An important goal in molecular biology is to understand at a molecular level how protein molecules perform their native functions. Understanding the mechanism of protein function can help develop novel reagents for use in biotechnology, research, and medicine. We use modeling (homology modeling, molecular dynamics simulation, bioinformatics) and experiments (biochemistry, yeast display, directed evolution, high throughput screening) to design proteins with novel molecular properties.
The current projects include:
1. Engineering and application of monomeric streptavidin
2. Epitope specific binders for vaccine development
3. In vivo and in vitro applications of split inteins
4. Development of therapeutic peptides/proteins
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 engineered to slow the dissociation of bound biotin. These mutants have slower dissociation kinetics (koff) and produce more consistent binding over time, which is useful for detection and labeling. mSA was reported in Lim et al, Biotech Bioeng (2013). mSA2 is discussed in Demonte et al, Proteins (2013). To learn about mSA3, see Lee et al, eLife (2017).
* 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. 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 use a combination of positive and negative selections to identify rare 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 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 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 simplify the construction of novel enzymes comprised of multiple modules, such as polyketide synthases and nonribosomal peptide synthetases.
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 therapeutic 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 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.