An important goal in molecular biology is to understand at a molecular level how protein molecules perform their native functions. On the one hand, understanding the mechanism of protein function can help model how living organisms operate at a molecular level. On the other hand, understanding structure-function relationship can help develop novel reagents for use in biotechnology, research, and medicine. We apply computational (homology modeling, molecular dynamics simulation, bioinformatics) and experimental tools (biochemistry, yeast display, directed evolution, high throughput screening) to design proteins with novel molecular properties.
The current projects in the group are:
1. Engineering and application of monomeric streptavidin
2. Epitope specific binders for vaccine development
3. Novel intein mutants for in vivo and in vitro applications
4. Therapeutic peptides for the treatment of Crohn’s disease
Monomeric streptavidin (PDB: 4JNJ)
* The plasmids for bacterial, yeast and mammalian expression are available through Addgene.
Streptavidin monomer lacks critical intersubunit interactions that are important for stability and affinity. Engineering a useful monomer thus requires introducing mutations that can increase the stability of the molecule and stabilize the interaction with biotin. The engineered monomer can be used as a fusion protein to allow interaction with biotinylated ligands.
* The protein (mSA) was further engineered to slow the dissociation of bound biotin. These mutants have slower dissociation kinetics (koff) and produce more consistent labeling over time. This is useful to achieve stable labeling over time.
HEK293 cells displaying singly biotinylated CFP labeled with Alexa488-conjugated mSA2 or mSA3 and imaged over 50 min at room temperature.
* mSA conjugated to agarose resin can be used in affinity purification. Bound biotionylated ligand can be eluted by addition of free biotin.
Designing epitope specific interaction
Novel protein binders are straightforward to engineer by directed evolution. These molecules are then used to elucidate biological mechanism or to induce therapeutic effects by interfering with the endogenous function of the target protein. However, ensuring that the engineered binders interact with the target protein at a specific surface patch is not simple. Epitope specific binders are more likely to disrupt the activity of the target molecule. For example, an antibody that binds the influenza hemagglutinin at the conserved receptor binding site is potential broadly neutralizing and active against multiple subtypes of the flu virus. We have developed a high throughput screening protocol based on a combination of positive and negative selections using wild type and mutant target proteins that predictably leads to the engineering of epitope specific binders. The technique should be applicable against any target molecule for which a suitable mutant can be designed.
We engineered fibronectin derived monobodies (Fn3) that bind the Erk-2 kinase at a protein-protein interaction site and thereby inhibit its function. The engineered monobodies were shown to inhibit Erk-2 signaling in vitro, in mammalian cells, yeast, and C. elegans.
Modulation of protein activity by temperature
Inteins are structurally independent domains that can autocatalytically remove themselves from the precursor proteins by splicing the flanking exteins together. They appear in microbial genomes and are potential molecular targets for therapy. In recent years, they have also been used in biotechnology for protein purification and biosensor design. We are using a yeast based screen to design temperature sensitive (ts) intein mutants for use in in vivo study of protein function. We also engineer split intein with improved activity for modular assembly of protein domains in vitro, which can vastly simplify the construction of novel enzymes comprised of multiple modules, such as polyketide synthase.
Intein domains splice two exteins to a single mature polypeptide and have potential use in biotechnology and research. We are engineering temperature sensitive inteins to understand the structural basis of temperature sensitivity and to develop a novel tool to study protein function inside and outside the cell.
A peptide based drug for Crohn's disease
Inflammatory bowel diseases, including Crohn's disease (CD), affect nealry 1 in 200 individuals in the country. Given that the existing treatment options for CD have side effects, new drugs that are more specific and effective are urgently needed. We are developing peptide based drugs to treat the disease. Homology modeling and directed evolution approaches are used to design the peptides, which are then tested using cell lines and patient derived cells.
Nod2 is a germline pathogen recognition receptor that binds and is activated by muramyl dipeptide (MDP), which is a component in the cell wall of both gram negative and positive bacteria. The molecule undergoes a conformational change upon MDP binding and forms a large complex that initiates the innate immune response through the NF-kB pathway. (left) The crystal structure of a Nod2 homolog (NLRC4) and (right) modeled Apaf-1 heptamer based on EM images.