Green-lighting green fluorescent protein: faster and more efficient folding by eliminating a cis-trans peptide isomerization event

Abstract

Wild-type green fluorescent protein (GFP) folds on a time scale of minutes. The slow step in folding is a cis-trans peptide bond isomerization. The only conserved cis-peptide bond in the native GFP structure, at P89, was remodeled by the insertion of two residues, followed by iterative energy minimization and side chain design. The engineered GFP was synthesized and found to fold faster and more efficiently than its template protein, recovering 50% more of its fluorescence upon refolding. The slow phase of folding is faster and smaller in amplitude, and hysteresis in refolding has been eliminated. The elimination of a previously reported kinetically trapped state in refolding suggests that X-P89 is trans in the trapped state. A 2.55 Å resolution crystal structure revealed that the new variant contains only trans-peptide bonds, as designed. This is the first instance of a computationally remodeled fluorescent protein that folds faster and more efficiently than wild type.

Keywords: GFP; cis; folding kinetics; protein design; trans isomerization.

Figure 1

The loop connecting the central helix with strand 4 of GFP as it appears in sfGFP (orange), in the designed model (pink), and in the AT-GFP crystal structure (green). Boxed numbers are strand labels. A thin green circle marks the cis-peptide bond in sfGFP.

Figure 2

2F_o–F_c electron density contoured at 1σ for the designed loop in chain A. All of the peptide bonds are trans. The electron density and coordinates are indistinguishable in the other four chains, which were refined with noncrystallographic symmetric restraints.

Figure 3

Comparison of rates (k) and amplitudes of the multiple phases of GuHCl dilution refolding for AT-GFP (closed circles), and OPT-GFP (open circles), with error bars showing the standard deviation over six replicates. Previously published multiphase kinetic measurements are shown for cycle3 GFP (open squares), and cycle3 GFP in the presence of CyPA (closed squares). Amplitudes are normalized to sum to 100%. Labels on the top are used in the text.

Figure 4

Fluorescence recovery versus time for AT-GFP (red) and OPT-GFP (cyan). For each protein, six experimental traces (thin black lines) are shown superposed on the least-squares fit to a four-phase kinetic model (thick lines). The traces were fit individually and the six rates and amplitudes. The curves were calculated using the averaged rates and amplitudes. The sum of the amplitudes was then scaled to the percent recovery of fluorescence. The inset shows a blow-up of the early portion of the same data.

Figure 5

(a) Hysteresis experiment. Protein starts at 0M GuHCl, pH8. FWD arrow represents titration to pH 5.5 or 1.8 M GuHCl, and incubation for 96 h. REV arrow represents titration to pH 2 or 8 M GuHCl, incubation for 1 h, followed by titration to pH 5.5 or 1.8 M GuHCl, and incubation for 96 h. Blue arrow: magnitude of the hysteresis effect. (b) Results of hysteresis experiment. Blue lines: OPT-GFP. Orange lines: AT-GFP. Solid lines: pH unfolding/refolding. Dashed lines: GuHCl unfolding/refolding. Error bars are from triplicate measurements.