Engineered switch protein inspired by novel protein affinity transition mode

Author

Liang Fang, University of Tennessee, KnoxvilleFollow

Date of Award

5-2021

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Chemical Engineering

Major Professor

Eric T. Boder

Committee Members

Eric T. Boder, Paul Dalhaimer, Cong T. Trinh, Todd Reynolds

Abstract

Many natural proteins involved in complex biological processes such as ligand binding and protein folding demonstrate multiple, allosterically-regulated conformational states, with protein activity regulated by effector molecules. The alpha L integrin and its inserted domain (I domain) is one example of such a protein. The binding of the effector molecule such as talin or filamin to the cytoplasmic domain of the integrin increases the binding affinity between I- domain and its ligand intercellular adhesion molecule-1, known as ICAM-1.There are multiple models attempting to describe the mechanism responsible for the change in binding affinity. According to research conducted by our lab, we proposed a new “ligand-like stabilizer” model. We successfully engineered a new chimeric protein by fusing EF3 and EF4 hands of calmodulin to Id to create an Id switch (IdCaM) based on the proposed “ligand-like stabilizer” mechanism. We characterize the interaction of IdCaM with two different peptides and affinity of monomeric IdCaM with Intercellular adhesion molecule 1. We also tried to find out new IdCaMs with EF hands domains binding specificity changed by screening error-prone PCR and saturation mutagenesis yeast surface display libraries. We expected the new IdCaMs could show switch activity when a new peptide (225D9.2+) was present. Results emphasize the potential limitation of a novel allosteric system, which could be leveraged to design environmentally-responsive targeting agents and therapeutics. And the results also demonstrate the directed evolution combining with yeast display is really power tool to help us to get better understanding of Id.

Recommended Citation

Fang, Liang, "Engineered switch protein inspired by novel protein affinity transition mode. " PhD diss., University of Tennessee, 2021.
https://trace.tennessee.edu/utk_graddiss/6705