Background

Deciphering the folding pathway of proteins is one of the most explored topics in biology to date. Once synthesized, a nascent polypeptide chain is folded into its native structure, which is essential for its biological function. These polypeptide chains, especially the unfolded chains of small single-domain proteins, often fold spontaneously without any active assistance from other proteins. In contrast, larger multi-domain proteins frequently require folding assistance from proteins, commonly known as molecular chaperones, to fold into their native structure. The process of protein folding from nascent chains to correctly folded native states is extremely fast on a biologically relevant time scale. Thus, in order to study the steps of the folding pathway, we need to monitor the folding process using a slow-folding protein in a cell-free system, which is achieved by mimicking the cellular environment in vitro using suitable buffers. In an in vitro experiment, we unfold the test protein using a suitable denaturant and then refold it in a folding-favorable environment. Currently, the available protocols for measuring refolding rates are either based on measuring the enzymatic activity of proteins after they are refolded or monitoring the intrinsic tryptophan fluorescence of the protein (Weissman et al., 1994; Zahn et al., 1994; Makio et al., 1999; Doyle et al., 2003; Taniguchi et al., 2004; Kerner et al., 2005; Sharma et al., 2008; Malhotra et al., 2014; Jain et al., 2018). While measuring the refolding efficiency by estimating the enzymatic activity is one of the most suitable ways of checking the folded and active states of the protein, it largely depends on its well-known enzymatic activity. In the case of proteins without well-described enzymatic activity or activity that depends on the functional form of the protein, which may not form immediately after it is refolded, the estimation of refolding efficiency becomes difficult (Rospert et al., 1993). Another way of evaluating in vitro refolding is by measuring the tryptophan fluorescence, which again is largely dependent on the number of tryptophan residues and their location within the native structure (Chakraborty et al., 2010). We have devised an in vitro refolding assay of a recently identified in vivo substrate of the GroEL/ES chaperonin system (Sadat et al., 2020). In this protocol, we describe the methodology to monitor the refolding of a slow-folding yeGFP mutant protein, hereafter referred to as sGFP, in real time. We unfold the sGFP protein using guanidine hydrochloride as a chemical denaturant and subsequently refold the unfolded protein in a suitable refolding buffer in the presence and absence of chaperones (Sadat et al., 2020). The potential issues that are frequently encountered during in vitro refolding experiments are: (a) achieving complete unfolding of the protein in a reversible manner; (b) measuring the exact time taken by the unfolded protein to fold into its native form; and (c) accurately measuring the refolding rate, since most proteins fold very fast and this becomes a potential problem for measuring the in vitro refolding rate of a protein. Using sGFP successfully evades these potential problems in monitoring the refolding process in real time; therefore, our protocol can be a very useful guide for measuring the in vitro refolding rate of fluorescent proteins possessing similar characteristics to GFP.