Characterization of Engineered Protein Switches Using Intramolecular Disulfide Bridges

Authors

  • Darren Gemmill* University of Lethbridge, Alberta RNA Research and Training Institute (ARRTI), Department of Chemistry and Biochemistry
  • Dylan Girodat University of Lethbridge, Alberta RNA Research and Training Institute (ARRTI), Department of Chemistry and Biochemistry
  • Hans-Joachim Wieden University of Lethbridge, Alberta RNA Research and Training Institute (ARRTI), Department of Chemistry and Biochemistry

Abstract

Protein engineering is the process of developing proteins that can perform functions that often are novel and do not occur in nature. Development of proteins with new properties is critical for a wide range of biotechnology applications including new metabolic pathways, biosensors, and molecular scaffolds. Engineered proteins are frequently used in the food industry, medicine, nanobiotechnology, and environmental applications. One of the limits of protein engineering is the lack of ability to rationally design molecular switches. Here we report on the development of a molecular switch which utilizes cysteine disulfide bridges.

Our model system for the development of a designer molecular switch is the translation elongation factor (EF) G. EF-G stimulates translocation of tRNA through the ribosome, a process that requires a conformational change in EF-G. We have developed and implemented a Molecular Dynamics simulation pipeline to design 3 EF-G variants (G163C-T650C, P121C-K675C, and T125C-S679C) with disulfide bridges that limit the conformational changes of EF-G to specific functional states. To assess correct folding of these conformationally restricted EF-G variants, we have used circular dichroism and determined key enzymatic properties such as nucleotide binding and GTP hydrolysis rates for these EF-G variants. The reported data validates the ability of our Molecular Dynamics pipeline to rationally design novel molecular switches.

*Indicates presenter

Published

2018-06-20

Issue

Section

Presentation Abstracts