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Current Projects
Probing ubiquitination specificity with redesigned
ubiquitin conjugation enzymes and ligases.
Tagging specific proteins with ubiquitin is one of the
primary methods used by cells to target particular
proteins for degradation, and therefore this
modification is essential for many cellular processes
including cell cycle control and cellular stress
response. Ubiquitin is attached to proteins by a cascade
of enzymatic reactions involving the E1 ubiquitin-activating
enzyme, the E2 ubiquitin-conjugating enzymes, and the E3
ubiquitin ligases. The substrate specificity of the
pathway is conveyed by the E3s of which there are many
in the human genome. A key question in the field is
which E3s target which proteins for ubiquitination. The
goal of this project is to develop a new method for
probing the specificity of E3s. The interface between
the E3, E6AP, and its E2 partner, UbcH7, will be
redesigned so that the new variants bind each other, but
no longer have appreciable affinity for the wild type
proteins. The substrate specificity of E6AP will then be
probed by loading the redesigned E2 with labeled
ubiquitin in vitro and then adding the loaded E2 to
cellular extract containing the redesigned E6AP. Only
proteins that are substrates for E6AP should be modified
with the labeled ubiquitin.
Design of protein conformational switches.
One
amazing property of proteins is that they are often able
to adopt multiple specific structures, each with its own
functional importance. The relative stability of these
states is typically regulated by ligand binding or
post-translational modification. To design new proteins
that switch conformation we have modified Rosetta so
that it can search for a sequence that is simultaneously
good for multiple target structures. We have used this
algorithm do design a protein that can switch between a
coiled-coil and a zinc finger. To add functionality to
this switch we are now adding unique DNA binding
properties to each of the two structures.
De Novo design of β -sheet proteins
Despite the large number of all-β proteins in nature,
the de novo design of a β -sheet protein has alluded
protein designers. To create a totally novel β -protein
we are using a protocol that iterates between structure
refinement and sequence optimization. The challenge with
β -sheet design is creating proteins that do not
aggregate. We are currently focused on adding negative
design elements to our sequences that prevent
aggregation.
De Novo design of protein-protein interfaces.
Protein-protein interactions are essential to life. The
ability to rationally design proteins that bind to
target proteins would allow for the creation of new
therapeutics, biosensors and tools for regulating cell
biology. The de novo design of interfaces is an
extremely challenging goal because it requires creating
proteins that interact favorably with the polar amino
acids typically found on protein surfaces. To meet this
goal we are developing protocols that iterate between
sequence design, backbone refinement and rigid body
docking.
Peptide-protein interface design with non-natural
amino acids.
Peptide-protein interactions are of great therapeutic
interest, because high-affinity peptides can modulate
bioactivity when targeted toward binding sites normally
occupied by other proteins or peptides. For many
peptide-protein interfaces evolution has probably
selected for sequences that are already close to
optimal. To enhance peptide binding, therefore, it may
be advantageous to consider amino acids that nature did
not have at its disposal. Hydrophobic amino acids with
novel geometries should help create new packing
configurations while the inclusion of non-natural polar
amino acids may help create more ideal hydrogen bonds.
An additional feature of some non-natural amino acids is
that they are more resistant to proteases, and peptides
made from them may have better bioavailability.
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