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 Here we present a computationally designed leave-one-out GFP in which wild-type strand 7 has been replaced by a 12-residue peptide (HA) from the H5 antigenic region of the Thailand strain of H5N1 influenza virus hemagglutinin.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 × 105 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.

Split Green Fluorescent Proteins: Scope, Limitations, and Outlook

“…While this particular assay is limited to in vitro studies, the phenomenon of chromophore maturation within a split FP fragment may be more prevalent than discussed in the BiFC literature. If a 158-residue EGFP fragment maintains enough structure and sufficient contacts with neighboring residues, however transiently, to form a mature chromophore in the absence of its complementary fragment, then it is plausible that similar split protein fragments, especially larger ones that contain most of the β-strands, generate mature chromophores during other in vivo and in vitro experiments (33, 45, 46). Alternatively, chromophore maturation within a single FP fragment may be facilitated inadvertently through stabilizing intramolecular (affinity tags, fusion proteins) or intermolecular (host proteins) interactions.…”

“…Split GFP also serves as a sensor for protein kinase and phosphatase activity (158) as well as cytoplasmic delivery of cell-penetrating peptides or other cargo (86, 101, 123). To create a sensor for a unique target peptide sequence, Huang et al (45) evolved a circularly permuted GFP variant missing its final strand to bind a peptide from influenza’s hemagglutinin protein; efforts are underway to optimize the fluorescent response of this sensor upon peptide binding. The tripartite split GFP system has also been engineered to report on enzymatic activity of small GTPases and sortase (72, 161).…”

Mispacking and the Fitness Landscape of the Green Fluorescent Protein Chromophore Milieu

“…3,4 It has also been implemented as an intracellular pH sensor, 5 a calcium sensor, 6 and a biosensor for influenza virus. 7 Alterations to the chemical structure and environment of its chromophore result in shifted excitation and emission maxima, such as those observed in red fluorescent protein variants. 8 However, our ability to engineer GFP is dependent on our knowledge of the sequence determinants of its fluorescent function.…”

Live-cell imaging of single mRNA dynamics using split superfolder green fluorescent proteins with minimal background

“…Therefore, we cannot simply attribute the better performance of the tripartite sfGFP system to the difference between binary and ternary interactions. It is possible that other improved bipartite split FP systems (Huang et al 2015;Feng et al 2017;Köker et al 2018) may perform as well as the tripartite sfGFP system tested in this report.…”