellular targets will be identified.