Barnase as a model for the denatured state of proteins

Abstract

Protein folding is one of the major thrusts of biochemical research today. It is the study of how proteins fold into their tertiary structure based solely on their sequence of amino acids. It was once believed that no interactions were made in the denatured state and it contributes to the stability of the native state. It has become apparent that new techniques are needed to probe the denatured state and learn about its properties. It was proposed to use chemical modification of cysteine mutant of Barnase to disrupt the hydrophobic core and denature the protein under physiological conditions. Then a series of size exclusion chromatography experiments would then be carried out to characterize the extent to which the protein unfolds. These experiments would be done in increasing osmolyte concentration, which will cause an open conformation to become more compact while an already compact conformation will change very little. Unfortunately, high Barnase yields could not be obtained with the first method of preparation and work was delayed significantly while a new method was investigated. A new expression vector and protocol was obtained from Bob Hartley at the NIG and implemented successfully two weeks ago. Work is ongoing now that problems with the expression system have been solved.