The Rough Energy Landscape of Superfolder GFP Is Linked to the Chromophore

Abstract

Many GFP variants have been developed for use as fluorescent tags, and recently a superfolder GFP (sfGFP) has been developed as a robust folding reporter. This new variant shows increased stability and improved folding kinetics, as well as 100% recovery of native protein after denaturation. Here, we characterize sfGFP, and find that this variant exhibits hysteresis as unfolding and refolding equilibrium titration curves are non-coincident even after equilibration for more than eight half-lives as estimated from kinetic unfolding and refolding studies. This hysteresis is attributed to trapping in a native-like intermediate state. Mutational studies directed towards inhibiting chromophore formation indicate that the novel backbone cyclization is responsible for the hysteresis observed in equilibrium titrations of sfGFP. Slow equilibration and the presence of intermediates imply a rough landscape. However, de novo folding in the absence of the chromophore is dominated by a smoother energy landscape than that sampled during unfolding and refolding of the post-translationally modified polypeptide.

Denaturant-dependent folding of GFP

“…The inset in Fig. 1C shows that f INT ðTÞ is very small, which explains the apparent two-state folding of Citrine in the absence of the chromophore (see also (34)). The finding that I EQL are populated during the folding process (see also Fig.…”

“…Denaturant Induced Unfolding: To make a direct comparison with ensemble experiments (33,34) we simulated the effects of Guanidinium chloride (GdmCl) using the MTM (see Methods and SI Text for details). Following our previous work (21), we choose a simulation temperature, T s , at which the free energy differences between the NBA and the unfolded state, ΔG NU ð¼ G N ðT s Þ− G U ðT s ÞÞ calculated from simulations agree with the measured value of ΔG E NU ¼ −16.5 kcal∕mole for R96A mutant (34) at denaturant concentration, ½C ¼ 0.…”

“…Following our previous work (21), we choose a simulation temperature, T s , at which the free energy differences between the NBA and the unfolded state, ΔG NU ð¼ G N ðT s Þ− G U ðT s ÞÞ calculated from simulations agree with the measured value of ΔG E NU ¼ −16.5 kcal∕mole for R96A mutant (34) at denaturant concentration, ½C ¼ 0. We chose R96A as a reference GFP for fixing T s because it exhibits a clear equilibrium two-state transition (34). Since the absolute values of the effective interaction energies in CG models cannot be determined, we used T s as an adjustable parameter to get accurate estimate of ΔG NU .…”

“…Folding mechanisms of Green Fluorescent Protein (GFP), with predominantly β-sheet native structures stabilized by contacts between residues that are well separated along the sequence, have been extensively investigated (32)(33)(34)(35)(36)(37)(38)(39)(40). However, the details of the folding kinetics including the structures in the states that are visited along the folding routes are unexplored.…”

“…1A and 1B). The chromophore, surrounded by the barrel and the capping helices, causes hysteresis (denaturant-induced equilibrium folding and unfolding do not coincide even on time scales that exceed hours) in the folding of a number of variants of GFP (34,35). Mutants that inhibit chromophore formation fold without hysteresis, and could form the basis of describing the de novo folding of the complex barrel structure.…”