The leave-one-out (LOO) green fluorescent protein (GFP) approach to biosensor design combines computational protein design with split protein reconstitution. LOO-GFPs reversibly fold and gain fluorescence upon encountering the target peptide, which can be redefined by computational design of the LOO site. Such an approach can be used to create reusable biosensors for the early detection of emerging biological threats. Enlightening biophysical inferences for nine LOO-GFP biosensor libraries are presented, with target sequences from dengue, influenza, or HIV, replacing beta strands 7, 8, or 11. An initially low hit rate was traced to components of the energy function, manifesting in the over-rewarding of over-tight side chain packing. Also, screening by colony picking required a low library complexity, but designing a biosensor against a peptide of at least 12 residues requires a high-complexity library. This double-bind was solved using a “piecemeal” iterative design strategy. Also, designed LOO-GFPs fluoresced in the unbound state due to unwanted dimerization, but this was solved by fusing a fully functional prototype LOO-GFP to a fiber-forming protein, Drosophila ultrabithorax, creating a biosensor fiber. One influenza hemagglutinin biosensor is characterized here in detail, showing a shifted excitation/emission spectrum, a micromolar affinity for the target peptide, and an unexpected photo-switching ability.

The autocatalytic maturation of the chromophore in green fluorescent protein (GFP) was thought to require the precise positioning of the side chains surrounding it in the core of the protein, many of which are strongly conserved among homologous fluorescent proteins. In this study, we screened for green fluorescence in an exhaustive set of point mutations of seven residues that make up the chromophore microenvironment, excluding R96 and E222 because mutations at these positions have been previously characterized. Contrary to expectations, nearly all amino acids were tolerated at all seven positions. Only four point mutations knocked out fluorescence entirely. However, chromophore maturation was found to be slower and/or fluorescence reduced in several cases. Selected combinations of mutations showed nonadditive effects, including cooperativity and rescue. The results provide guidelines for the computational engineering of GFPs.

Supplementary Figure 1 from Aurora-A Kinase Regulates Breast Cancer–Associated Gene 1 Inhibition of Centrosome-Dependent Microtubule Nucleation

Split fluorescent proteins have been engineered for various purposes, in each case signaling their spontaneous reconstitution by fluorescence. By combining split protein reconstitution and computational protein design, we have constructed a circularly permuted and truncated variant of green fluorescent protein (GFP) in which the seventh beta strand has been left out and the sites around it computationally designed to accommodate a peptide from influenza hemagglutinin. We call this a "leave-one-out" GFP biosensor (LOO-GFP). A LOO-GFP was designed using DEEdesign, selected by plate screening a bacterial library in the presence of the influenza peptide target, and was found to have seven point mutations. But binding was weak (9μM) and was at the expense of stability. The weakened, partially folded protein aggregated in the absence of its target. Furthermore, the aggregated biosensor fluoresced more than its monomeric peptide-bound form. In this work, the LOO-GFP was rationally redesigned to fold more robustly and bind the target tighter. Modeling of the GFP folding pathway suggested that one of the seven mutations, F83W, interfered with the closing of the beta barrel. Mutating this residue back to a F indeed, along with several other rationally justified changes followed by re-screening, produced several biosensor sequences with slower unfolding rates, a positive binding signal, and higher chromophore maturation efficiency. We also observed a blue-shift in the excitation spectrum, and lower background fluorescence in the unbound state. Kd was unchanged. In parallel experiments, LOO-GFP biosensors were genetically fused to fibers formed by the Drosophila protein ultrabithorax (Ubx), and were found to be absent any background fluorescence in the unbound state, but recovered fluorescence when exposed to the target peptide. Implications for the design of biosensing materials are discussed.

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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.

We have introduced two disulfide crosslinks into the loop regions on opposite ends of the beta barrel in superfolder green fluorescent protein (GFP) in order to better understand the nature of its folding pathway. When the disulfide on the side opposite the N/C-termini is formed, folding is 2× faster, unfolding is 2000× slower, and the protein is stabilized by 16 kJ/mol. But when the disulfide bond on the side of the termini is formed we see little change in the kinetics and stability. The stabilization upon combining the two crosslinks is approximately additive. When the kinetic effects are broken down into multiple phases, we observe Hammond behavior in the upward shift of the kinetic m-value of unfolding. We use these results in conjunction with structural analysis to assign folding intermediates to two parallel folding pathways. The data are consistent with a view that the two fastest transition states of folding are "barrel closing" steps. The slower of the two phases passes through an intermediate with the barrel opening occurring between strands 7 and 8, while the faster phase opens between 9 and 4. We conclude that disulfide crosslink-induced perturbations in kinetics are useful for mapping the protein folding pathway.

Green fluorescent protein (GFP) is a 27 kD protein consisting of 238 amino acid residues [1]. GFP was first identified in the aquatic jellyfish Aequorea victoria by Osamu Shimomura et al. in 1961 while studying aequorin, a Ca2+-activated photoprotein.Aequorin and GFP are local‐ ized in the light organs of A. victoria and GFP was accidentally discovered when the energy of the blue light emitted by aequorin excited GFP to emit green light.Unlike most fluores‐ cent proteins which contain chromophores distinct from the amino acid sequence of the pro‐ tein, the chromophore of GFP is internally generated by a reaction involving three amino acid residues [2]. This unique property allows GFP to be easily cloned into numerous bio‐ logical systems, both prokaryotic and eukaryotic, which has paved the way for its utilisation in a variety of biological applications, most notably in biosensing.

In animal cells, microtubules are organized by centrosomes, which are 1-2 mu m diameter organelles. The generation of functional centrosome fragments in-vitro through ultrasonication is presented along with microtubule assembly directed by the patterned centrosome fragments. While centrosome fragments are smaller than the fully constituted centrosomes, their microtubule organization function is retained. The centrosome fragment templates offer greater flexibility and better coverage in both patterning and assembly of microtubules when compared with intact centrosomes. This work provides the rationale and potential for the large-area assembly of microtubules and should expand the application of centrosomes and centrosome components for the creation of microtubule-based nanoscale devices.

In animal cells, microtubules are organized by centrosomes, which are 1-2 microm diameter organelles. The generation of functional centrosome fragments in-vitro through ultrasonication is presented along with microtubule assembly directed by the patterned centrosome fragments. While centrosome fragments are smaller than the fully constituted centrosomes, their microtubule organization function is retained. The centrosome fragment templates offer greater flexibility and better coverage in both patterning and assembly of microtubules when compared with intact centrosomes. This work provides the rationale and potential for the large-area assembly of microtubules and should expand the application of centrosomes and centrosome components for the creation of microtubule-based nanoscale devices.

The overall mechanisms governing the role of laminins during osteogenic differentiation of human mesenchymal stem cells (hMSC) are poorly understood. We previously reported that laminin-332 induces an osteogenic phenotype in hMSC and does so through a focal adhesion kinase (FAK) and extracellular signal-related kinase (ERK) dependent pathway. We hypothesized that this is a result of integrin-ECM binding, and that it occurs via the known alpha3 LG3 integrin binding domain of laminin-332. To test this hypothesis we cultured hMSC on several different globular domains of laminin-332. hMSC adhered best to the LG3 domain, and this adhesion maximally activated FAK and ERK within 120 min. Prolonged culturing (8 or 16 days) of hMSC on LG3 led to activation of the osteogenic transcription factor Runx2 and expression of key osteogenic markers (osterix, bone sialoprotein 2, osteocalcin, alkaline phosphatase, extracellular calcium) in hMSC. LG3 domain binding did not increase matrix mineralization, demonstrating that the LG3 domain alone is not sufficient to induce complete osteogenic differentiation in vitro. We conclude that the LG3 domain mediates attachment of hMSC to laminin-332 and that this adhesion recapitulates most, but not all, of the osteogenic differentiation associated with laminin-5 binding to hMSC.