The extremely slow-exchanging core and acid-denatured state of green fluorescent protein

Green fluorescent protein (GFP) is a large protein with a complex eleven-stranded beta-barrel structure. Previous studies have shown that it has a complex energy landscape for folding on which there are several intermediate states and a denatured state with significant residual structure. Here, we use two different types of H/D exchange measurement and nuclear magnetic resonance (NMR) techniques to probe the energy landscape for folding of GFP in further detail. H/D exchange experiments were performed over a wide range of conditions including different concentrations of denaturant. Results show that the penetration model dominates the exchange mechanism, consistent with the known stability and slow unfolding kinetics of GFP. H/D exchange experiments at high pH establish that there is an extremely slow-exchanging superstable core of amide protons in GFP that are clustered and located in beta-strands 1, 2, 4, 5, and 6. These residues form part of a mini-beta-sheet which we propose constitutes a folding nucleus. Using a pulsed-labeling strategy, the acid-denatured state has been investigated and the residual structure observed in earlier studies shown to locate to beta-strands 1 and 3. There is some evidence that this residual structure is stabilized by a localized hydrophobic collapse of the polypeptide chain. ©2008 HFSP Publishin