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Sheldon J. Park Assistant Professor 905 Furnas Hall (716) 645-1199 Fax: (716) 645-3822 sjpark6@buffalo.edu

Topics

• Protein Engineering
• Yeast Surface Display
• Bioinformatics
• Molecular dynamics simulations

Research Themes

Protein engineering is a discipline inspired by evolution at a molecular level. As life has evolved, the molecular basis of living organisms—including the protein molecules that make up living organisms—has also undergone huge diversification. As a result, there are virtually a limitless number of different protein molecules in nature, many of which have fascinating structural and functional properties. It is an important goal in biological sciences to study how these molecules work in their natural milieu to understand how living organisms operate. Additionally, once we have studied the way these molecules function in nature, some of them can be further developed to serve useful roles in research, medicine and biotechnology. Our understanding of the physical basis of protein function has significantly advanced in the past. There have also been important progresses in molecular biology to allow the construction and experimental testing of various mutants. As a result, we can now construct new protein molecules and systems in vitro with interesting molecular properties that can be useful in a variety of applications.

In our lab, we use both computational and experimental tools to design protein molecules with novel physical and biological properties. We use biochemical and biophysical techniques to characterize the designed molecules. Finally, the designed molecules are tested in various applications to confirm they have expected molecular properties and to demonstrate their utility. We currently have several projects that use computational modeling, directed evolution, biochemistry, and structural biology that exemplify this protein engineering practice. The ongoing projects include:

1. Design and characterization of monomeric streptavidin for biotechnology applications
2. Engineer antibody analogs that specifically target protein enzymes for research and therapeutic applications
3. Design temperature sensitive intein mutants for structure-function study and in vivo cell biology applications

Project 1: Monomeric streptavidin
Streptavidin is widely used in biotechnology and molecular research for detection, purification, crosslinking, and labeling of biotinylated targets. However, wild type (wt) streptavidin is an obligate tetramer and can crosslink biotinylated targets. Target aggregation is a significant obstacle in some potential applications, including the labeling of biotinylated cell surface receptors, where crosslinking can perturb protein stability and function. To address this problem, we are using a combination of homology modeling, rational design, and directed evolution to design a streptavidin monomer that has high thermal stability and biotin affinity.

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 monomer subunit is shown in black ribbon.

Project 2: Antibody analog inhibitors of protein enzymes
Proteins are far more engineerable than small molecules because their structure and function can be manipulated by changing the underlying DNA sequence. Proteins often interact with other molecules with high specificity and affinity. For example, novel antibodies can be engineered to bind their respective antigens with high specificity. The interaction, which can be optimized to distinguish subtle structural differences, can be used to design highly specific inhibitors against protein enzymes, such as cell surface receptor tyrosine kinases. We are engineering antibody mimetics, called monobodies, that are smaller and simpler than an antibody but are yet capable of binding and inhibiting the function of a target enzyme. Currently, we are using yeast display and biochemical assays to design monobody molecules that specifically inhibit the activity of Erk-2. The inhibitors are then tested in vitro, in cultured cells, yeast, and a model organism to evaluate their activities.

The MAP kinase networks mediate cellular response to various extracellular inputs, including growth factors, GPCR and stress. Inhibiting specific MAPK pathways with engineered inhibitors can be useful as a therapy against aberrant kinase signaling, which result in various human diseases, including cancer.


Project 3: Temperature sensitive intein
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 yeast surface display and yeast based functional assays to design temperature sensitive (ts) intein mutants to understand the physical basis of temperature sensitivity. Ts mutants are frequently used in genetics to study protein function but their construction is time consuming because they need to be engineered separately for individual proteins. Instead, we seek to use the engineered ts intein mutants to significantly expedite the discovery of other ts mutants so that they can be used to characterize protein function inside the cell.

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Last Updated: June 2012