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
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
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.
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.
Discovery of New Bio-Orthogonal Reactions
Through Genetically Encoded
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.
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
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.