The Folding and Long Range Communication within a Beta-Sheet Protein : Chromophore Packing Leads to Hysteresis in GFP

In an effort to more fully investigate the folding of all beta-sheet proteins we initiated studies on the folding of GFP. Graduate student Ben Andrews in my laboratory was fully trained in both the experimental and theoretical methods used to investigate the folding of this protein. Recent experimental studies suggest that the mature GFP has an unconventional landscape composed of an early folding event with a typical funneled landscape, followed by a very slow search and rearrangement step into the locked, active chromophore-containing structure. GFP possesses a unique folding landscape with a dual basin leading to the hysteretic folding behavior observed in experiment. While theoretical data do not have the resolution necessary to observe details of the chromophore during refolding, experimental results point to the chromophore as the cause of the observed hysteresis. With the use of NMR spectroscopy, which probes at the level of the individual residue, the hysteretic intermediate state is further characterized in the context of the loosely folded isomerized native-like state Niso predicted in simulation. In the present study, several residues located in the lid of GFP indicate heterogeneity of the native states. Some of these residues show chemical shifts when the native-like intermediate Niso responsible for GFP's hysteretic folding behavior is trapped. Observed changes in the chromophore are consistent with increased flexibility or isomerization in Niso as predicted in recent theoretical work. Here, we observed that multiple chromophore environments within the native state are averaged in the trapped intermediate, linking chromophore flexibility to mispacking in the trapped intermediate. The present work is experimental evidence for the proposed final "locking" mechanism in GFP folding forming an incorrectly or loosely packed barrel under intermediate (hysteretic) folding conditions.