Toward Computationally Designed Self-Reporting Biosensors Using Leave-One-Out Green Fluorescent Protein

Abstract

Leave-one-out green fluorescent protein (LOOn-GFP) is a circularly permuted and truncated GFP lacking the nth β-strand element. LOO7-GFP derived from the wild-type sequence (LOO7-WT) folds and reconstitutes fluorescence upon addition of β-strand 7 (S7) as an exogenous peptide. Computational protein design may be used to modify the sequence of LOO7-GFP to fit a different peptide sequence, while retaining the reconstitution activity. Here we present a computationally designed leave-one-out GFP in which wild-type strand 7 has been replaced by a 12-residue peptide (HA) from the H5 antigenic region of the Thailand strain of H5N1 influenza virus hemagglutinin. The DEEdesign software was used to generate a sequence library with mutations at 13 positions around the peptide, coding for approximately 3 × 10(5) sequence combinations. The library was coexpressed with the HA peptide in E. coli and colonies were screened for in vivo fluorescence. Glowing colonies were sequenced, and one (LOO7-HA4) with 7 mutations was purified and characterized. LOO7-HA4 folds, fluoresces in vivo and in vitro, and binds HA. However, binding results in a decrease in fluorescence instead of the expected increase, caused by the peptide-induced dissociation of a novel, glowing oligomeric complex instead of the reconstitution of the native structure. Efforts to improve binding and recover reconstitution using in vitro evolution produced colonies that glowed brighter and matured faster. Two of these were characterized. One lost all affinity for the HA peptide but glowed more brightly in the unbound oligomeric state. The other increased in affinity to the HA peptide but still did not reconstitute the fully folded state. Despite failing to fold completely, peptide binding by computational design was observed and was improved by directed evolution. The ratio of HA to S7 binding increased from 0.0 for the wild-type sequence (no binding) to 0.01 after computational design (weak binding) and to 0.48 (comparable binding) after in vitro evolution. The novel oligomeric state is composed of an open barrel.

Figure 1

Leave-one-out method for biosensor design. Omitting a secondary structural element eliminates this signal. Adding back the left-out piece recovers the signal. Designing the site to bind a different sequence creates a biosensor.

Figure 2

Fluorescence trajectory upon adding peptide in solution to LOO7-WT and LOO7-DS2. Lines are least-squares fits to the data. Flat residual (lower lines) shows the quality of the fit.

Figure 3

SEC traces for LOO7-WT and LOO7-HA4. (a) Purified LOO7-WT with (green) and without (purple) the addition the S7 peptide. Gray trace are the molecular weight standards, marked by kD. (b) Purified LOO7-HA4 with (green) and without (purple) addition of synthetic HA peptide. The sharp peak at approximate 27kD corresponds to monomers. The broad peaks at higher MW correspond to nonmonodisperse mixtures of oligomeric states which are in slow equilibrium with monomer. LOO7-WT forms higher order and a greater fraction of oligomers than LOO7-HA4.

Figure 4

Peptide binding assay. (a) Example of fluorescent traces for varying concentrations of S7 peptide (0.12–15 µM) added at time zero to LOO7-DS2. Kinetic traces all fit single exponential decays. Residuals of the Excel/Solver fits are shown along the bottom of the plot. Data were logarithmically downsampled for better behavior in Solver. (b, c) Eadie–Hofstee plots for S7 peptide binding to each of the three variants discussed. Data plotted are relative amplitude of fluorescence signal (positive or negative) over peptide concentration, versus relative amplitude of signal in arbitrary units. Error bars are standard deviations for triplicate measurements. Linear fits are shown. Slopes represent dissociation constants, K_d.

Figure 5

Proposed working model for LOO7-GFP oligomerization, binding, barrel closing, and nonproductive binding. Open arcs represent LOO7-GFPs in open β barrel configuration. Triangles are peptides (S7 or HA). QY = quantum yield relative to (a) the unbound, oligomeric state. Peptide binding (a–c, b–d) is followed by (e) barrel closing to form a more fluorescent state.

Figure 6

Computationally designed mutation F83W creates clashes with residues on β strand 4 and 10. Opening the β barrel would probably relieve these clashes. Reverting W83 to F might result in a closed barrel.