Quantitative in vivo solubility and reconstitution of truncated circular permutants of green fluorescent protein

Abstract

Several versions of split green fluorescent protein (GFP) fold and reconstitute fluorescence, as do many circular permutants, but little is known about the dependence of reconstitution on circular permutation. Explored here is the capacity of GFP to fold and reconstitute fluorescence from various truncated circular permutants, herein called ''leave-oneouts'' using a quantitative in vivo solubility assay and in vivo reconstitution of fluorescence. Twelve leave-one-out permutants are discussed, one for each of the 12 secondary structure elements. The results expand the outlook for the use of permuted split GFPs as specific and self-reporting gene encoded affinity reagents.

GFP-Based Biosensors

“…When synthesized directly via the genetic approach, LOO-GFPs are non-fluorescent or weakly fluorescent. However, if co-expressed with the omitted piece, fluorescence sometimes develops in vivo, depending on which of the secondary structure elements was left out [39,40]. Expressing the full-length GFP and removing the beta strand by proteolysis, denaturation and gel filtration produces similar results [41].…”

“…In general, protein folding is an all-or-none process and leaving out any whole secondary structure element leads to an unfolded protein which aggregates in the cell. Yet, [40] has shown that it is possible to reconstitute LOO-GFP after truncation at several positions in the sequence. The key to understanding why LOO is sometimes possible is in the protein folding pathway.…”

“…SSEs that fold late and not required for most of the folding pathway, leaving behind a mostly-folded protein which is more soluble. Leave-One-Out solubility analysis provides a unique insight into the folding pathway of GFP [40]. Omitting strand 7 (LOO7-GFP) appears to be the least detrimental to the overall structure of GFP, suggesting that strand 7 folds last.…”

“…This resulted in GFP1-10 OPT which has an 80% increased solubility over the corresponding superfolder GFP1-10.GFP OPT contains 7 new mutations (N39I, T105K, E111V, I128T, K166T, I167V and S205T) in addition to the superfolder mutations [50].Directed evolution of GFP11 resulted in GFP11 optima tag that had the dual properties of 1) complementation with GFP1-10 OPT and 2) minimized perturbation of the protein of interest. Note that full-length GFP OPT was subsequently found to be more tolerant of circular permutation and truncation than superfolder GFP [40]. In addition to providing a less laborious method for detecting protein variants and reaction conditions for generating soluble recombinant protein, the split GFP complementation assay also serves as an assay of aggregation in living cells.…”

“…Fluorescence is recovered in LOO-GFP when the left-out piece is encountered in the analyte. A promising application of LOO-GFP, knowing that it binds to the left-out segment and fluoresces [39,40], is to engineer novel LOO-GFP molecules that recognize and sense desired peptides derived from other sources such as virus, bacteria and parasites. By modifying the sites of one of the eleven β-strands to complement shapes of given target peptides, the engineered LOO-GFP molecules will report the presence of specific target proteins, and therefore their host organism, through simple fluorescence readout (Figure 10).…”