Nathan Zelt

Program: Biochemistry Biophysics and Structural Biology

Current advisor: Janice L. Robertson, PhD

Undergraduate university: Indiana University-Bloomington, 2019

Enrollment year: 2020

Research summary
Using fluorescent techniques, I will investigate the changes in the kinetic and thermodynamic stability of CLC-ec1 dimers containing interfacial tryptophan residues.

The CLC superfamily are transmembrane anion/proton transporters or channels commonly found as dimers throughout all branches of biology. CLC-ec1, a transporter from E. coli, forms dynamic but thermodynamically strong dimers via an entirely hydrophobic and highly complimentary dimer interface. Tryptophan residues located along the dimer interface of CLC-ec1 can disrupt dimer stability in detergent micelles, but how tryptophan mutations affect the kinetic and thermodynamic stability of CLC-ec1 dimers in lipid membranes has yet to be investigated. Further, understanding whether specific tryptophan positions effect the dimer stability can help guide understanding on how tryptophan might affect dimerization in natural or disease causing CLC in biology. CLC-ec1 constructs will be expressed and purified from e. coli prior to fluorescent labeling and reconstitution into E. coli Polar Lipid extract (EPL) membranes. We can then use bulk Förster Resonance Energy Transfer (FRET) to measure changes in subunit exchange kinetics and single-molecule photobleaching analysis with TIRF microscopy to measure the equilibrium dimerization and thermodynamic stability. Finally, functional chloride dump assays are able to decern whether CLC-ec1 remains functional after labeling and/or incubation.

Graduate publications