Chapter 7 Protein Folding and Intracellular Transport: Evaluation of Conformational Changes in Nascent Exocytotic Proteins

Abstract

This chapter describes various assays that have been developed to analyze the in vivo process of protein folding or oligomerization within the ER. These assays are divided into two groups—namely, (1) assays that probe conformational changes in the nascent polypeptide chain and (2) assays that follow the assembly of the polypeptides into oligomers. To determine the time course in vivo of folding and assembly of membrane or secretory proteins, it is necessary to label the nascent polypeptides with a radioactive amino acid, usually [35S]methionine or [35S]cysteine. Optimally, the period of incorporation of isotope should be short relative to the time course of folding so that only the completely unfolded polypeptide will be labeled at the start of the chase period. However, in practice it may be difficult to achieve, because initial folding of the polypeptide may commence even before translation and translocation of the chain is completed. The assays for analysis of protein folding using antibodies specific for different conformational states depend on the availability of antibodies that recognize different conformational forms of the protein of interest, and in particular antibodies that discriminate between unfolded and fully folded forms of the polypeptide chain. Polyclonal or monoclonal antibodies raised against reduced and alkylated polypeptides are likely to recognize the fully unfolded conformations and may not recognize the folded molecule.