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Maltose-binding protein: a versatile platform for prototyping biosensing
Maltose-binding protein: a versatile platform for prototyping biosensing Igor L Medintz and Jeffrey R Deschamps The bacterial periplasmic-binding protein (PBP) superfamily members, in particular the maltose-binding protein, have been used extensively to prototype a variety of biosensing platforms. Although quite diverse at the primary sequence level, this protein superfamily retains the same basic twodomain structure, and upon binding a recognized ligand almost all PBPs undergo a conformational change to a closed structure. This process forms the basis for most, but not all, PBP-based biosensor signal transduction. Many direct detection or reagentless sensing modalities have been utilized with maltose-binding protein for both in vitro and in vivo detection of target compounds. Signal transduction modalities developed to date include direct fluorescence, electrochemical detection, fluorescence resonance energy transfer (FRET)based detection, surface-tethered FRET sensing, hybrid quantum dot FRET sensing, and enzymatic detection, each of which have different benefits, potential applications and limitations. Addresses Center for Bio/Molecular Science and Engineering, Code 6900, Laboratory for the Structure of Matter, Code 6812, US Naval Research Laboratory, WA 20375–5320, USA Corresponding author: Medintz, Igor L ([email protected])
Current Opinion in Biotechnology 2006, 17:17–27 This review comes from a themed issue on Analytical biotechnology Edited by Jan Roelof van der Meer and J Colin Murrell Available online 18th January 2006 0958-1669/$ – see front matter Published by Elsevier Ltd. DOI 10.1016/j.copbio.2006.01.002
Introduction The bacterial periplasmic-binding proteins (PBPs), most commonly cloned out of E. coli, are part of a large diversified protein superfamily that is found even in eukaryotes [1,2,3]. Although unrelated at the primary sequence level, almost all PBPs share a common structural motif that consists of two domains, joined by a ‘hinge’ region, which surround a central ligand-binding site [1,2,3], see Figure 1. A common functional conformational change is also found in this protein family, which involves switching from the ‘open’ ligand-free form to a ‘closed’ or ligand-bound form. This transition is accomplished through a bending and swiveling twist www.sciencedirect.com
motion about the hinge region, which is analogous to a ‘venus-fly-trap’ closing, see Figure 1b [4]. The interest in exploiting PBPs for biosensing arises both from their specific recognition of myriad analytes approaching nanomolar concentrations in some cases and from the ligand-induced conformational changes that they undergo [4]. It is believed that the common functional mechanism and wide specificity of PBPs originated during evolution by positive selective pressure on their hosts as they mediated solute recognition and uptake in the periplasm and directed chemotaxis [1]. The analytes targeted by bacterial (b)PBPs include: various sugars such as glucose, maltose, ribose and arabinose; amino acids such as glutamine, glutamate/aspartate and histidine; di- and tripeptides; and metals and ions such as Fe(III), Ni(II), phosphate and sulphate, to name but a few [2,5]. Furthermore, Hellinga and co-workers have shown that PBPs can be computationally remodeled and mutated to recognize non-natural analytes or to function as enzymes (see below) [6,7]. Owing to the extensive understanding of its structure and function, maltose-binding protein (MBP) is the prototypical member of this superfamily. MBP specificity is well known, the crystal structure in both the open and ligand-bound form has been solved [8–10], and a variety of mutants that have different maltose sensitivities (Kds that range from nM to mM) have been created [2]. Moreover, MBP has been extensively utilized for recombinant protein purification over amylose resin [11]. This thorough understanding of structure–function in MBP initially, and now in other PBPs, has spurred research into exploitation of the ligand-induced conformational rearrangements [12] and the application of diverse analytical methods to create viable biosensors. A variety of analytical or signal transduction mechanisms have been used to monitor PBP binding and conformational changes, including direct and environmentally sensitive fluorescence, electrochemical and fluorescence resonance energy transfer (FRET), see Figure 1a. Although not discussed here, there have been other sensing modalities prototyped on PBPs including surface plasmon resonance, quartz crystal microbalance analysis and potentiometry [13–15]. Cumulatively, the results indicate that PBPs, and especially MBP, are an excellent platform not only for biosensing but also for prototyping a variety of diverse sensing modalities for eventual use in various environmental, clinical and security applications [16–18]. This review highlights many of the direct detection or reagentless sensing modalities that have been utilized Current Opinion in Biotechnology 2006, 17:17–27
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Figure 1
PBP structure and function. (a) A prototypic PBP structure is depicted; this has two lobes (green) that create the ligand-binding pocket and are joined by the hinge-binding region (pink). Upon binding the ligand, the PBP undergoes transition to the closed structure. PBPs present two intrinsic properties available for biosensor exploitation, either separately or when functioning in concert: recognition specificity and conformational change. Recognition specificity has been utilized for fluorescence- and FRET-based sensing. Conformational changes have been utilized for FRET-, electrochemical-, fluorescent- and enzymatic-based sensing. (b) Ribbon and string rendering of MBP structure in the open form (right) [8,9] and ligandbound closed form (left) [10]. MBP dimensions are 30 40 65 A˚ [9,23]. The two domains (lobes) are highlighted in green and grey. Upon binding maltose (purple), the lobes rotate 358and twist laterally 88 relative to each other [9,23]. Overall the amino- and carboxy-termini move 7 A˚ closer to each other after binding [4]. Note the change in conformation of the overall structure upon binding maltose.
with MBP for both in vitro and in vivo detection of target compounds. The benefits, potential applications and limitations of each sensing method are discussed along with recent developments in this field.
Direct fluorescent monitoring of dye-labeled periplasmic-binding proteins The early work in adaptation of PBPs for biosensing focused on direct fluorescent monitoring of dye-labeled proteins. In a pioneering study, Cass and co-workers [19] demonstrated that MBP could be mutated to express a cysteine residue near the binding site, which could act as a site for subsequent dye conjugation. Upon maltose addition, the fluorescence intensity of the dyes attached at this site increased from 60% to 180% as MBP underwent ligand-induced conformational changes. Similar sensing strategies are still in use today. Kobatake and Current Opinion in Biotechnology 2006, 17:17–27
co-workers [20] coupled glutamine-binding protein (QBP) to a designer hydrophobic polypeptide to allow adherence onto unmodified hydrophobic surfaces. The conjugation of dyes to a unique cysteine site on QBP also demonstrated that fluorescence intensity increases upon ligand binding. This study indicates that it might be feasible to prepare arrays of PBP sensors on unmodified surfaces. However, the work in the laboratories of both Cass [19] and Kobatake [20] relies on environmentally sensitive fluorophores that respond appropriately to the binding-induced environmental changes in the surrounding protein. These dyes can be relatively insoluble and unstable when in aqueous environments for long periods of time. The monograph of De Lorimier, Hellinga and coworkers [2] is perhaps the best resource on designing PBP-based sensors for fluorescent monitoring. This reference includes strategies on designing, engineering and www.sciencedirect.com
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characterizing PBP-based biosensors. It can be considered a ‘how-to’ guide for anyone interested in this field. Although both genetic and protein structural data can be used to optimize sensor design, specific dye–protein interactions are still not fully understood. Results have shown that not all choices of mutation sites and fluorophores function for sensing, and multiple sites typically have to be tested [2].
Fluorescent protein–periplasmic-binding protein fusions Using the same concept as the Ca2+-sensing ‘cameleons’ of Tsien and co-workers [21,22], in which fluorescent proteins flank a central recognition sequence and their relative positions change upon Ca2+ binding, the Frommer laboratory [4,23,24] has constructed an elegant series of reversible FRET-based sensors that consist of fluorescent proteins fused to the amino- and carboxy-termini of various PBPs, see Figure 2a. The intrinsic sensitivity of FRET to molecular scale rearrangements lends itself to monitoring the relative nanoscale re-arrangements of the fluorescent proteins in these sensors during ligand binding. Frommer’s maltose-sensing prototype, termed ‘FLIPmal’, consisted of MBP that had an enhanced cyan fluorescent protein (CFP) donor (D) fused to the amino terminus and an enhanced yellow fluorescent protein (YFP) acceptor (A) fused to the carboxy terminus, see Figure 2a. Upon binding maltose, the sensor undergoes the obligatory conformational rearrangement, bringing the two fluorescent proteins into proximity and altering the efficiency of FRET between them in a concentrationdependent manner. A series of mutants with Kds of 2, 25 and 225 mM were created to increase the dynamic range of sensing from <1 mM to >1 mM. The utility of this nanosensor was demonstrated by measurement of the maltose concentration in a complex beer sample and the maltose uptake in live yeast cells that endogenously express the sensor [23], see Figure 2b. Additional sensors targeted glucose and ribose by use of glucose-binding protein (GBP) and ribose-binding proteins (RBPs) as backbones [24,25]. However, in these nanosensors the fluorescent proteins move apart in response to binding, see Figure 2a, which results in a decrease in FRET donor/acceptor fluorescent ratio. This is owing to placement of the fluorescent proteins near the hinge region of the proteins as opposed to at the opposite end of each lobe as in the FLIPmal series [4,24]. A FLIPglucose sensor that expresses three copies of the simian virus 40 large T nuclear localization signal allowed almost exclusive targeting to the nucleus in COS-7 cells; this demonstrates cell compartmental monitoring specificity, see Figure 2c [26]. Recently, Frommer and co-workers [27] have adapted the glutamate/aspartate-binding protein to enable it to measure glutamate release from neurons; the same design was used in conjunction with the platelet-derived growth factor receptor transmemwww.sciencedirect.com
brane domain to create a sensor that is surface-displayed in hippocampal cells and directly monitors glutamate metabolism. The ability to express these nanosensors in plants, yeast and eukaryotes in vivo is probably the single biggest advantage of this sensing strategy. Perhaps, the only drawback is the small dynamic range in FRET donor/acceptor ratio, which might necessitate sensitive equipment and spectral deconvolution. An evolved CFP–YFP FRET pair that exhibits a 20-fold ratiometric change in FRET signal has been reported, which might represent a possible solution [28]. Given the potential pool of analytes that can be targeted by the PBP superfamily, these biosensors will see much use owing to the increased research on how cells monitor and respond to signaling and nutrients in vivo [29,30]. Perhaps a ‘cassette’ version will become available that will allow one to pick from a variety of sensors for targeted analysis within a cell line of choice.
Periplasmic-binding protein surface-tethered sensing assemblies We have focused on creation of reagentless sensing assemblies by the exploitation of several PBP properties including recognition specificity and sensitivity, ease of recombinant engineering, the ability to site-specifically implant fluorophores within the protein structure, and their overall size [31,32,33]. Figure 3a highlights the key features incorporated into this design and tested using MBP as a prototype [33]. The sensing assembly consists of dye-labeled MBP that interacts with a co-functional modular DNA arm, both of which are surface-tethered. Sensing in a microtiter-well format allows access to fluorescent plate readers — a technology common to many biological laboratories. Surface tethering and orienting of all components relies on biotin-avidin chemistry, which allows the entire sensor to be self-assembled yet robust. MBP is attached to a NeutrAvidin microtiter well surface by an engineered carboxy-terminal 5-histidine, HIS5, sequence (originally incorporated for metal-affinity purification) using a bifunctional biotin–Ni2+–nitriloacetic acid moiety, engendering a quasi-fixed ‘face-up’ surface orientation to the MBP. Prior to immobilization, MBP is site-specifically labeled with either a fluorophore or a dark quenching dye. The co-functional modular arm consists of a flexible biotinylated DNA oligonucleotide attached to a central dye and terminates in b-cyclodextrin (b-CD), a known MBP ligand, see Figure 3b [8]. During surface self-assembly of both components, MBP binds the proximal b-CD ligand bringing the two dyes into proximity and establishing efficient FRET. Addition of maltose displaces the b-CD-linked dye altering FRET in a concentration-dependent manner and provides fluorescent signaling, see Figure 3c–e. The assembly is then washed free of analyte and is regenerated for subsequent sensing events. Up to eight consecutive sensing/regenerations have been demonstrated for the same assembly. Two methods were tested to adjust the sensitivity. The Current Opinion in Biotechnology 2006, 17:17–27
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Figure 2
Fluorescent protein–PBP fusion sensors. (a) Schematic design of PBP–fluorescent protein FRET sensors. CFP, depicted in blue, and YFP, in yellow, are fused to the amino and carboxy termini of the proteins. The differing results achieved using GBP, MBP and RBP are shown. Owing to the differing positions of the termini in the proteins, the binding of maltose by MBP moves the fluorescent proteins together, whereas binding of sugar by RBP and GBP move the fluorescent proteins apart. Their relative movement upon ligand binding is monitored by FRET between the two fluorescent proteins. Ostermeier uses a nomenclature (FLIPmal) to describe his constructs whereby ‘FLIP’ designates a construct of two fluorescent proteins and ‘mal’ indicates the central protein and target analyte. (b) Visualization of dynamic maltose concentration changes in the cytosol of yeast that expresses the FLIPmal sensor with a Kd of 25 mm. The graph indicates the change in uptake emission intensity ratio for a single yeast cell. Figure copyright National Academy of Sciences USA [23]. The colors that range from blue to red are a scale reflecting the increasing internal maltose concentration. The plotted squares show the data as the difference in FLIPmal ratio. These results demonstrate that this sensor effectively allows one to monitor the increasing concentration of internal maltose. (c) Specific targeting of sensors to subcellular compartments. In COS7 cells the protein is distributed in the cytosol, however, sensors that carry the triple simian virus 40 large T nuclear localization signal are targeted almost exclusively to the nucleus. Panels (a) and (c) adapted from [4] with permission from Elsevier Ltd.
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Maltose-binding protein Medintz and Deschamps 21
Figure 3
Surface-tethered sensing assemblies. (a) Schematic description of a tethered MBP biosensor. The modular arm (b-CD–Dye–DNA) is attached indirectly to the NeutrAvidin (NA) surface by way of DNA hybridization to a biotinylated (B) complimentary DNA that is directly tethered to the NA surface (red). Dye-labeled MBP (Dye 1) is attached to the same surface by a Bio-X (NTA) moiety that binds to the HIS5 sequence of MBP. Controlled self-assembly allows attachment of both MBP and modular arm to the NA. MBP binding of b-CD–dye–DNA arm assembles the final sensor by bringing Dye 1 and 2 into proximity, which allows FRET quenching when excited at the appropriate wavelengths. Maltose (the target analyte) displaces the b-CD, which disrupts FRET in a concentration-dependent manner. The sensor assembly is then washed and regenerated in buffer for a subsequent detection event. Use of MBP-binding mutants or rigidifying the DNA linker with a complementary DNA modulator alters useful sensing ranges. (b) Structure of the modular arm that functions with MBP to form the sensing assembly. (c) The sensor schematically depicted in (a) was self-assembled using an MBP95C mutant labeled with a QSY7 dark quencher. The resulting titration versus maltose is shown. The apparent binding constant (Kapp) is shown along with the estimate of useful sensing range — defined as 10–90% sensor saturation [23]. (d) Variant of the sensor is presented using MBP80C QSY7 substituted for the MBP95C protein. (e) Variant of the MBP95C sensor in (a) with the addition of a complimentary modulator DNA that rigidifies the DNA linker arm. Figures are reproduced with permission from the American Chemical Society [33].
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first utilizes MBP mutants that have differing binding constants (Kapp), by substituting the MBP95C mutant (Kapp 5 mM) with a MBP80C mutant (Kapp 575 mM), see Figure 3c and d. This also adjusted the useful sensing range (defined as 10–90% sensor saturation) from low mM to low mM. The second method uses complimentary ‘modulator’ DNA to rigidify the DNA arm to adjust sensitivity, specifically by increasing the useful sensing range, see Figure 3e. Besides being regenerable and having adjustable sensitivity, several other benefits of this design have been noted. Sensors could be pre-assembled on microtiter well plates, tested, washed, dried, stored in a refrigerator and then reconstituted for reuse several weeks later. This has implications for pre-packing such sensors for later commercial use or for deployment in the field. The modular nature of this sensing assembly suggested that it could target other analytes; this was proven by the development of a sensor assembly that targeted the explosive 2,4,6-trinitrotoluene (TNT). This was accomplished by the substitution of an antibody fragment specific for TNT in place of MBP and the synthesis of an appropriate co-functional arm that terminates in a TNT analog [34]. This strategy might, however, be limited to smaller target analytes owing to FRET size considerations as well as finding appropriate pairs of protein recognition elements and synthesizing cofunctional dye-labeled analogs amenable to use in such a scheme. Overall, the concept demonstrates a way to incorporate into a reagentless sensor ensemble a PBP that can be attached to a surface and that does not rely on conformational coupling for signal transduction. This also shows that MBP can function in concert with complex chemical entities to achieve sensing.
Quantum dot–MBP sensor Luminescent quantum dots (QDs) are colloidal semiconductor nanocrystal-based fluorophores that have interesting photophysical properties including broad absorption spectra, molar extinction coefficients 10–100 times those of organic dyes, high quantum yield (QY), high resistance to chemical degradation and photobleaching, and, most impressively, have size-tunable photoluminescent emission [35–37]. Their large effective Stokes shift (in this context the spectral difference between wavelength of excitation and emission) means that mixed QD populations can be effectively excited at wavelengths far removed from their respective emissions. In particular, for FRET, QD photoluminescent spectra can be size-tuned or ‘dialed in’ to improve overlap with a particular acceptor [36]. These unique properties are stimulating a great deal of interest in the adaptation of these nanocrystalline fluorophores for biosensing [35,37]. Using the same criteria that made MBP an attractive prototype for the surface-tethered sensing assembly above, we designed and tested a prototype hybrid nanoCurrent Opinion in Biotechnology 2006, 17:17–27
crystal–protein or QD–MBP based FRET sensor. The sensor consisted of MBP self-assembled onto the surface of 560 nm-emitting CdSe–ZnS core-shell QDs, see Figure 4a [38]. The MBP’s carboxy-terminal HIS5 tract allows the protein to self-assemble and coordinate to Zn on the QD shell by way of metal-affinity coordination. Prior to being immobilized on the QD surface, the MBP is allowed to pre-bind a b-CD maltose analog that is covalently attached to a QSY-9 dark quencher or non-emissive acceptor dye. Subsequent attachment of the b-CD quencher that carries MBP to the QD surface brings the quencher into proximity to the QD exciton-emitting core and establishes efficient FRET quenching. Addition of maltose displaces the b-CD-quencher analog, disrupting FRET, and recovers the QD emission in a concentration-dependent manner, see Figure 4b and c. MBP specificity and sensitivity were maintained in this format, with low micromolar recognition of only those sugars that contain a1–4 glucosidic linkages. The orientation of the final QD–MBP conjugate was also modeled based upon FRET between the QD donor and fluorophores attached to unique sites within the MPB structure, using a strategy analogous to a nanoscale global positioning system [39]. In this case, MBP allowed elucidation of a QD–protein bioconjugate structure. Following on from this work, we have recently prototyped a reagentless version of the QD–MBP biosensor that is a functional hybrid between the above QD–FRET displacement sensing format and the previously described conformational-dependent approach, see Figure 4d and e. MBP labeled with a Cy3 at a peristeric site (close to the binding pocket but does not interfere with substrate binding) was self-assembled onto the surface of QDs using HIS5–Zn coordination [40]. This brings the Cy3 acceptor dye in proximity to the QD donor, again resulting in efficient FRET between the two. Upon binding maltose, MBP undergoes the obligatory conformational change that alters the environment around the dye, changing the assembly photoluminescence (PL) signature in a concentration-dependent manner. Signal transduction could be monitored from assembly PL directly or from changes in fluorescent lifetimes. In this configuration the QD acts both as a nanoscaffold for attachment of multiple copies of the MBP-dye and as an energy donor that harvests shorter wavelength excitation to drive the sensor via FRET [40]. Several important lessons were learnt from the maltosesensing demonstrations. Although the QD is much larger than MBP, by using the QD as a nanoplatform to array multiple FRET acceptor-carrying proteins, a higher FRET efficiency is achieved as opposed to the single donor–single acceptor scenario through the proportional increase in acceptor cross-section [35,38]. Moreover, potential donor–acceptor distance constraints imposed by the size of QDs in conjunction with the less than www.sciencedirect.com
Maltose-binding protein Medintz and Deschamps 23
Figure 4
Quantum dot–MBP sensor. (a) Schematic of 560QD–MBP nanosensor function. Each 560 nm-emitting QD is surrounded by an average of 10 MBP moieties; a single MBP is shown for simplicity. Specific binding of QD–MBP–b-CD–QSY-9 (maximum absorption 565 nm) results in effective quenching of QD emission. Addition of maltose displaces b-CD–QSY9 from the sensor assembly, resulting in a concentration-dependent increase in direct QD emission. (b) Results from titrating a 560 QD–10MBP–QD conjugate (quantum yield [QY] 39%) preassembled with 1 mM b-CD–QSY-9 with increasing concentrations of maltose. The graph shows that the added maltose competes with and displaces the b-CD–QSY-9 in a concentration-dependent manner allowing effective sensing of maltose concentration. (c) Transformation of titration data. The right axis shows photoluminescence (PL in arbitrary units, AU) at 560 nm, and fractional saturation is shown on the left axis. The point that corresponds to 50% saturation was used to derive the apparent maltose dissociation constant (Kapp) value. The useful sensing range corresponds to 500 nm to 100 mM maltose (reprinted by permission of the Nature Publishing Group [38]). (d) Structural model of MBP41C–Cy3 as it self-assembles on the QD surface. The MBP (white) orientation is based upon the model described in [39]. The Cy3 maleimide (red) is attached to a cysteine at residue 41. Cross-section projection along the QD–protein polar axis is shown. The point closest to the QD surface is indicated in green. This is the location of attachment for the HIS5 sequence that interacts with the QD surface. The distance from the QD center to the Cy3 center is 74 A˚. The sphere represents a 530 nm QD with a diameter of 6 nm. (e) Top view (looking into the QD center), showing the open MBP conformation with the closed ligand-bound MBP (yellow) superimposed onto it. Protein interaction upon closing with the Cy3 dye located across the binding pocket is also shown. Figure is adapted from [40].
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optimal acceptor absorbance could be overcome by use of a two-step FRET-sensing strategy that utilizes a midway or relay-station fluorophore [38]. The same QD-sensing strategy was applied to detection of TNT by substituting an anti-TNT antibody fragment for the MBP and using an appropriate dye-labeled TNT analog [41]. This, again, highlights the ability to prototype with MBP and then to apply the same strategy to another target.
Electrochemical detection with appropriately modified periplasmic-binding proteins Hellinga and co-workers [42] have demonstrated a viable strategy to exploit the conformational changes of PBPs to allow electrochemical detection of analyte. MBPs that express the carboxy-terminal HISn sequence and unique cysteine residues were again utilized for prototyping. The HISn allowed MBP labeled with a ruthenium redox reporter on the cysteine to self-assemble onto the surface of nitrilotriacetic acid (NTA) modified gold electrodes, see Figure 5. As MBP binds maltose, the conformational coupling changes the relative distance of the ruthenium reporters to the gold electrode in a systematic manner, which allows electrical monitoring and reporting of current changes, see Figure 5. Mutations in the MBP binding pocket created variants that had altered binding isotherms that covered a broad range of concentrations. Hellinga and co-workers [42] further demonstrated that this strategy could be extended to target zinc, glucose and glutamate by the utilization of appropriately modified PBPs. Benson and co-workers [43] recently reported a modification of this strategy that is coupled to QDs. A ruthenium-labeled MBP that expresses a poly-cysteine metallothionein domain was attached to core-only CdSe QDs. The conformational changes caused by maltose binding result in an increase in relative QD emission. Benson argues that this is caused by changes in charge transfer from the Ru–MBP complex to the QDs, as QD emission can be altered by charge state [44]. However,
similar ruthenium complexes are also used as relatively long-lifetime dyes [45] and therefore it is not clear if charge-transfer alone is responsible or if FRET and fluorescence inactivation processes are also contributing.
Periplasmic-binding protein–chimeric switching proteins In an exciting development, Ostermeier and co-workers [46,47,48] demonstrated that an iterative approach of circular permutation of fused genetic sequences could create molecular switches in which the recognition and binding of ligand by one protein can allosterically ‘switch on’ or modulate the activity of another conjoined protein in an expressed chimera. The considerable understanding of MBP structure–function made it the choice for prototyping in this case [46,47,48]. The gene for MBP was recombined with the gene for TEM1 b-lacatamase (BLA) to create a family of MBP–BLA hybrids. Binding of maltose to MBP in the chimeric proteins could act as both a positive and a negative regulator of b-lactam hydrolysis, depending upon which construct was used [46]. Furthermore, MBP–BLA switches functioned effectively in an ‘on–off’ manner, with maltose inducing BLA hydrolytic activity by as much as 600-fold in some switches [46], see Figure 6. Interestingly, BLA activity could also modulate MBP function. More importantly, when transformed into E. coli, a library of 4 106 variants could be rapidly screened to identify new switches that responded to sucrose, an epimer of maltose not normally recognized by MBP, which demonstrates the power of this technique to select for switches that have new properties [46]. Mechanistically, it appears that the switches are more apt to be functional through fusion of the two participating proteins at their termini. Ostermeier indicates that almost 50% of single-domain proteins might be amenable to the same fusion and permutation process to create new
Figure 5
Schematic function of electrochemical-sensing with MBP. A gold (Au) electrode is functionalized with both hydroxyl-terminated and nickel charged NTA-terminated thioalkanes. The carboxy-terminal HISn sequence self-assembles MBP onto the NTA by way of metal-affinity coordination. MBP is functionalized at a unique cysteine that has a Ru(II) redox reporter group, which positions the reporter between the protein and the monolayers. Upon binding the ligand — maltose — the binding-induced protein conformational changes alter the interaction between the reporter and the electrode, which is monitored electrically. Sensors for glucose, zinc and glutamine using GBP, ZBP and QBP, respectively, were also demonstrated [42]. Current Opinion in Biotechnology 2006, 17:17–27
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Maltose-binding protein Medintz and Deschamps 25
Figure 6
Ribbon and string structures of the MBP–BLA RG13 fusion. Maltose-bound MBP and BLA bound to an active-site inhibitor are oriented such that the fusion sites are proximal. MBP residues 1–316 are shown in dark blue and 319–370 in green (317 and 318 deleted); BLA residues are 227–286 shown in yellow and 24–226 in red. A GSGGG linker is shown in white. Figure kindly provided by M Ostermeier and published with permission from Elsevier Ltd [47].
switches as these proteins have their amino and carboxy termini in proximity. If this selection process can be shown to create other designer switches, it will create a technology that has a variety of applications, both in vitro and in vivo. Of particular interest is the creation of programmable cells for biotechnological applications, in which exposure to one effector will allow control over an enzymatic or binding event. The wide number of PBPs and their analytes might further allow the generation of a library of switch effectors to choose from.
Computational redesign of periplasmicbinding protein specificity The Hellinga laboratory has taken the lead in engineering PBPs to recognize and to bind a variety of other substrates. This approach is based on the computationally intensive iterative redesign of the ligand-binding sites of several PBPs to recognize disparate and/or non-natural ligands [1,6,49,50]. This approach is highlighted by the re-engineering of MBP to become a zinc biosensor [50], converting GBP and RBP to recognize pinacolyl methyl phosphonic acid — the predominant hydrolytic product of the nerve agent soman [49] — and re-engineering RBP to bind TNT [6]. Hellinga and co-workers [7] have also shown that RBP could be reconfigured into an enzymatically active triose phosphate isomerase that could support bacterial growth. The lessons learnt here were later applied to the convertion of a non-PBP cell adhesion protein to recognize calcium [51]. Such demonstrations, in conjunction with the switch work of Ostermeier and cowww.sciencedirect.com
workers, also highlight the emerging paradigms in biological research — namely the intersection of computational biology and recombinant engineering. They also, however, highlight the specialized disciplines needed, unique combinations of which are not currently found in many ‘classic’ research environments.
Conclusions Looking at all these demonstrations cumulatively, we see common themes. The PBPs, in particular MBP, are an elegant platform for prototyping a variety of diverse reagentless sensing modalities. Moreover, the sensing strategies do not necessarily have to rely on conformational coupling. The HISn and unique cysteines within the structure can be used orthogonally for attachment and reporter labeling. Once prototyped, the lessons learnt can be applied to using other non-PBP proteins for targeted biosensing. It is worth noting that each sensing modality has its own particular focus and thus might be more suitable for a specialized use. The fluorescent fusion protein approach of Frommer and co-workers [4,23–27] is particularly suited to in vivo monitoring, whereas electrochemical sensing might lead to microfabricated addressable arrays of biosensors. There is a huge array of ligand targeting PBPs to choose from, but the redesign strategy of Hellinga and co-workers [1,6,49,50] offers to expand this even more. If a signal transduction mechanism or biosensing strategy needs testing, MBP probably offers one of the best-characterized protein platforms to work with. Current Opinion in Biotechnology 2006, 17:17–27
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Acknowledgements IM and JRD acknowledge the Office of Naval Research and the US Naval Research Laboratory.
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Looger LL, Dwyer MA, Smith JJ, Hellinga HW: Computational design of receptor and sensor proteins with novel functions. Nature 2003, 423:185-190.
7. Dwyer MA, Looger LL, Hellinga HW: Computational design of a biologically active enzyme. Science 2004, 304:1967-1971. The authors utilize computational design to alter a PBP into an enzyme that is able to support bacterial growth. This offers a glimpse into how designer proteins might be created in the future. Such proteins might combine elements of and benefits from many different proteins into the final product. 8.
Sharff AJ, Rodseth LE, Quiocho FA: Refined 1.8-Angstrom structure reveals the mode of binding of b-cyclodextrin to the maltodextrin binding-protein. Biochemistry 1993, 32:10553-10559.
9.
Sharff AJ, Rodseth LE, Spurlino JC, Quiocho FA: Crystallographic evidence of a large ligand-induced hinge-twist motion between the 2 domains of the maltodextrin binding-protein involved in active-transport and chemotaxis. Biochemistry 1992, 31:10657-10663.
25. Lager I, Fehr M, Frommer WB, Lalonde SW: Development of a fluorescent nanosensor for ribose. FEBS Lett 2003, 553:85-89. 26. Fehr M, Lalonde S, Ehrhardt DW, Frommer WB: Live imaging of glucose homeostasis in nuclei of COS-7 cells. J Fluoresc 2004, 14:603-609. An important contribution that demonstrates how to monitor glucose levels both in the cytosol and the nucleus using a fluorescent protein– bPBP construct. 27. Okumoto S, Looger LL, Micheva KD, Reimer RJ, Smith SJ, Frommer WB: Detection of glutamate release from neurons by genetically encoded surface-displayed FRET nanosensors. Proc Natl Acad Sci USA 2005, 102:8740-8745. This study continues the work from [23] and demonstrates a new use for genetically encoded FRET-based PBP-sensors. 28. Nguyen AW, Daugherty PS: Evolutionary optimization of fluorescent proteins for intracellular FRET. Nat Biotechnol 2005, 23:355-360. 29. Miyawaki A: Visualization of the spatial and temporal dynamics of intracellular signaling. Dev Cell 2003, 4:295-305.
10. Spurlino JC, Lu GY, Quiocho FA: The 2.3-a resolution structure of the maltose-binding or maltodextrin-binding protein, a primary receptor of bacterial active-transport and chemotaxis. J Biol Chem 1991, 266:5202-5219.
30. Ross SA, Srinivas PR, Clifford AJ, Lee SC, Philbert MA, Hettich RL: New technologies for nutrition research. J Nutr 2004, 134:681-685.
11. Riggs P: Expression and purification of recombinant proteins by fusion to maltose-binding protein. Mol Biotechnol 2000, 15:51-63.
31. Medintz IL, Mauro JM: Use of a cyanine dye as a reporter probe in reagentless maltose sensors based on E. coli maltose binding protein. Anal Lett 2004, 37:191-202.
12. Marvin JS, Hellinga HW: Manipulation of ligand binding affinity by exploitation of conformational coupling. Nat Struct Biol 2001, 8:795-798.
32. Medintz IL, Goldman ER, Lassman ME, Mauro JM: A fluorescence resonance energy transfer sensor based on maltose binding protein. Bioconjug Chem 2003, 14:909-918.
13. Hsieh HV, Pfeiffer ZA, Amiss TJ, Sherman DB, Pitner JB: Direct detection of glucose by surface plasmon resonance with bacterial glucose/galactose-binding protein. Biosens Bioelectron 2004, 19:653-660. 14. Kubo I, Satoh D: Preparation of phosphate-binding-proteinmodified electrode and its application to reagentless phosphate sensor. Sensors and Materials 2004, 16:391-399.
33. Medintz IL, Anderson GP, Lassman ME, Goldman ER, Bettencourt LA, Mauro JM: General strategy for biosensor design and construction employing multifunctional surface-tethered components. Anal Chem 2004, 76:5620-5629. This study highlights a strategy for the creation of self-assembled surface-tethered reagentless biosensors that allow repetitive sensing events. It demonstrates an alternate PBP-based sensing format that does not involve conformational coupling.
15. Takada K, Naal Z, Park JH, Shapleigh JP, Bernhard S, Batt CA, Abruna HD: Study of specific binding of maltose binding protein to pyrrole-derived bipyridinium film by quartz crystal microbalance. Langmuir 2002, 18:4892-4897.
34. Medintz IL, Goldman ER, Lassman ME, Hayhurst A, Kusterbeck AW, Deschamps JR: Self-assembled TNT biosensor based on modular multifunctional surface-tethered components. Anal Chem 2005, 77:365-372.
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Maltose-binding protein Medintz and Deschamps 27
35. Medintz I, Uyeda H, Goldman E, Mattoussi H: Quantum dot bioconjugates for imaging, labeling and sensing. Nat Mater 2005, 4:435-446. 36. Clapp AR, Medintz IL, Mauro JM, Fisher BR, Bawendi MG, Mattoussi H: Fluorescence resonance energy transfer between quantum dot donors and dye-labeled protein acceptors. J Am Chem Soc 2004, 126:301-310. 37. Michalet X, Pinaud FF, Bentolila LA, Tsay JM, Doose S, Li JJ, Sundaresan G, Wu AM, Gambhir SS, Weiss S: Quantum dots for live cells, in vivo imaging, and diagnostics. Science 2005, 307:538-544. 38. Medintz IL, Clapp AR, Mattoussi H, Goldman ER, Fisher B, Mauro JM: Self-assembled nanoscale biosensors based on quantum dot FRET donors. Nat Mater 2003, 2:630-638. 39. Medintz IL, Konnert JH, Clapp AR, Stanish I, Twigg ME, Mattoussi H, Mauro JM, Deschamps JR: A fluorescence resonance energy transfer derived structure of a quantum dot-protein bioconjugate nanoassembly. Proc Natl Acad Sci USA 2004, 101:9612-9617. Describes a FRET-based method for elucidation of the structure of protein–nanoparticle bioconjugates by utilizing MBP and QDs. 40. Medintz IL, Clapp AR, Melinger JS, Deschamps JR, Mattoussi H: A reagentless biosensing assembly based on quantum dot donor fo¨rster resonance energy transfer. Adv Mater 2005, in press. 41. Goldman E, Medintz I, Whitley J, Hayhurst A, Clapp A, Uyeda H, Deschamps J, Lassman M, Mattoussi H: A hybrid quantum dot-antibody fragment fluorescence resonance energy transfer-based TNT sensor. J Am Chem Soc 2005, 127:6744-6751. 42. Benson DE, Conrad DW, de Lorimer RM, Trammell SA, Hellinga HW: Design of bioelectronic interfaces by exploiting
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hinge-bending motions in proteins. Science 2001, 293:1641-1644. 43. Sandros MG, Gao D, Benson DE: A modular nanoparticle-based system for reagentless small molecule biosensing. Journ Am Chem Soc 2005, 127:12198-12199. 44. Wang C, Shim M, Guyot-Sionnest P: Electrochromic nanocrystal quantum dots. Science 2001, 291:2390-2392. 45. Clapp AR, Medintz IL, Fisher BR, Anderson GP, Mattoussi H: Can luminescent quantum dots be efficient energy acceptors with organic dye donors. J Am Chem Soc 2005, 127:1242-1250. 46. Guntas G, Mansell TJ, Kim JK, Ostermeier M: Directed evolution of protein switches and their applications to the creation of ligand-binding proteins. Proc Natl Acad Sci USA 2005, 102:11224-11229. A fascinating demonstration of how to select for novel bi-functional chimeric proteins, whereby the binding of ligand by one protein, MBP, ‘switches’ on the activity of the other — b-lactamase. 47. Guntas G, Mitchell SF, Ostermeier M: A molecular switch created by in vitro recombination of nonhomologous genes. Chem Biol 2004, 11:1483-1487. 48. Guntas G, Ostermeier M: Creation of an allosteric enzyme by domain insertion. J Mol Biol 2004, 336:263-273. 49. Allert M, Rizk SS, Looger LL, Hellinga HW: Computational design of receptors for an organophosphate surrogate of the nerve agent soman. Proc Natl Acad Sci USA 2004, 101:7907-7912. 50. Marvin JS, Hellinga HW: Conversion of a maltose receptor into a zinc biosensor by computational design. Proc Natl Acad Sci USA 2001, 98:4955-4960. 51. Yang W, Wilkins AL, Ye YM, Liu ZR, Li SY, Urbauer JL, Hellinga HW, Kearney A, van der Merwe PA, Yang JJ: Design of a calcium-binding protein with desired structure in a cell adhesion molecule. J Am Chem Soc 2005, 127:2085-2093.
Current Opinion in Biotechnology 2006, 17:17–27
Current Opinion in Biotechnology 2006, 17:17–27 This review comes from a themed issue on Analytical biotechnology Edited by Jan Roelof van der Meer and J Colin Murrell Available online 18th January 2006 0958-1669/$ – see front matter Published by Elsevier Ltd. DOI 10.1016/j.copbio.2006.01.002
Introduction The bacterial periplasmic-binding proteins (PBPs), most commonly cloned out of E. coli, are part of a large diversified protein superfamily that is found even in eukaryotes [1,2,3]. Although unrelated at the primary sequence level, almost all PBPs share a common structural motif that consists of two domains, joined by a ‘hinge’ region, which surround a central ligand-binding site [1,2,3], see Figure 1. A common functional conformational change is also found in this protein family, which involves switching from the ‘open’ ligand-free form to a ‘closed’ or ligand-bound form. This transition is accomplished through a bending and swiveling twist www.sciencedirect.com
motion about the hinge region, which is analogous to a ‘venus-fly-trap’ closing, see Figure 1b [4]. The interest in exploiting PBPs for biosensing arises both from their specific recognition of myriad analytes approaching nanomolar concentrations in some cases and from the ligand-induced conformational changes that they undergo [4]. It is believed that the common functional mechanism and wide specificity of PBPs originated during evolution by positive selective pressure on their hosts as they mediated solute recognition and uptake in the periplasm and directed chemotaxis [1]. The analytes targeted by bacterial (b)PBPs include: various sugars such as glucose, maltose, ribose and arabinose; amino acids such as glutamine, glutamate/aspartate and histidine; di- and tripeptides; and metals and ions such as Fe(III), Ni(II), phosphate and sulphate, to name but a few [2,5]. Furthermore, Hellinga and co-workers have shown that PBPs can be computationally remodeled and mutated to recognize non-natural analytes or to function as enzymes (see below) [6,7]. Owing to the extensive understanding of its structure and function, maltose-binding protein (MBP) is the prototypical member of this superfamily. MBP specificity is well known, the crystal structure in both the open and ligand-bound form has been solved [8–10], and a variety of mutants that have different maltose sensitivities (Kds that range from nM to mM) have been created [2]. Moreover, MBP has been extensively utilized for recombinant protein purification over amylose resin [11]. This thorough understanding of structure–function in MBP initially, and now in other PBPs, has spurred research into exploitation of the ligand-induced conformational rearrangements [12] and the application of diverse analytical methods to create viable biosensors. A variety of analytical or signal transduction mechanisms have been used to monitor PBP binding and conformational changes, including direct and environmentally sensitive fluorescence, electrochemical and fluorescence resonance energy transfer (FRET), see Figure 1a. Although not discussed here, there have been other sensing modalities prototyped on PBPs including surface plasmon resonance, quartz crystal microbalance analysis and potentiometry [13–15]. Cumulatively, the results indicate that PBPs, and especially MBP, are an excellent platform not only for biosensing but also for prototyping a variety of diverse sensing modalities for eventual use in various environmental, clinical and security applications [16–18]. This review highlights many of the direct detection or reagentless sensing modalities that have been utilized Current Opinion in Biotechnology 2006, 17:17–27
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Figure 1
PBP structure and function. (a) A prototypic PBP structure is depicted; this has two lobes (green) that create the ligand-binding pocket and are joined by the hinge-binding region (pink). Upon binding the ligand, the PBP undergoes transition to the closed structure. PBPs present two intrinsic properties available for biosensor exploitation, either separately or when functioning in concert: recognition specificity and conformational change. Recognition specificity has been utilized for fluorescence- and FRET-based sensing. Conformational changes have been utilized for FRET-, electrochemical-, fluorescent- and enzymatic-based sensing. (b) Ribbon and string rendering of MBP structure in the open form (right) [8,9] and ligandbound closed form (left) [10]. MBP dimensions are 30 40 65 A˚ [9,23]. The two domains (lobes) are highlighted in green and grey. Upon binding maltose (purple), the lobes rotate 358and twist laterally 88 relative to each other [9,23]. Overall the amino- and carboxy-termini move 7 A˚ closer to each other after binding [4]. Note the change in conformation of the overall structure upon binding maltose.
with MBP for both in vitro and in vivo detection of target compounds. The benefits, potential applications and limitations of each sensing method are discussed along with recent developments in this field.
Direct fluorescent monitoring of dye-labeled periplasmic-binding proteins The early work in adaptation of PBPs for biosensing focused on direct fluorescent monitoring of dye-labeled proteins. In a pioneering study, Cass and co-workers [19] demonstrated that MBP could be mutated to express a cysteine residue near the binding site, which could act as a site for subsequent dye conjugation. Upon maltose addition, the fluorescence intensity of the dyes attached at this site increased from 60% to 180% as MBP underwent ligand-induced conformational changes. Similar sensing strategies are still in use today. Kobatake and Current Opinion in Biotechnology 2006, 17:17–27
co-workers [20] coupled glutamine-binding protein (QBP) to a designer hydrophobic polypeptide to allow adherence onto unmodified hydrophobic surfaces. The conjugation of dyes to a unique cysteine site on QBP also demonstrated that fluorescence intensity increases upon ligand binding. This study indicates that it might be feasible to prepare arrays of PBP sensors on unmodified surfaces. However, the work in the laboratories of both Cass [19] and Kobatake [20] relies on environmentally sensitive fluorophores that respond appropriately to the binding-induced environmental changes in the surrounding protein. These dyes can be relatively insoluble and unstable when in aqueous environments for long periods of time. The monograph of De Lorimier, Hellinga and coworkers [2] is perhaps the best resource on designing PBP-based sensors for fluorescent monitoring. This reference includes strategies on designing, engineering and www.sciencedirect.com
Maltose-binding protein Medintz and Deschamps 19
characterizing PBP-based biosensors. It can be considered a ‘how-to’ guide for anyone interested in this field. Although both genetic and protein structural data can be used to optimize sensor design, specific dye–protein interactions are still not fully understood. Results have shown that not all choices of mutation sites and fluorophores function for sensing, and multiple sites typically have to be tested [2].
Fluorescent protein–periplasmic-binding protein fusions Using the same concept as the Ca2+-sensing ‘cameleons’ of Tsien and co-workers [21,22], in which fluorescent proteins flank a central recognition sequence and their relative positions change upon Ca2+ binding, the Frommer laboratory [4,23,24] has constructed an elegant series of reversible FRET-based sensors that consist of fluorescent proteins fused to the amino- and carboxy-termini of various PBPs, see Figure 2a. The intrinsic sensitivity of FRET to molecular scale rearrangements lends itself to monitoring the relative nanoscale re-arrangements of the fluorescent proteins in these sensors during ligand binding. Frommer’s maltose-sensing prototype, termed ‘FLIPmal’, consisted of MBP that had an enhanced cyan fluorescent protein (CFP) donor (D) fused to the amino terminus and an enhanced yellow fluorescent protein (YFP) acceptor (A) fused to the carboxy terminus, see Figure 2a. Upon binding maltose, the sensor undergoes the obligatory conformational rearrangement, bringing the two fluorescent proteins into proximity and altering the efficiency of FRET between them in a concentrationdependent manner. A series of mutants with Kds of 2, 25 and 225 mM were created to increase the dynamic range of sensing from <1 mM to >1 mM. The utility of this nanosensor was demonstrated by measurement of the maltose concentration in a complex beer sample and the maltose uptake in live yeast cells that endogenously express the sensor [23], see Figure 2b. Additional sensors targeted glucose and ribose by use of glucose-binding protein (GBP) and ribose-binding proteins (RBPs) as backbones [24,25]. However, in these nanosensors the fluorescent proteins move apart in response to binding, see Figure 2a, which results in a decrease in FRET donor/acceptor fluorescent ratio. This is owing to placement of the fluorescent proteins near the hinge region of the proteins as opposed to at the opposite end of each lobe as in the FLIPmal series [4,24]. A FLIPglucose sensor that expresses three copies of the simian virus 40 large T nuclear localization signal allowed almost exclusive targeting to the nucleus in COS-7 cells; this demonstrates cell compartmental monitoring specificity, see Figure 2c [26]. Recently, Frommer and co-workers [27] have adapted the glutamate/aspartate-binding protein to enable it to measure glutamate release from neurons; the same design was used in conjunction with the platelet-derived growth factor receptor transmemwww.sciencedirect.com
brane domain to create a sensor that is surface-displayed in hippocampal cells and directly monitors glutamate metabolism. The ability to express these nanosensors in plants, yeast and eukaryotes in vivo is probably the single biggest advantage of this sensing strategy. Perhaps, the only drawback is the small dynamic range in FRET donor/acceptor ratio, which might necessitate sensitive equipment and spectral deconvolution. An evolved CFP–YFP FRET pair that exhibits a 20-fold ratiometric change in FRET signal has been reported, which might represent a possible solution [28]. Given the potential pool of analytes that can be targeted by the PBP superfamily, these biosensors will see much use owing to the increased research on how cells monitor and respond to signaling and nutrients in vivo [29,30]. Perhaps a ‘cassette’ version will become available that will allow one to pick from a variety of sensors for targeted analysis within a cell line of choice.
Periplasmic-binding protein surface-tethered sensing assemblies We have focused on creation of reagentless sensing assemblies by the exploitation of several PBP properties including recognition specificity and sensitivity, ease of recombinant engineering, the ability to site-specifically implant fluorophores within the protein structure, and their overall size [31,32,33]. Figure 3a highlights the key features incorporated into this design and tested using MBP as a prototype [33]. The sensing assembly consists of dye-labeled MBP that interacts with a co-functional modular DNA arm, both of which are surface-tethered. Sensing in a microtiter-well format allows access to fluorescent plate readers — a technology common to many biological laboratories. Surface tethering and orienting of all components relies on biotin-avidin chemistry, which allows the entire sensor to be self-assembled yet robust. MBP is attached to a NeutrAvidin microtiter well surface by an engineered carboxy-terminal 5-histidine, HIS5, sequence (originally incorporated for metal-affinity purification) using a bifunctional biotin–Ni2+–nitriloacetic acid moiety, engendering a quasi-fixed ‘face-up’ surface orientation to the MBP. Prior to immobilization, MBP is site-specifically labeled with either a fluorophore or a dark quenching dye. The co-functional modular arm consists of a flexible biotinylated DNA oligonucleotide attached to a central dye and terminates in b-cyclodextrin (b-CD), a known MBP ligand, see Figure 3b [8]. During surface self-assembly of both components, MBP binds the proximal b-CD ligand bringing the two dyes into proximity and establishing efficient FRET. Addition of maltose displaces the b-CD-linked dye altering FRET in a concentration-dependent manner and provides fluorescent signaling, see Figure 3c–e. The assembly is then washed free of analyte and is regenerated for subsequent sensing events. Up to eight consecutive sensing/regenerations have been demonstrated for the same assembly. Two methods were tested to adjust the sensitivity. The Current Opinion in Biotechnology 2006, 17:17–27
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Figure 2
Fluorescent protein–PBP fusion sensors. (a) Schematic design of PBP–fluorescent protein FRET sensors. CFP, depicted in blue, and YFP, in yellow, are fused to the amino and carboxy termini of the proteins. The differing results achieved using GBP, MBP and RBP are shown. Owing to the differing positions of the termini in the proteins, the binding of maltose by MBP moves the fluorescent proteins together, whereas binding of sugar by RBP and GBP move the fluorescent proteins apart. Their relative movement upon ligand binding is monitored by FRET between the two fluorescent proteins. Ostermeier uses a nomenclature (FLIPmal) to describe his constructs whereby ‘FLIP’ designates a construct of two fluorescent proteins and ‘mal’ indicates the central protein and target analyte. (b) Visualization of dynamic maltose concentration changes in the cytosol of yeast that expresses the FLIPmal sensor with a Kd of 25 mm. The graph indicates the change in uptake emission intensity ratio for a single yeast cell. Figure copyright National Academy of Sciences USA [23]. The colors that range from blue to red are a scale reflecting the increasing internal maltose concentration. The plotted squares show the data as the difference in FLIPmal ratio. These results demonstrate that this sensor effectively allows one to monitor the increasing concentration of internal maltose. (c) Specific targeting of sensors to subcellular compartments. In COS7 cells the protein is distributed in the cytosol, however, sensors that carry the triple simian virus 40 large T nuclear localization signal are targeted almost exclusively to the nucleus. Panels (a) and (c) adapted from [4] with permission from Elsevier Ltd.
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Maltose-binding protein Medintz and Deschamps 21
Figure 3
Surface-tethered sensing assemblies. (a) Schematic description of a tethered MBP biosensor. The modular arm (b-CD–Dye–DNA) is attached indirectly to the NeutrAvidin (NA) surface by way of DNA hybridization to a biotinylated (B) complimentary DNA that is directly tethered to the NA surface (red). Dye-labeled MBP (Dye 1) is attached to the same surface by a Bio-X (NTA) moiety that binds to the HIS5 sequence of MBP. Controlled self-assembly allows attachment of both MBP and modular arm to the NA. MBP binding of b-CD–dye–DNA arm assembles the final sensor by bringing Dye 1 and 2 into proximity, which allows FRET quenching when excited at the appropriate wavelengths. Maltose (the target analyte) displaces the b-CD, which disrupts FRET in a concentration-dependent manner. The sensor assembly is then washed and regenerated in buffer for a subsequent detection event. Use of MBP-binding mutants or rigidifying the DNA linker with a complementary DNA modulator alters useful sensing ranges. (b) Structure of the modular arm that functions with MBP to form the sensing assembly. (c) The sensor schematically depicted in (a) was self-assembled using an MBP95C mutant labeled with a QSY7 dark quencher. The resulting titration versus maltose is shown. The apparent binding constant (Kapp) is shown along with the estimate of useful sensing range — defined as 10–90% sensor saturation [23]. (d) Variant of the sensor is presented using MBP80C QSY7 substituted for the MBP95C protein. (e) Variant of the MBP95C sensor in (a) with the addition of a complimentary modulator DNA that rigidifies the DNA linker arm. Figures are reproduced with permission from the American Chemical Society [33].
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first utilizes MBP mutants that have differing binding constants (Kapp), by substituting the MBP95C mutant (Kapp 5 mM) with a MBP80C mutant (Kapp 575 mM), see Figure 3c and d. This also adjusted the useful sensing range (defined as 10–90% sensor saturation) from low mM to low mM. The second method uses complimentary ‘modulator’ DNA to rigidify the DNA arm to adjust sensitivity, specifically by increasing the useful sensing range, see Figure 3e. Besides being regenerable and having adjustable sensitivity, several other benefits of this design have been noted. Sensors could be pre-assembled on microtiter well plates, tested, washed, dried, stored in a refrigerator and then reconstituted for reuse several weeks later. This has implications for pre-packing such sensors for later commercial use or for deployment in the field. The modular nature of this sensing assembly suggested that it could target other analytes; this was proven by the development of a sensor assembly that targeted the explosive 2,4,6-trinitrotoluene (TNT). This was accomplished by the substitution of an antibody fragment specific for TNT in place of MBP and the synthesis of an appropriate co-functional arm that terminates in a TNT analog [34]. This strategy might, however, be limited to smaller target analytes owing to FRET size considerations as well as finding appropriate pairs of protein recognition elements and synthesizing cofunctional dye-labeled analogs amenable to use in such a scheme. Overall, the concept demonstrates a way to incorporate into a reagentless sensor ensemble a PBP that can be attached to a surface and that does not rely on conformational coupling for signal transduction. This also shows that MBP can function in concert with complex chemical entities to achieve sensing.
Quantum dot–MBP sensor Luminescent quantum dots (QDs) are colloidal semiconductor nanocrystal-based fluorophores that have interesting photophysical properties including broad absorption spectra, molar extinction coefficients 10–100 times those of organic dyes, high quantum yield (QY), high resistance to chemical degradation and photobleaching, and, most impressively, have size-tunable photoluminescent emission [35–37]. Their large effective Stokes shift (in this context the spectral difference between wavelength of excitation and emission) means that mixed QD populations can be effectively excited at wavelengths far removed from their respective emissions. In particular, for FRET, QD photoluminescent spectra can be size-tuned or ‘dialed in’ to improve overlap with a particular acceptor [36]. These unique properties are stimulating a great deal of interest in the adaptation of these nanocrystalline fluorophores for biosensing [35,37]. Using the same criteria that made MBP an attractive prototype for the surface-tethered sensing assembly above, we designed and tested a prototype hybrid nanoCurrent Opinion in Biotechnology 2006, 17:17–27
crystal–protein or QD–MBP based FRET sensor. The sensor consisted of MBP self-assembled onto the surface of 560 nm-emitting CdSe–ZnS core-shell QDs, see Figure 4a [38]. The MBP’s carboxy-terminal HIS5 tract allows the protein to self-assemble and coordinate to Zn on the QD shell by way of metal-affinity coordination. Prior to being immobilized on the QD surface, the MBP is allowed to pre-bind a b-CD maltose analog that is covalently attached to a QSY-9 dark quencher or non-emissive acceptor dye. Subsequent attachment of the b-CD quencher that carries MBP to the QD surface brings the quencher into proximity to the QD exciton-emitting core and establishes efficient FRET quenching. Addition of maltose displaces the b-CD-quencher analog, disrupting FRET, and recovers the QD emission in a concentration-dependent manner, see Figure 4b and c. MBP specificity and sensitivity were maintained in this format, with low micromolar recognition of only those sugars that contain a1–4 glucosidic linkages. The orientation of the final QD–MBP conjugate was also modeled based upon FRET between the QD donor and fluorophores attached to unique sites within the MPB structure, using a strategy analogous to a nanoscale global positioning system [39]. In this case, MBP allowed elucidation of a QD–protein bioconjugate structure. Following on from this work, we have recently prototyped a reagentless version of the QD–MBP biosensor that is a functional hybrid between the above QD–FRET displacement sensing format and the previously described conformational-dependent approach, see Figure 4d and e. MBP labeled with a Cy3 at a peristeric site (close to the binding pocket but does not interfere with substrate binding) was self-assembled onto the surface of QDs using HIS5–Zn coordination [40]. This brings the Cy3 acceptor dye in proximity to the QD donor, again resulting in efficient FRET between the two. Upon binding maltose, MBP undergoes the obligatory conformational change that alters the environment around the dye, changing the assembly photoluminescence (PL) signature in a concentration-dependent manner. Signal transduction could be monitored from assembly PL directly or from changes in fluorescent lifetimes. In this configuration the QD acts both as a nanoscaffold for attachment of multiple copies of the MBP-dye and as an energy donor that harvests shorter wavelength excitation to drive the sensor via FRET [40]. Several important lessons were learnt from the maltosesensing demonstrations. Although the QD is much larger than MBP, by using the QD as a nanoplatform to array multiple FRET acceptor-carrying proteins, a higher FRET efficiency is achieved as opposed to the single donor–single acceptor scenario through the proportional increase in acceptor cross-section [35,38]. Moreover, potential donor–acceptor distance constraints imposed by the size of QDs in conjunction with the less than www.sciencedirect.com
Maltose-binding protein Medintz and Deschamps 23
Figure 4
Quantum dot–MBP sensor. (a) Schematic of 560QD–MBP nanosensor function. Each 560 nm-emitting QD is surrounded by an average of 10 MBP moieties; a single MBP is shown for simplicity. Specific binding of QD–MBP–b-CD–QSY-9 (maximum absorption 565 nm) results in effective quenching of QD emission. Addition of maltose displaces b-CD–QSY9 from the sensor assembly, resulting in a concentration-dependent increase in direct QD emission. (b) Results from titrating a 560 QD–10MBP–QD conjugate (quantum yield [QY] 39%) preassembled with 1 mM b-CD–QSY-9 with increasing concentrations of maltose. The graph shows that the added maltose competes with and displaces the b-CD–QSY-9 in a concentration-dependent manner allowing effective sensing of maltose concentration. (c) Transformation of titration data. The right axis shows photoluminescence (PL in arbitrary units, AU) at 560 nm, and fractional saturation is shown on the left axis. The point that corresponds to 50% saturation was used to derive the apparent maltose dissociation constant (Kapp) value. The useful sensing range corresponds to 500 nm to 100 mM maltose (reprinted by permission of the Nature Publishing Group [38]). (d) Structural model of MBP41C–Cy3 as it self-assembles on the QD surface. The MBP (white) orientation is based upon the model described in [39]. The Cy3 maleimide (red) is attached to a cysteine at residue 41. Cross-section projection along the QD–protein polar axis is shown. The point closest to the QD surface is indicated in green. This is the location of attachment for the HIS5 sequence that interacts with the QD surface. The distance from the QD center to the Cy3 center is 74 A˚. The sphere represents a 530 nm QD with a diameter of 6 nm. (e) Top view (looking into the QD center), showing the open MBP conformation with the closed ligand-bound MBP (yellow) superimposed onto it. Protein interaction upon closing with the Cy3 dye located across the binding pocket is also shown. Figure is adapted from [40].
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optimal acceptor absorbance could be overcome by use of a two-step FRET-sensing strategy that utilizes a midway or relay-station fluorophore [38]. The same QD-sensing strategy was applied to detection of TNT by substituting an anti-TNT antibody fragment for the MBP and using an appropriate dye-labeled TNT analog [41]. This, again, highlights the ability to prototype with MBP and then to apply the same strategy to another target.
Electrochemical detection with appropriately modified periplasmic-binding proteins Hellinga and co-workers [42] have demonstrated a viable strategy to exploit the conformational changes of PBPs to allow electrochemical detection of analyte. MBPs that express the carboxy-terminal HISn sequence and unique cysteine residues were again utilized for prototyping. The HISn allowed MBP labeled with a ruthenium redox reporter on the cysteine to self-assemble onto the surface of nitrilotriacetic acid (NTA) modified gold electrodes, see Figure 5. As MBP binds maltose, the conformational coupling changes the relative distance of the ruthenium reporters to the gold electrode in a systematic manner, which allows electrical monitoring and reporting of current changes, see Figure 5. Mutations in the MBP binding pocket created variants that had altered binding isotherms that covered a broad range of concentrations. Hellinga and co-workers [42] further demonstrated that this strategy could be extended to target zinc, glucose and glutamate by the utilization of appropriately modified PBPs. Benson and co-workers [43] recently reported a modification of this strategy that is coupled to QDs. A ruthenium-labeled MBP that expresses a poly-cysteine metallothionein domain was attached to core-only CdSe QDs. The conformational changes caused by maltose binding result in an increase in relative QD emission. Benson argues that this is caused by changes in charge transfer from the Ru–MBP complex to the QDs, as QD emission can be altered by charge state [44]. However,
similar ruthenium complexes are also used as relatively long-lifetime dyes [45] and therefore it is not clear if charge-transfer alone is responsible or if FRET and fluorescence inactivation processes are also contributing.
Periplasmic-binding protein–chimeric switching proteins In an exciting development, Ostermeier and co-workers [46,47,48] demonstrated that an iterative approach of circular permutation of fused genetic sequences could create molecular switches in which the recognition and binding of ligand by one protein can allosterically ‘switch on’ or modulate the activity of another conjoined protein in an expressed chimera. The considerable understanding of MBP structure–function made it the choice for prototyping in this case [46,47,48]. The gene for MBP was recombined with the gene for TEM1 b-lacatamase (BLA) to create a family of MBP–BLA hybrids. Binding of maltose to MBP in the chimeric proteins could act as both a positive and a negative regulator of b-lactam hydrolysis, depending upon which construct was used [46]. Furthermore, MBP–BLA switches functioned effectively in an ‘on–off’ manner, with maltose inducing BLA hydrolytic activity by as much as 600-fold in some switches [46], see Figure 6. Interestingly, BLA activity could also modulate MBP function. More importantly, when transformed into E. coli, a library of 4 106 variants could be rapidly screened to identify new switches that responded to sucrose, an epimer of maltose not normally recognized by MBP, which demonstrates the power of this technique to select for switches that have new properties [46]. Mechanistically, it appears that the switches are more apt to be functional through fusion of the two participating proteins at their termini. Ostermeier indicates that almost 50% of single-domain proteins might be amenable to the same fusion and permutation process to create new
Figure 5
Schematic function of electrochemical-sensing with MBP. A gold (Au) electrode is functionalized with both hydroxyl-terminated and nickel charged NTA-terminated thioalkanes. The carboxy-terminal HISn sequence self-assembles MBP onto the NTA by way of metal-affinity coordination. MBP is functionalized at a unique cysteine that has a Ru(II) redox reporter group, which positions the reporter between the protein and the monolayers. Upon binding the ligand — maltose — the binding-induced protein conformational changes alter the interaction between the reporter and the electrode, which is monitored electrically. Sensors for glucose, zinc and glutamine using GBP, ZBP and QBP, respectively, were also demonstrated [42]. Current Opinion in Biotechnology 2006, 17:17–27
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Maltose-binding protein Medintz and Deschamps 25
Figure 6
Ribbon and string structures of the MBP–BLA RG13 fusion. Maltose-bound MBP and BLA bound to an active-site inhibitor are oriented such that the fusion sites are proximal. MBP residues 1–316 are shown in dark blue and 319–370 in green (317 and 318 deleted); BLA residues are 227–286 shown in yellow and 24–226 in red. A GSGGG linker is shown in white. Figure kindly provided by M Ostermeier and published with permission from Elsevier Ltd [47].
switches as these proteins have their amino and carboxy termini in proximity. If this selection process can be shown to create other designer switches, it will create a technology that has a variety of applications, both in vitro and in vivo. Of particular interest is the creation of programmable cells for biotechnological applications, in which exposure to one effector will allow control over an enzymatic or binding event. The wide number of PBPs and their analytes might further allow the generation of a library of switch effectors to choose from.
Computational redesign of periplasmicbinding protein specificity The Hellinga laboratory has taken the lead in engineering PBPs to recognize and to bind a variety of other substrates. This approach is based on the computationally intensive iterative redesign of the ligand-binding sites of several PBPs to recognize disparate and/or non-natural ligands [1,6,49,50]. This approach is highlighted by the re-engineering of MBP to become a zinc biosensor [50], converting GBP and RBP to recognize pinacolyl methyl phosphonic acid — the predominant hydrolytic product of the nerve agent soman [49] — and re-engineering RBP to bind TNT [6]. Hellinga and co-workers [7] have also shown that RBP could be reconfigured into an enzymatically active triose phosphate isomerase that could support bacterial growth. The lessons learnt here were later applied to the convertion of a non-PBP cell adhesion protein to recognize calcium [51]. Such demonstrations, in conjunction with the switch work of Ostermeier and cowww.sciencedirect.com
workers, also highlight the emerging paradigms in biological research — namely the intersection of computational biology and recombinant engineering. They also, however, highlight the specialized disciplines needed, unique combinations of which are not currently found in many ‘classic’ research environments.
Conclusions Looking at all these demonstrations cumulatively, we see common themes. The PBPs, in particular MBP, are an elegant platform for prototyping a variety of diverse reagentless sensing modalities. Moreover, the sensing strategies do not necessarily have to rely on conformational coupling. The HISn and unique cysteines within the structure can be used orthogonally for attachment and reporter labeling. Once prototyped, the lessons learnt can be applied to using other non-PBP proteins for targeted biosensing. It is worth noting that each sensing modality has its own particular focus and thus might be more suitable for a specialized use. The fluorescent fusion protein approach of Frommer and co-workers [4,23–27] is particularly suited to in vivo monitoring, whereas electrochemical sensing might lead to microfabricated addressable arrays of biosensors. There is a huge array of ligand targeting PBPs to choose from, but the redesign strategy of Hellinga and co-workers [1,6,49,50] offers to expand this even more. If a signal transduction mechanism or biosensing strategy needs testing, MBP probably offers one of the best-characterized protein platforms to work with. Current Opinion in Biotechnology 2006, 17:17–27
26 Analytical biotechnology
Acknowledgements IM and JRD acknowledge the Office of Naval Research and the US Naval Research Laboratory.
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