Green fluorescent protein (GFP) is a 27 kD protein consisting of 238 amino acid residues . GFP was first identified in the aquatic jellyfish
1.1. The three dimensional structure
The molecular structure of GFP was first determined in 1996 using X-ray crystallography .One of the most obvious features of its tertiary structure is a beta-barrel composed of 11 mostly-antiparallel beta strands. The molecular structure of GFP is illustrated in Figure 1 along with a cartoon representation showing the organization of the secondary structure elements that compose the beta barrel.Each beta strand is 9 to 13 residues in length and hydrogen bonds with adjacent beta strands to create an enclosed structure.The bottom of the barrel contains both termini and two distorted helical crossover segments, and the top has one short crossover and one distorted helical crossover segment.The beta-barrel (sometimes referred to as a “beta can” because it contains a central alpha-helical segment) consists of three anti-parallel three-stranded beta-meander units and a two-stranded beta-hairpin (shown in blue, green, and yellow, and red in Figure 1 respectively).The very distorted central alpha helix contains three residues which participate in an auto-catalyzed cyclization/oxidation chromophore maturation reaction which generates the
The interior of the GFP beta barrel is unusually polar.There is an interior cavity filled with four water molecules on one side of the central helix, while the other side contains a cluster of hydrophobic side chains which is more typical of a protein core.Several polar side chains interact with and stabilize the GFP chromophore.Three of these, His148, Thr203, and Ser205, form hydrogen bonds with the phenolic hydroxyl group of the chromophore.Arg96 and Gln94 interact with the carbonyl group of the imidazolidone ring. Figure 2 depicts these stabilizing hydrogen bonding interactions with the chromophore.Additionally, a number of internal residues interact with and stabilize Arg96, a side chain that is known to be required for the maturation of the chromophore.Specifically, Thr62 and Gln183 form hydrogen bonds with the protonated form of Arg96 stabilizing a buried positive charge within the GFP beta barrel, which in turn stabilizes a partial negative charge on the carbonyl oxygen of the imidazolidone ring.
1.2. Thermodynamic and kinetic properties
Wild type GFP has a number of interesting characteristics that can potentially complicate its applicability to biosensing.One is its tendency to aggregate in the cell, especially when expressed in high concentrations. Aggregation is typically caused by exposed hydrophobicity, which may be due to either the presence of hydrophobic patches on the surface of the protein, or to low thermostability, or to slow folding.Surface hydrophobic-to-hydrophilic mutations decrease the aggregation tendency of GFP , but some biosensing applications require surface mutations that may increase aggregation. Most likely, GFP’s low
1.3. Maturation of the chromophore
The chromophore of the native GFP structure is generated by an internal, autocatalytic reaction involving three residues on the interior alpha helix.Cyclization and oxidation of internal residues of Ser65, Tyr66, and Gly67, generate a
The three-step mechanism for the spontaneous generation of the chromophore consists of cyclization, oxidation, and dehydration . Figure 3 illustrates the mechanism, beginning with the original triplet of amino acids. The slow step in chromophore maturation is the diffusion of molecular oxygen into the active site of the closed beta barrel (step 3). The positioning of side chains surrounding the chromophore is crucial for stabilizing the intermediates in the process of chromophore maturation,especially Arg96, which stabilizes the enolate form of intermediate 1 by forming a salt bridge with the negatively-charged oxygen atom, and Glu222, which receives protons from the water molecules to cycle between the protonated and deprotonated states.The two coplanar aromatic rings of the chromophore adopt the
The two spectral absorbances of the GFP chromophore have been found to be highly sensitive to pH changes .At physiological pH, GFP exhibits maximal absorption at 395 nm while absorbing lesser amounts of light at 475 nm.However, increasing the pH to about 12.0 causes the maximal absorption of light to occur around 475 nm while diminishing the absorption at 395 nm.The two absorption maxima correspond to different protonated states of the chromophore.The pKa for the side chain hydroxyl group of Tyr66 is about 8.1  and therefore, the maximal absorbance for the neutral chromophore occurs at 395 nm while maximal absorbance occurs at 470 nm for the anionic form of the chromophore.At acidic pHs lower than 6 or alkaline pHs above 12, fluorescence is diminished as GFP is denatured and the chromophore is quenched.
1.4. Wavelength variants and FRET
Starting with homologous green and red fluorescent proteins, a rainbow of different-colored fluorescent proteins have been developed. Mutating Tyr66 of the GFP chromophore to a tryptophan produces cyan fluorescence, while a histidine mutation produces blue fluorescence. Mutating a threonine on beta strand 10 to a tyrosine introduces a pi-stacking interaction which produces yellow fluorescence. See  for more details. At the other end of the color spectrum, the coral-derived DsRed fluorescent protein, a structural homolog of GFP, was diversified into the mFruits library, producing eight fluorescent proteins with emission maxima ranging from 537 to 610 nm . Far-red fluorescent proteins, which have potential for use in deep tissue imaging due to the penetration of these wavelengths, have been discovered [14-16], while others have been developed in the lab  and even using computational approaches . Further enhancement of these wavelength-shifted variants has improved their biophysical properties and made them available to more applications.
GFP and its derivatives have seen significant use as fluorescent pairs for Förster Resonance Energy Transfer (FRET) experiments. FRET emission arises when the emission spectrum of one chromophore overlaps with the excitation spectrum of another chromophore. If the two chromophores are physically close (on the order of a few nanometers) and in the correct orientation, then excitation of the first chromophore will excite the second chromophore through non-radiative energy transfer and produce fluorescence at the second chromophore's emission wavelength (Figure4). This phenomenon can be used to detect when two fluorescent proteins (FPs) are within a certain distance, which may be induced by a ligand-dependent conformational change in a linking domain between the two fluorescent proteins, or by binding of interacting domains fused to fluorescent proteins. The canonical pairing for FRET using fluorescent proteins is cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) ; but this pairing has issues concerning overlapping emission spectra, stability to photobleaching, and sensitivity to the chemical environment. The study in  had the goal of producing a cyan fluorescent protein more suitable for use in FRET experiments. Other pairings, such as GFP and the the DsRed-based variant mCherry red fluorescent protein, have been proposed as consistent, reliable alternatives . A full review of the development and usage of fluorescent proteins as tools for FRET can be found in . The genetic and physical ease of use of GFP-derived fluorescent proteins, in conjunction with their wide range of colors and spectral overlaps, makes them ideal molecules for the design of FRET-based biosensors.
1.5. Mutants with improved features
Because of the aforementioned slow folding, low solubility and slow chromophore maturation, a significant effort has been put forth to improve these properties in GFP. These strategies range from specific, directed rational mutations based on structural and biophysical information to fully randomized approaches such as error-prone PCR  and DNA shuffling . By mutating the chromophore residue serine 65 to a threonine (S65T) and phenylalanine 64 to a leucine (F64L), an “enhanced” GFP (EGFP, gi:27372525) was produced with the excitation maximum shifted from ultraviolet to blue and with better folding efficiency in
Mutating surface polar residues to increase the net charge, called “supercharging”, may be one solution to the problem of aggregation. Armed with the knowledge that the net surface charge does not often affect protein folding or activity,  demonstrated that mutating the surface residues either to majority positive or to majority negative side chains does not significantly affect fluorescence. Furthermore, these “supercharged” variants of GFP showed increased resistance to both thermally and chemically-induced aggregation with a minimal decrease in thermal stability. The only side effects are the unwanted binding of positively supercharged GFP to DNA, and the formation of a fluorescent precipitate when oppositely supercharged variants are mixed.
Disulfide bonds have been known to confer additional stability to proteins. Two externally-placed disulfides were engineered into cycle3 GFP,one predicted to have no effect on stability, the other predicted to have a stabilizing effect . The predictions, based on estimations of local disorder, were correct. Adding a disulfide where the chain is more disordered improved stability the most.
In recent, unpublished work in our lab , a faster-folding GFP has been made by eliminating a conserved cis-peptide bond. The slowest phase of folding of superfolder GFP has been known to be related to cis/trans isomerization of a peptide bond preceding a proline . We targeted Pro89 for mutation, since the peptide bond is cis at that position in the crystal structure, but modeling studies suggested that a simple point mutation would not have worked. Instead, we added two residues creating a longer loop, and then selected new side chains for four residues based on modeling. The new variant, called “all-trans” or AT-GFP, folds faster, lacking the slow phase. A 2.7Å crystal structure, in progress, shows clearly that the backbone is indeed composed of all trans peptide bonds in the new loop region.
All of the variants discussed so far are derived from
A novel fluorescent protein was developed using the consensus engineering approach, synthesizing a consensus sequence gene from 31 homologs of the monomeric Azami green protein, a distant homolog of
1.6. Sequential rearrangements and truncations
Circular permutation is the repositioning of the N and C-termini of the protein to different regions of the sequence, connecting the original termini with a flexible peptide linker to produce a continuous, shuffled polypeptide. Many proteins retain their structure and function after permutation, provided the permutation site is not disruptive to secondary structural elements. This process demonstrates the tolerance of the protein's overall structure to significant rearrangements of primary sequence , enabling the design of biosensors based on split GFPs as discussed later.
GFP's rigid structure, extreme stability and unique post-translational chromophore formation reaction do not seem to suggest that it would tolerate circular permutation, and for the most part, it does not. All permutations that disrupt beta strands do not form the chromophore, and about half of the permutations in loop regions cannot form the chromophore. However, one particular permutation, starting the protein at position 145 (just before beta strand 7) expresses and fluoresces well, although it is less stable and less bright than the wild type GFP . This circular permutation can also tolerate protein fusions to its new termini (positions 145 and 144 in wild type numbering), and position 145 in the wild type can accept a full protein insertion, such as calmodulin or a zinc finger binding domain . The “superfolder” GFP reported in  was able to fluoresce after 13 of the 14 possible circular permutations, whereas the folding-reporter GFP only tolerated 3 of those 14 permutations.Figure 5summarizes permutation and loop insertion results.
Circular rearrangements preserve the overall “ordering” of the secondary structural elements; however, non-circular rearrangement of the secondary structural elements is also possible. Using rational computational modeling and knowledge about GFP's folding pathway,  designed a “rewired” GFP with identical fluorescence properties and stability as a variant of superfolder GFP, but with the beta strands connected in a different order. These experiments demonstrate the selective robustness of GFP's structure to large-scale rearrangements in sequence, which has implications for deciphering the GFP folding pathway, as well as for design of split-GFP biosensors.
1.7. “Leave-One-Out” GFP
GFP can also be engineered to omit one of its secondary structural elements, either at one end or in the middle of the sequence by truncating a circular permutant. Truncation may be accomplished either at the genetic level or at the protein level, the latter by using proteolysis and gel filtration. Constructs missing one secondary structure element have been named “Leave-One-Out” or LOO, borrowing the term from a method for statistical cross-validation. 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
That Leave-One-Out works is non-intuitive. 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,  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. Although folding appears to be an all-or-none process by most experimental metrics, it proceeds along a loosely defined sequence of nucleation and condensation events called a folding pathway . If the sequence segment that is removed is in the part of the protein that folds last, then a kinetic intermediate exists whose structure closely resembles the native state with one piece removed. This intermediate need not be the lowest energy state and may not be visible by equilibrium measurements, but its minute presence diminishes the energetic barrier of folding enough that the addition of a peptide can push the protein to the folded state. In short, Leave-One-Out uses the idea that some cyclically permuted, truncated proteins are natural sensors of the part left out.
Some LOO-GFPs also show interesting reactions to ambient light. LOO11-GFP (beta strand 11 omitted) does not bind strand 11 when kept completely in the dark, but does bind it upon irradiation with light . Raman spectroscopy showed that, in the dark, the chromophore assumes a
2. GFP-based biomarkers
The term biomarker has accumulated a variety of definitions over the years. Herein, biomarkers are defined as genetically encoded molecular indicators of state that are linked to specific genes.The utility of GFP as a biomarker was first demonstrated using GFP reporter constructs . When GFP is used as a transcription reporter, a cellular promoter drives expression of the fluorescent protein resulting in fluorescent signal that temporally and locally reflects expression from the promoter
GFP fusion proteins (generated by combining the fluorescent protein coding region with the coding region of the cellular protein) are used as markers for visualization of intracellular protein tracking and interactions (reviewed in [47-49]).The GFP moiety may be N-terminal, C-terminal or even internal to the cellular protein.The availability of a color palette of fluorescent proteins allows multicolor imaging of distinct fluorescent protein fusions in the cellular environment. GFP fusion proteins are a major component of the molecular toolkit in cell biology.
2.1. Using GFP as an
in vivo solubility marker
GFP has been used as a genetically encoded reporter for folding of expressed proteins.Expression of recombinant proteins in
Split GFP may also be used to assay folding and solubility of a protein of interest in vivo by “tagging” the recombinant protein with the smaller portion of the split GFP sequence, and expressing the larger portion separately or adding it exogenously. The small size of a protein tag makes it less likely to interfere with the folding and function of the protein of interest.In the split GFP complementation assay a large fragment of GFP folding reporter (GFP1-10 ) is coexpressed with tagged GFP protein (GFP11-protein x) . As shown in Figure 6, neither GFP1-10 nor the GFP11-tagged protein fluoresce alone; however, if both components are soluble,GFP1-10 and the GFP11-tagged protein reconstitute the native structure and fluorescence.For successful implementation of the assay, directed evolution of superfolder GFP1-10 was required. 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 .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 .
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. For example, aggregates of the microtubule associated protein tau are found in neurofibrillary tangles but their role in the pathology of Alzheimer disease and Parkinson disease is not clear .The split GFP complementation assay enables monitoring of the aggregation process in living mammalian cells [52,53] and was validated using GFP1-10 and GFP11-tau variants.Cells containing soluble tagged protein show visible fluorescence but aggregates have little or no fluorescence. Protein aggregates of GFP11-tau sequestered the GFP11 tag, leading to decreased complementation of GFP1-10 and decreased fluorescence. Thus the split GFP complementation assay using tagged-GFP tau showed that it could be used as an
2.2. GFP biomarkers for single molecule imaging
It is also possible to utilize GFP biomarkers for single-molecule localization, a form of super-resolution microscopy. High affinity single chain camelid antibodies (nanobodies) to GFP can be used to deliver organic fluorophores to GFP tagged proteins that are in turn used in single molecule “nanoscopy.” [54, 55]. This novel approach combines the molecular specificity of genetic tagging with the high photon yield of the organic dyes. Additionally, by varying the buffer conditions used, many organic dyes can become photoswitchable. The small size of camelid antibodies and their high affinity allow for access to regions that are generally inaccessible to conventional antibodies and targets that are expressed at very low levels .
One should caution that the overexpression of FRET biomarkers in transgenic animals carries some concerns that this could lead to the perturbation of endogenous signaling pathways and even retardation of animal development . Additionally, in compact tissue, such as the brain tissue, cell type identification is particularly tedious due the diffused expression of the biomarkers.
3. GFP biosensors
Biosensors are distinct from biomarkers in that they are not linked to the expression of a specific gene product. Biosensors may function
In vivo pH biosensors
Within the cell, pH varies from the neutral pH of the cytosol to the acidic pH of the lysosome lumen and protons may serve as cellular signals.Genetically encoded pH biosensors enable subcellular detection of pH and can provide insight into the regulation of cellular activities by pH. Addition of intracellular targeting tag directs the pH biosensor to particular subcellular compartments.
Many GFP variants show sensitivity to pH which results from protonation and deprotonation of the chromophore (see
Two classes of GFP pH indicators have been described: ratiometric and nonratiometric [58, 64].In the ratiometric pH indicators, the chromophore environment is such that the GFP biosensor has two sets of excitation/ emission spectra, one that varies with pH and another that does not.For these GFP variants, a calibration curve can be generated for the ratio of the spectra versus the pH.Nonratiometric GFP variants,such as EGFP  or ecliptic GFP , have pH dependent emission from the anionic chromophore (deprotonated) but almost no fluorescence of the neutral chromophore (protonated). These variants are used for reporting pH changes within cells when used as single molecule pH sensor or used in tandem with pH insensitive fluorescent partner (described below).
Ratiometric GFP pH biosensors have been generated by modification of a few key amino acids in the vicinity of the chromophore.Ratiometric pHluorin (RaGFP), the first ratiometric GFP described,contains a key S202H mutation and shows pH dependent change in excitation ratio between pH 5.5 and pH 7.5 .TheS202H mutation was shown to be important for the ratiometric property; pHlourins lacking the S202H were non ratiometric.Another class of GFP ratiometric pH sensors, deGFPs were generated from mutagenesis of the S65T GFP variant  resulting in substitutions H148G (deGFP1) or H148C (deGFP4) and T203C.The deGFPs are dual emission ratiometric GFPs emitting both blue and green light; blue light emission decreases with increase pH while green light emission increases with increased pH.
Variants pH GFP (H148D)  and E2GFP (F64L/S65T/T203Y/L231H) function as dual excitation ratiometric pH indicators with pH-dependent excitation at 488 nm and relatively pH-independent excitation at458 nm).In addition to its pH sensing properties, fluorescence emission from E2GFP is affected by the concentration of certain ions, including Cl-. The chloride ion sensitivity of E2GFP is a key component of the GFP–based chloride ion and pH sensor ClopHensor  (discussed in section
In addition to single molecule based pH biosensors, ratiometric pH biosensors using tandem fluorescent protein variants have been constructed in which a pH sensitive GFP variant is linked to a less sensitive or pH insensitive GFP.GFpH and YFpH are tandem FRET pairsfor the detection of pH changes in the cytosol and nucleus of living cells. GFpH combines GFPuv, which has low pH sensitivity, with pH sensitive EGFP and YFpH combines GFPuv and EYFP [58, 69].Not all tandem GFP biosensors are FRET pairs, however. pHusion is a ratiometric tandem GFP biosensor in whichmRFP (pH insensitive) is tethered to EGFP (pH sensitive) via a linker.pH measurements are determined from the ratio of EGFP to mRFP fluorescence. pHusion biosensor was developed for analysis of intracellular and extracellular pH in developing plants .
In vivoFRET-based biosensors
Genetically-encoded FRET-based biosensors can be applied in a variety of capacities to visualize intracellular spatiotemporal changes in real time. The evolution of these applications has progressed from cell culture systems that transiently express FRET biosensors to transgenic mouse models that express them in a heritable manner . Production of transgenic mice with FRET biosensors arose in an effort to enhance our understanding of the differences that exist between tissue culture and living systems. Transgenic FRET GFP biosensor systems are very efficient and their fluorescence signals are easily distinguished from autofluorescence, which is analyte-independent fluorescence. The sensors themselves can be used to probe a variety of pathways for the activity of signaling enzymes as well as a number of post translational modifications.
3.2.1. Detection of enzyme activity
In transgenic animal models, FRET biosensors can be used to study PKA activation by cAMP, ERK activation by TPA and their association with various physiological changes . PKA and ERK areenzymes that transfer the γ-phosphate of ATP to a number of protein substrates thereby affecting a conformational change. Kinase induced conformational changes are important because they are involved in the control a number of critical cellular processes that include glycogen synthesis, hormonal response, and ion transport . A number of signaling cascades that involve kinases require a means of dynamic control and spatial compartmentalization of the kinase activity; a requirement highlights the need for a mechanism to continuously track kinase activity in different compartments and signaling microdomains
Traditional methods of assaying kinase activity fail to capture its dynamicity; a void that is filled by genetically encoded FRET-based biosensors. These sensors are constructed so that the substrate protein of the kinase of interest is flanked with a fluorescent protein pair in such a way that the conformational change imparted by phosphorylation translates into a change in the FRET signal (Figure7) . These biosensors can be localized to particular sites of interest with the aid of appropriate targeting signal sequences, allowing the imaging of site-specific kinase activity.G-protein coupled receptors, when used in a biosensor, provide a mechanism for transducingdrug mediated effects on PKA activity into a light signal. Transgenic mice expressing FRET based biosensors provide an ideal system for studying the pharmacodynamics of these drugs.
When used to study the signaling events in wound healing, the strength and duration of the fluorescent signals that are generated by these biosensors are dependent on the location within the tissue (tissue depth has a negative impact on the intensity of the fluorescent signal), its vicinity in relation to the site of injury, as well as the contributions made by chemical mediators (drugs) in sustaining kinase activity . These model systems provide a means of visualizing in real-time the agonist/antagonist pharmacodynamics associated with a plethora of signaling molecules that do not necessarily have to be limited to PKA and ERK activity. They also provide a tool for resolving the maze of upstream signaling pathways that contribute to chemotaxis in the animals.
Genetically encodable FRET GFP biosensors have proven to be useful in characterizing the dynamic phosphorylation dependent regulation of small GTPases . Ras GTPases play essential roles in regulating cell growth, cell differentiation, cell migration, and lipid vesicle trafficking. Upon binding GFP, the G-protein Ras recruits the serine/threonine kinase Raf. FRET biosensors for GTPase activity such as Raichu-Ras (Ras and Interacting protein CHimeric Unit for RAS) use this Ras-Raf interaction as the basis for the molecular switch. Raichu-Ras functions by using H-Ras as the sensor domain and the Ras Binding Domain (RBD) of Raf as the ligand domain in constructing a molecular switch that in turn is sandwiched by the FRET pair CFP/YFP (Figure 7). Such a design allows for the monitoring of Ras activation in living cells on the basis of fluctuations in the FRET signals generated.
3.2.2. Detection of antioxidant activity and reactive oxygen species
FRET-based GFP biosensors can also be employed in
3.2.3. Detection of calcium ions
FRET-based and single domain Ca2+ sensors have been constructed using the allosteric effect of calcium binding to receptors calmodulin or troponin . In one construct, the CFP/YFP pairing is separated by a linker containing a calmodulin domain and a calmodulin ligand peptide called M13.When Ca2+ is present, it binds to the calmodulin domain, inducing a conformational change and binding of the proximal M13 peptide sequence. The M13 binding results in shortening of the linker, bringing CFP within FRET distance of YFP and changing the emission wavelength from cyan to yellow. The Ca2+ binding affinity was found to be highly variable, around 0.3 uM with a Hill coefficient of n=4, depending on conditions. When used
In vitro applications
GFP has great potential to work as an
4.1. GFP-antibody chimeric proteins
The goal of GFP-antibody chimeric proteins (GFPAbs) is to convert a multi-step experimental process for locating molecules via antibodies and enzyme-linked secondary antibodies, into a one-step process using a GFPAbs.This molecule could then work as a detection reagent in flow cytometry, for intracellular targeting, or fluorescence-based ELISAs .However, in order to replace antibodies in these techniques, it is important to achieve the same nanomolar sensitivity that is found in the natural antibodies.To do this,  inserted two antigen-binding loops into the GFP structure, counting on cooperativity in binding to enhance affinity.
It became clear that adding loops impinges on the integrity of the native GFP structure.The binding loops must be placed such that their presence in the fluorescent protein does not jeopardize its structural fidelity, or that of the chromophore.There are only a few locations in the molecule that are amenable to such insertions:turn regions β4/β5 (residue 102), β7/β8 (residue 172) and β8/β9 (residue 157).The latter two are too far apart in three-dimensional space to provide for cooperative binding (see Figure 5). The β4/β5 and β8/β9 loop regions are in close proximity, but these do not easily accommodate random loop insertions.
 used directed evolution with yeast surface display  to find sequences that stabilized the folded conformation in the context of loop insertions.The yeast secretory pathway does not allow unfolded protein to reach the surface of the cell, thus only mutants that yield fully folded GFP were displayed by yeast cells. Directed evolution revealed several mutations that conferred additional stability and increased fluorescence in the context of inserted loops: D19N, F64L, A87T, Y39H, V163A, L221V, and N105T. The F64L mutation has been shown to increase fluorescence of GFP and also to shift the excitation maximum to 488 nm.Y39H and N105T have been shown to improve refolding kinetics and refolding stability, respectively.V163A is linked to improved folding as a result of its increased expression in yeast surface display . These mutations accommodated the insertions of antigen-binding loops from antibodies raised against streptavidin-phycoerythrin, biotin-phycoerythrin, TrkB, or GADPH, all while maintaining 40% of the fluorescence and 60% of the expression of wild type GFP.With dual loop insertion, dissociation constants as low as 3.2 nM have been achieved . The success of this construct means that molecules such as GADPH can be located within cells without having to engineer a second round of antibodies, saving both time and resources.
4.2. A chimeric fluorescent biosensor based on allostery
A general method for developing a biosensor for a specific receptor-ligand interaction has been described  in which a receptor protein is inserted into the GFP sequence between strand 8 and strand 9. The insertion puts enough of a strain on GFP that its fluorescence is reduced. Binding of the ligand to the GFP-receptor chimera may then impart enough of a change in its conformation that it causes a change in fluorescence, since the b8/b9 loop is fairly close in space to the chromophore. This change may be found by plate screening for fluorescence. In , the receptor Bla1 was cloned into the loop, and random mutations were made to this construct. Mutant constructs that detected the Bla1 ligand BLIP were identified by a visual screen of colonies before and after the induced expression of BLIP. Using this method, a double mutant was found that was shown to detect BLIP
4.3. FRET-based biosensors using quantum dots
FRET-based in vitro biosensors may be constructed by linking fluorescent proteins to quantum dots (QDs).QDs are inorganic molecular nano-crystals whose absorption and emission spectra are dictated by the size of the QD.For example, a QD may be engineered to absorb ultraviolet light and emit light at 550 nm, which overlaps well with the excitation spectrum of mCherry, a variant of GFP , and produces FRET when the two fluorophores are in close proximity.
In order to make the FRET emission analyte-dependent, the QD was linked to the mCherry via an N-terminal linker peptide that contained a protease cleavage site and a 6 histidine tag.The imidazole side chains of the histidines electronically coordinate with the zinc atoms of the CdSe—ZnS core-shell semiconductor of the QD . Multiple mCherry molecules can be coordinated with each QD. Splitting of mCherry from the QD by a protease may be detected by the loss of FRET.By placing the caspase-3 cleavage sequenceinto the linker between GFP and the QD, the FRET complex becomes a biosensor for the presence of caspase-3, glowing red at 610 nm in the absence of the protease, and reverting to the yellow fluorescence of the QD at 550 nm when the protease is present (Figure 8).
GFP/QD FRET emission may be also be manipulated by pH-induced changes in the spectral overlap, without having to spatially separate the QD from the fluorescent protein.It has been shown that fluorescent proteins such as GFP and mOrange experience a shift in excitation and emission spectra with changes in pH .At a slightly acidic pH, there is very little spectral overlap between the QD emission and the mOrange excitation, which means that the QD emission is seen, in this case around 520 nm.However, as the pH increases, the excitation spectrum of mOrange shifts such that there is more overlap with the QD emission, which subsequently causes an increase in FRET.The result is an upward shift in the emission wavelength with increasing pH.It is important to note that since there is a fluctuating hydrogen ion concentration, the histidine-QD coordination complex becomes unstable.In order to remedy this problem, a covalently linked quantum dot must be used.
4.4. Fluorescent proteins as intrinsic ion sensors
Fluorescent proteins, especially E2GFP, have been shown to be sensitive not only to pH changes but also to the concentration of certain ions, particularly chloride ions.E2GFP provides an avenue for single domain ratiometric analysis of pH because it contains two excitation and emission peaks. Only the longer wavelength emission peak is pH dependent .Therefore analysis of pH based on the ratio of green fluorescence to cyan.By coupling E2GFP to another fluorescent protein in a fusion construct, it is also possible to measure other intracellular chloride ion concentration.For example, DsRed is neither pH nor chloride ion sensitive, so it can be used to measure chloride ion concentration based on the ratio of its fluorescence to the cyan emission of E2GFP.
Making a few modifications can make GFP sensitive to the concentration of other ions.For example, superfolder GFP can be made sensitive to copper ions by mutating the arginine at position 146 to a histidine, which, as previously mentioned, coordinates well with metal ions . GFP can also become sensitive to ions by creating channels in the structure through which small molecules can pass through and access the chromophore (Figure 9).By mutating position 165 from a phenylalanine to a glycine, a channel is opened that is about 4 Å wide.This allows small molecules such as copper ions to enter the hydrophobic core of the protein and quench fluorescence . GFP, thanks to its stability, has shown a remarkable ability to be modified, and thus shows great promise in visualizing a large variety of intracellular and extracellular substances.
5. Computationally designed LOO-GFPs
Recent work in the Bystroff lab has focused on programming GFP to accept any desired protein as a binding partner, like an antibody, and to switch on fluorescence only when the targeted protein is bound. The strategy combines Leave-One-Out split reconstitution with computational design and high throughput screening.
Leave-One-Out (LOO) was described earlier (
Theoretically, this goal could be achieved by random mutation followed by high throughput screening to find mutants that glow in the presence of a peptide. However, random mutation would be extremely inefficient. Computational protein design methods offer a much better alternative for rationally generating sequence diversity before the labor-intensive experimental screen.
5.1. Computer-aided protein design
Computational protein design predicts protein sequences that fold into predefined protein structures. Proteins are described as a set of atoms with 3D spatial coordinates and physical/chemical properties [81-84]. Instead of mutating residues experimentally, mutations are explored
5.2. Protein biosensors versus other methods for detecting pathogens
Biosensors for specific proteins and pathogens offer potential advantages over the current state of the art, notably speed and simplicity. Laboratory diagnostics of infections commonly includes pathogen isolation using culture, direct antigen detection, or detection of pathogen specific DNA and/or RNA by polymerase chain reaction (PCR). The isolation method requires a culture system to inoculate a specimen, followed by the examination of specific characteristics produced by pathogens, such as the cytopathogenic effect of virus and the distinct metabolism of bacteria. Although culture-based methods have higher detection sensitivity, they generally take three to ten days for diagnosis. Alternatively, immunoassays utilize pathogen specific antibodies and secondary anti-antibodies to detect and report a pathogen. Most of the rapid diagnostic tests only take 15 to 30 min for diagnosis, but raising specific antibodies against pathogens is time-consuming and expensive. Thirdly, molecular diagnosis using PCR takes the advantage of the gene amplification and provides a highly sensitive detection in diagnosis from minute amounts of pathogen genome within a short time. However, the need for real-time PCR and gel electrophoresis apparati and reagents means it will not be possible in all settings, where a simple biosensor test would be possible. PCR assumes that DNA is present, but some pathogens such as anthrax toxin, snake venom and bovine spongiform encephalopathy contain no genetic material. All these point to a need for developing a diagnostic tool for proteins that is fast and easy to use, and suitable for rural, point-of-care facilities in developing nations.
The following describes how the computer-aided design of LOO-GFP was done, and the encouraging but preliminary results. The process has three steps: (1) the selection of a target peptide sequence from the genome of the pathogen, (2) the computational design of the LOO-GFP• target complex, and (3) the experimental screening of a library of potential biosensor sequences.
5.3. Target peptide selection
A target peptide for detection must be unique in order to avoid false positives, and must be conformable to the LOO-GFP binding site, which is the site of one of the eleven β-strands of GFP. From the examination of GFP and homolog fluorescent protein structures and sequences, we defined a set of signature patterns for each β-strand. These patterns define the limits of mutation. For example, no position within a target peptide may be a proline, since it must be hydrogen bonded on both sides to the neighboring β-strands. Cysteines are also disallowed, for experimental reasons.Target peptides are selected by searching the sequences of the target organism for a match to the signature pattern.Other considerations including the location of protease recognition sites, cellular location, and protein expression levels.
In the case study described here, a twelve-residue peptide (SSHEVSLGVSSA) was selected from hemagglutinin (HA) sequence of avian influenza virus H5N1, using the signature pattern of GFP β-strand 7. The target peptide retains the sequence pattern of the wild type β-strand 7, and it can be released by the chymotrypsin digestion of HA protein. A BLAST search of all known protein sequences confirmed that the HA target sequence occurs only in hemagglutinin from influenza virus type A.
5.4. Computational pre-screening of candidate biosensor sequences
To engineer customized LOO-GFP biosensors that sense a given peptide we developed a set of software called DEEdesign. DEEdesign uses a combination of physical properties and statistical knowledge to energetically evaluate the fitness of rotamers in protein structures, along with sampling algorithms to search the space of all possible mutations. The parameters used in the fitness scoring system are trained by a machine learning technique to reproduce the true sidechain conformations in high-resolution crystal structures . Sequence space is searched using one of two methods, either using Monte Carlo , with random mutations accepted or rejected based on the calculated energy, or using the dead-end elimination theorem (DEE), which holds that if energies can be decomposed into pairwise terms, then a solution to the problem of finding the lowest energy set of mutations can be found by a process of successive elimination .
However, inaccuracies in design due to the imperfect scoring system, the use of discretized side chains, and the lack of precise modeling of backbone flexibility, affect the reliability of the method. Therefore, instead of relying on the accuracy of the single lowest energy protein sequence, DEEdesign provides an ensemble of plausible mutants, all with reasonably low calculated energy scores. These are assembled into a single amino acid profile, from which a library of nucleotide sequences is derived, employing degenerate codons for those positions in the sequence that have more than one possible amino acid.
In our case study, residues 143-154 NSHNVYITADKQ of β-strand 7 were mutated
5.5. Experimental screening and diversity generation by
in vitro evolution
The computationally generated library for the H5N1 LOO-GFP biosensor had a complexity of around 10000 sequences and was relatively easy to screen in low to medium-throughput manner by looking for colonies that were fluorescent when co-expressed with its target peptide sequence. We fused the target peptide to intein  so that it would be cleaved immediately after expression and would exist as a free peptide.
However, potential mutations that are distant from the binding site of the target peptide (i.e. >10Å away from the binding site) may still have indirect effects on the binding of the target, or influence on LOO-GFP folding, are not easily captured in the computational design process because of time and memory limitations. To expand the screening, candidate mutant genes can be subjected to rounds of
We demonstrated the first proof-of-concept for designing LOO-GFP biosensors by combining computational protein design and
Potential candidates for LOO-GFP biosensors were plate-screened in
When co-expressed with the HA peptide, the DS1 mutant exhibited target-dependent maturation of chromophore, while in the absense of the peptide it showed barely detectable fluorescence even after 24 hours, indicating a specific interaction between DS1 mutant and the HA peptide. DS2 mutant showed faster recovery of fluorescence within four hours in the presence of the HA peptide; however, a higher degree of nonspecific auto-fluorescence was also observed after 24 hours. The DS1 mutant chromophore formation showed a greater dependency on the left-out peptide (i.e. the HA peptide), implying better folding of designed LOO-GFP molecule, than DS2 mutant
The unique physical properties of GFP have made it a gold mine for the development of biosensors and biomarkers. GFP is kinetically super-stable. Its sequence may be readily permuted and mutated. Its engineered variants fluoresce at wavelengths across the visual spectrum, and some pairs of variants can interact via FRET. GFP is quenched by unfolding, by certain ions, and sometimes by light, and variants of GFP are pH sensitive. With many ways of generating a signal, it is no surprise that many types of biosensors have been developed that use GFP and its homolog fluorescent proteins.GFP and its variants can be immobilized and even dried while retaining structure and biosensor function, leading to the promise of future GFP-biosensor microarrays capable of detecting a wide variety of analytes in a single assay. In addition to being broadly useful, such material should be very cheap to produce, and would also be easily stored, used, and read.Arrays of GFP-based biosensors on paper or film may someday become available for household use, so that infections may be rapidly diagnosed without a trip to the hospital, or may become integral parts of devices that continuously monitor the water and air, making the world a healthier and safer place.
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