title: Chromophore packing leads to hysteresis in GFP.
abstract: Green fluorescent protein (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 {N(iso)} 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 {N(iso)} responsible for GFP's hysteretic folding behavior is trapped. Observed changes in the chromophore are consistent with increased flexibility or isomerization in {N(iso)} 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.
authors: Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, 92093-0375, USA.
authors: Benjamin T
authors: BT
authors: Andrews
authors: Melinda
authors: M
authors: Roy
authors: Patricia A
authors: PA
authors: Jennings
mesh_terms: D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary
chemical_list: D049452:Green Fluorescent Proteins