Analysis of Disulfide Bond Formation

Abstract

In this unit, protocols are provided for detection of disulfide bond formation in cultures of intact cells and in an in vitro translation system containing isolated microsomes or semi-permeabilized cells. First, the newly synthesized protein of interest is biosynthetically labeled with radioactive amino acids in a short pulse. The labeled protein then is chased with unlabeled amino acids. At different times during the chase, a sample is collected, membranes are lysed with detergent, and the protein is isolated by immunoprecipitation, as described. A support protocol is provided for analysis of disulfide bonds in the immunoprecipitates by SDS-PAGE with and without prior reduction. The difference in mobility observed between the gels with nonreduced and reduced samples is due to disulfide bonds in the nonreduced protein. An additional support protocol is included that uses PEG-maleimide to modify free thiols and follow disulfide-bond formation by SDS-PAGE. © 2017 by John Wiley & Sons, Inc.

Keywords: disulfide bonds; endoplasmic reticulum; protein folding; pulse-chase; radiolabeling; secretory pathway.

Figure 14.1.1

In vitro translation/rough endoplasmic reticulum–derived microsome translocation and folding system. (A) For cotranslational folding, the protein is translocated into microsomes containing an oxidizing environment (GSSG). This provides an opportunity for the protein to fold vectorially during the translation and translocation processes. (B) For post-translational folding, oxidizing agent is added after translation and translocation, permitting synchronization and isolation of the folding process.

Figure 14.X.X

Assessing oxidative state using PEG-Maleimide modification. Cartoon representation of a protein possessing two cysteines involved in an intramolecular disulfide bond and two unpaired cysteines. The presence of free thiols is analyzed using sulfhydryl-reactive maleimide linked to a polyethylene glycol group (PEG-Mal). Cells expressing the protein of interest are treated with or without DTT to assess reducing and non-reducing states, respectively (1 and 2). Cells are lysed in the presence and absence of PEG-maleimide and protein samples are resolved by SDS-PAGE. Insert, reduced and unlabeled proteins are expected to migrate fastest through the SDS-PA gel (1), followed by nonreduced proteins labeled with PEG-maleimide (2), with reduced and fully labeled proteins being slowest (3).