Optical Biosensors: Present and Future F.S. Ligler and C.A. Rowe Taitt (editors) 9 2002 Elsevier Science B.V. All fights reserved CHAPTER 10 GENETIC ENGINEERING OF SIGNALING MOLECULES AGATHA FELTUS, PH.D., AND SYLVIA DAUNERT, PHARM.D., PH.D. Departments of Chemistry and Pharmaceutical Sciences University of Kentucky Lexington, KY, USA In order to expand the capabilities of biosensors, there is a need to develop new signaling molecules. This chapter focuses on molecules, produced through genetic engineering, that combine the recognition element with a signaling element (such as a fluorophore) in an effort to optimize the signal caused by the binding of the analyte to the recognition element. These systems, while not necessarily originally developed for an optical fiber, can be immobilized at the tip of the fiber either through chemical attachment or entrapment behind a membrane. Three different systems will be examined: fluorophore-labeled binding proteins, FRET-based systems, and bacteria-based sensors. These systems use optical signaling methods to reveal the binding event, taking advantage of molecular biological techniques to optimize the signal. The advantages and disadvantages of each system will be discussed, as well as the current state of the art of these biosensors. 1. Technical Concept In its simplest terms, a biosensor is a sensing system composed of a biological recognition element and a transducer. Under the strictest definition of the term, the transducer is responsible for converting the binding event into an electrical signal. Bacteria that fluoresce upon analyte binding and fluorophore-labeled binding proteins have been refered to as "reagentless biosensors" or "reagentless biosensing systems" even though they are used as assays, rather than 307 Feltus and Daunert immobilized in a sensor. There is no reason these sensing systems cannot be used as the recognition/signaling element of a fiber optic biosensor, however. These systems will be discussed in both contexts, i.e., both free in solution as assays and after adapation to a fiber optic probe. In order to avoid confusion, we will refer to the source of the optical signal as the signaling molecule. I.I. Fluorophore-labeled binding proteins As the name implies, a fluorophore-labeled binding protein consists of two distinct moieties: the fluorophore and the binding protein. The fluorophore is covalently attached to the binding protein through the protein's amino acid side chains. Binding of the analyte to the binding protein causes a conformational change in the protein structure which may result in altered optical characteristics of the fluorophore (Figure 1). Depending upon the actual environmental change experienced by the fluorophore, this could result in an increase or decrease in the fluorescence intensity, a change in the emission wavelength, or a change in the lifetime of the fluorophore, depending upon the characteristic to be measured. These changes are generally caused by two separate phenomena: an increased or decreased polarity in the environment surrounding the fluorophore or rotational constraint of the fluorophore. For example, if the fluorophore moves from a position of high polarity (exposure to the buffer or the presence of local side chains from polar amino acids) to one of low polarity (a position inside the protein), the fluorescence will increase due to decreased quenching by the solvent molecules or dissolved oxygen in the solvent. Likewise, rotationally constraining the fluorophore's motion by trapping it inside the protein will increase fluorescence by removing frictional energy loss caused by fluorophore movement. These changes are difficult to predict beforehand and are often only revealed once the protein has actually been labeled. Having said this, even if the direction of fluorescence change cannot be predicted, knowledge of the protein structure serves as a good starting point for choosing the placement of the fluorophore. From the point of view of signal-to- noise ratio, it is most advantageous to have a single fluorophore placed in a location where a large environmental change can occur. Often, the most likely location for such a change is near the binding site. Therefore, most initial studies are conducted by labeling at a site near the binding site as determined from observations of the crystal structure or from mutational studies. Selective labeling of the binding protein is usually accomplished via labeling through cysteine residues using sulfhydryl-selective fluorophores. (For examples of commonly employed fluorophores, see Figure 2.) In order to create one-to- one conjugates of fluorophore to protein, molecular biology is often necessary to create recombinant proteins with unique cysteine residues. Using recombinant 308 Genetic Engineering of Signalling Molecules Figure 1. Schematic of a fluorescently labeled protein sensing system. The protein is labeled with an environmentally-sensitive fluorophore such that the binding of the analyte changes the conformation of the protein, altering the solvation of the fluorophore. a) In this example, amino acid 197 of phosphate binding protein (PBP) is located near the binding pocket and will undergo a change in environment as PBP closes around its ligand, phosphate. This can result in either b) an increase or c) a decrease in fluorescence upon ligand binding. In some cases, the emission wavelength of the fluorophore can also change. DNA techniques, such as site-directed mutagenesis, all other cysteines in the protein are removed and other residues that will be the site of attachment are individually changed to cysteines. In doing so, care must be taken not to alter any amino acids necessary for the proper functioning of the protein, such as those residues involved in analyte binding or in oligomerization of the protein. This entire process will be examined in greater detail in Section 3.1. 309 Feltus and Daunert H2C-' C~ ~C~ " II 0 N(CH 3)2 (Q-t 3(:1-t 2)2 N ~O H O_ O~ N~ II 0 IVD(X3 (OH 3OH 2)2 N ~ ) O O HN ~ N C CH21 8Doll 1 ,,54 ~/g,~ Figure 2. Structures of some cysteine-reactive environmentally sensitive fluorophores. The reaction with the protein takes place through the maleimide or iodoacetimide groups. 1.2. FRET-based systems Another method which has been used extensively to develop sensing systems, particularly in small volumes and inside living cells, is the use of FRET-based sensing systems (Giuliano and Post, 1995; Giuliano and Taylor, 1998) (Figure 3). Fluorescence resonance energy transfer (FRET) occurs when one fluorophore, a donor, nonradiatively transfers its energy to a second fluorophore, the acceptor. The acceptor then relaxes normally, producing light at its emission wavelength. In order for this to occur, there must be a significant overlap between the emission spectrum of the donor and the excitation spectrum of the acceptor. An important property of FRET is that the rate of energy transfer between the donor and the acceptor is proportional to the inverse sixth power of the distance between the two fluorophores (FRET ~ l/r6). For most pairs the F~3rster radius, 310 Genetic Engineering of Signalling Molecules Figure 3. FRET-based sensing system for cAMP based on protein kinase A (PKA). This system consists of a cell line transfected with a vector coding for two fusion proteins: PKA regulatory subunit-BFP (blue fluorescent protein) and PKA catalytic subunit-GFP (green fluorescent protein). In the absence of cAMP, the regulatory and catalytic subunits associate, bringing the BFP and GFP moieties in close proximity and allowing FRET. The presence of cAMP dissociates the complex of regulatory and catalytic subunits, disrupting FRET. Adapted from Zaccolo et al., 2000. the distance at which the efficiency of energy transfer is 50%, is between 20A and 50/~ (Lakowicz, 1983). This distance is comparable to the size of most proteins, which allows FRET to be used when the distance between the two fluorophores will be significantly changed by the binding event. This can occur if either both fluorophores are attached to the same protein molecule and binding of a ligand to the protein causes a conformational change that either shortens or lengthens the distance between them, or if the donor is attached to one of the binding molecules and the acceptor to the other. In the latter case, a donor fluorophore attached to one of the components can transfer its energy to an acceptor fluorophore attached to the other only while the two are closely associated. An example of this is given in Figure 3. FRET as a detection methodology has a number of advantages for biosenor applications. Because the system employs the excitation wavelength of the acceptor and the emission wavelength of the donor, the Stokes shift is more pronounced than for fluorescence, leading to a lower background. Another advantage of FRET is that the ratio of fluorescence intensities of the two 311 Feltus and Daunert Figure 4. Schematic of a bacteria-based sensing system. The bacteria are transformed with a plasmid containing the reporter gene under the control of an analyte-sensitive promoter. In the presence of the analyte, the regulatory protein is released from the promoter region, allowing transcription of the reporter gene. The mRNA is then translated into protein, which can be assayed. The amount of protein produced is proportional to the amount of analyte present, although there is amplification at each step so that there are many more proteins present than reporter genes. Sometimes it is necessary to also place the gene for the regulatory protein on the plasmid as well as the reporter gene, as the native levels of reporter protein within the bacteria are insufficient for proper regulation of transcription. fluorophores can be used; this ratiometric technique is more accurate than measuring just one fluorescence signal. 1.3. Bacteria-based sensing systems Amplification-based methods take advantage of the high turnover of substrates to produce a large number of product molecules. This is the basis of such techniques as PCR and RT-PCR. In these cases, DNA or RNA molecules are selectively amplified to quantify the numbers of their parent strands. Whole-cell sensing systems take this one step further by first producing DNA, which is then amplified again during the transcription to RNA, and finally amplified a third time by translation to protein. 312 Genetic Engineering of Signalling Molecules Table 1. Reporter proteins used in whole cell sensing systems. Detection Protein Gene Catalyzed reaction method* chloramphenicol acetyltransferase Cat acetylation of chloramphenicol RI, FL CR, FL, EC, [3-galactosidase LacZ hydrolysis of 13-galactosides CL firefly luciferase Luc bacterial luciferase LuxAB aequorin AQ440 green fluorescent protein GFP luciferin + O2 + ATP BL oxyluciferin + AMP + PPi + h v FMNI-I2 + R-CHO+ 02 -~ FMN + BL HE0 + RCOOH + h v coelenterazine + 02 + Ca 2+ ~ BL coelenteramide + CO2 + h t, posttranslational formation of an internal chromophore FL * RI, radioisotope; FL, fluorescence; CR, colorimetric; EC, electrochemical; CL, chemiluminescence; BL, bioluminescence. A typical whole-cell sensing system consists of an organism, generally a bacterium, that is transformed with a plasmid containing a reporter gene under the control of a promoter responsive to the analyte of interest (Daunert et al., 2000; Lewis et al., 1998; Ramanathan et al., 1997a). This plasmid may also contain genes that will produce the necessary accessory proteins for the promoter, such as the regulatory proteins. These additional genes are sometimes necessary, as the number of promoters on the plasmid may greatly outnumber the usual number of promoters; a larger number of regulatory proteins is necessary to regulate these plasmid-borne promoters. Once the cells are exposed to analyte, transcription of the reporter gene will begin (Figure 4). After transcription, the RNA molecules are translated into protein. Amplification occurs at each of these steps to produce many more protein molecules than there are reporter genes. If desired, an extra level of amplification can be achieved if the reporter protein is an enzyme that will turn over large numbers of substrate molecules. However, if this further amplification is not required, then a protein such as the green fluorescent protein (GFP) can be used. GFP does not require addition of an external substrate, as the protein itself emits green fluorescence upon excitation at 490 nm. Another way to obviate adding a substrate is to use the entire lux cassette, instead of just luxAB, to produce bacterial luciferase. In this way all the accessory proteins to produce the substrates necessary for bacterial luciferase activity are also transcribed (Manen et al., 1997). 313 Feltus and Daunert The sensitivity of these systems is determined by a number of factors. The response of the promoter must be taken into account, but the largest effect of the promoter/repressor protein is upon the selectivity of the system. The more controllable factor is the choice of reporter protein, since there is often only a limited choice of promoters for a given analyte. The ideal reporter protein will be easy to use, have an easily discernable signal over the background, and have a wide dynamic range and high sensitivity (Daunert et al., 2000). Examples of reporter proteins that have been used to develop whole-cell sensing systems are given in Table 1. Sensitivity can be a function of several factors, including the detection method, efficiency of expression, reporter protein turnover number (if the protein is an enzyme), and, if applicable, the endogeneous levels of the reporter protein. For this reason, bioluminescent reporter proteins are a popular choice because bioluminescence is not found in most organisms, and is a very sensitive method of detection. 2. History These three types of fluorescent signaling systems emerged from the need of researchers in the biological sciences to study protein response to the binding of various ligands. For example, bacteria-based sensing systems are the result of experiments on regulation of transcription at various promoters. 2.1. Fluorophore-labeled proteins and FRET-based systems These two systems share a common ancestry in studies of protein function. One way of examining the structural changes in proteins upon ligand binding, dimerization, or denaturation is by measuring in the native fluorescence of tryptophan residues. This approach since they might not be close can be used to measure binding only when the tryptophan is proximal to the active site. This limitation led to the use of fluorescent cofactors and substrates, such as flavin mononucleotide, to study changes occurring within the binding pocket. Later, proteins were labeled with extrinsic fluorophores. Such labeled proteins have been used for a number of applications, including microinjection into cells to study protein localization and solution studies of protein structural changes. Initially, biochemists used these fluorophore-labeled proteins to gain information about the alterations in size, shape, and binding properties of proteins. However, with the development of environmentally sensitive fluorophores and the ability to produce mutated recombinant proteins, the fluorophore-labeled sensing system as it stands today was born. Table 2 gives several examples of analytes that have been measured using these systems. Most of the currently-developed sensing systems of this type depend upon molecular biology to either create a unique site for fluorophore attachment, to translate the protein such that it incorporates non- native fluorescent amino acids, or to fuse a GFP to the protein. 314 Genetic Engineering of Signalling Molecules Table 2. Examples of fluorophore-labeled protein sensing systems. In vitro/in vivo refers to whether the protein is used in situ after being produced by the cells or whether the proteins are expressed, isolated, and purified prior to use, and used as a sensing system. In vitro~in Analyte Protein* vivo Reference P~ PBP In vitro Brune et al., 1994, 1998 fatty acids I-FABP In vitro Richieri et al., 1992 maltose MBP In vitro Gilardi et al., 1994 biotin Streptavidin In vitro Murakami et al., 2000 Ca2+ CaM-YFP In vitro~in Baird et al., 1999 fusion vivo Ca 2+ CaM-EGFP- in vitro~in Nakai et al., 2001 vivo M 13 fusion Ca z+ CaM in vitro Co 2+, Zn 2+, Carbonic in vitro Cu 1+ anhydrase Salins et al., 1998; Schauer-Vukasinovic et al., 1997 Thompson et al., 1998 Salins et al., 2001; glucose GGBP in vitro Tolosa et al., 1999 Dattelbaum and glutamine GlnBP in vitro Lakowicz, 2001 *Abbreviations: PBP, phosphate binding protein; I-FABP, intestinal fatty acid binding protein; MBP, maltose binding protein; CaM, calmodulin; YFP, yellow fluorescent protein; EGFP, enhanced green fluorescent protein; GGBP, galactose/glucose-binding protein; GlnBP, glutamine binding protein FRET-based systems can be considered as a subclass of the fluorophore-labeled proteins, different only because they depend upon the proteins being labeled with two fluorophores rather than one. Because FRET is a distance-dependent phenomenon, it was originally used to study assembly of multi-subunit protein complexes, such as ribosomes, or interaction between a protein and cellular membranes. In the 1990s, however, FRET-based systems started to be used for analytical purposes (Table 3). The most recent trend is to use GFP and its wavelength-shifted mutants as the donor and acceptor molecules. 315 Feltus and Daunert Table 3. FRET-based assays using labeled proteins.* Analyte Protein I)'onor Acceptor Reference factor Xa factor Xa site " BFP rsGfp Mitra et ai.',' 1996 caspase-3 Caspase-3 site BFP GFP Xu et., 1998 caspase-3 Caspase-3 site CFP YFP Jones et al., 2000 Zn 2+ Ca 2+ zinc finger Lissamine rhodamin Godwin and Ber, peptide e 1996 aequorin Aequorin GFP Baubet et al., 2000 Ca 2+ CaM BFP GFP Romoser et al., 1997 Ca 2+ PKA Cam/M13 BFP/CFP GFP/YFP Miyawaki et al., 1997 KID B FP GFP Nagai et al., 2000 CAMP PKA fluorescei rhodamin Adams et al., 1991 n e CAMP PKA BFP/CFP GFP/YFP Zaccolo et al., 2000 *Abbreviations: BFP, blue fluorescent protein; GFP, green fluorescent protein; CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; CaM, calmodulin; PKA, protein kinase A; KID, kinase inducible domain. Because these fluorophores are proteins themselves, plasmid constructs can be made that fuse the GFP to the sensing protein, allowing these proteins to be produced within a cell and used in situ as sensors. This is advantageous, since the analytes of interest are generally intracellular second messengers. Moreover, the need to microinject purified chemically-labeled proteins is avoided. 2.2. Bacteria-based sensing systems Sensing systems of this type trace their origin back to bioassays in which nutrient-deficient or antibiotic-resistant strains were plated on media containing various concentrations of the analyte and the surviving number of cells counted. From this methodology evolved the non-specific bacteria-based sensing systems. These bacteria constitutively express a reporter protein while alive, but as toxins begin tokill the bacteria, the protein is no longer produced, giving a lower signal. At the same time, the bacterial operons were discovered, and reporter genes were 316 [...]... Desvergne and Czamik, 1997; de Silva et al., 1997; Kimura and Koike, 1998a; Fabbrizzi et al., 1998b, 20 00; Granda-Vald6s et al., 20 00; Yamauchi and Hayashita, 20 00; Prodi et al., 20 00; Baragossi et al., 20 00) While innovative work on chemosensors for metals continues (e.g., Hayashita et al., 20 00; Bronson et al., 20 01; Baxter, 20 01; Raker and Glass, 20 01; McFarland and Finney, 20 01; Mello and Finney, 20 01),... Bonev and G Homeck, 1997, Mut Res 379, $20 6 Rettberg, P., C Baumstark-Khan, K Bandel, L.R Ptitsyn and G Horneck, 1999, Anal Chim Acta 387, 28 9 Richieri, G.V., R.T Ogata and A.M Kleinfeld, 19 92, J Biol Chem 26 7, 23 495 Romoser, V.A., P.M Hinkle and A Persechini, 1997, J Biol Chem 27 2, 1 327 0 Salins, L.L.E., V Schauer-Vukasinovic and S Daunert, 1998, Proc SPIE 327 0, 16 Salins, L.L.E., R.A Ware, C.M Ensor and. .. Taylor, R.Y Tsien and T Pozzan, 20 00, Nature Cell Biol 2, 25 329 Optical Biosensors: Present and Future F.S Ligler and C.A Rowe Taitt (editors) 9 20 02 Elsevier Science B.V All fights reserved CHAPTER 11 ARTIFICIAL RECEPTORS FOR CHEMOSENSORS THOMAS W BELL, PH.D AND NICHOLAS M HEXT, PH.D Department of Chemistry, University of Nevada, Reno Reno, NV 8955 7-0 020 USA Chemosensors are molecules of abiotic origin... Johansson and B Mattiasson, 1999, Anal Chim Acta 387, 23 5 Dattelbaum, J.D and J.R Lakowicz, 20 01, Anal Biochem 29 1, 89 Daunert, S., G Barrett, J.S Feliciano, R.S Shetty, S Shrestha and W SmithSpencer, 20 00, Chem Rev 100, 27 05 de Lorenzo, V., S Fernandez, M Herrero, U Jakubzik and K.N Timmis, 1993, Gene 130, 41 Finn, B.E., T Drakenberg and S Forsen, 1993, FEBS Lett 336, 368 Gilardi, G., L.Q Zhou, L Hibbert and. .. and Atwood, 20 00) has identified several intermolecular forces that play important roles The fundamental electrostatic (ion-ion, ion-dipole and dipole-dipole), hydrogen bonding, and van der Waals interactions are of course important, but more subtle forces have also been examined, including n-stacking (Hunter and Sanders, 1990; Hunter, 1993), cation-n interaction (Ma and Dougherty, 1997), CH-rc interaction... (Ca2+)4-CaM to the sensor increases the inter-fluorophore distance from ~25 /~ to ~65A, effectively eliminating FRET (Figure 6) The change in the fluorescence emission ratio is dose-dependent for both Ca 2 and (Ca2+)4-CaM and is shown to work well when microinjected into cells, as well as in vitro A similar system was developed by Miyawaki et al (1997) using BFP or CFP, CaM, CaM-binding peptide M13, and. .. Anal Chem 66, 3840 Giuliano, K.A and P.L Post, 1995, Annu Rev Biophys Biomol Struct 24 , 405 Giuliano, K.A and D.L Taylor, 1998, Trends Biotechnol 16, 135 Godwin, H.A and J.M Berg, 1996, J Amer Chem Soc 118, 6514 Guan, X., S Ramanathan, J.P Garris, R.S Shetty, C.M Ensor, L.G Bachas and S Daunert, 20 00, Anal Chem 24 23 Guzzo, J., A Guzzo and M.S DuBow, 19 92, Toxicol Lett 6 4-6 5 Spec No, 687 Heitzer, A., K... Salins, L.L.E., R.A Ware, C.M Ensor and S Daunert, 20 01, Anal Biochem 29 4, 19 Schauer-Vukasinovic, V., L.C Cullen and S Daunert, 1997, J Am Chem Soc 119, 111 02 Scott, D.L., S Ramanathan, W Shi, B.P Rosen and S Daunert, 1997, Anal Chem 69, 16 Selifonova, O.V and R.W Eaton, 1996, Appl Envir Microbiol 62, 778 Shetty, R.S., Y Liu, S Ramanathan and S Daunert, 20 00, The Pittsburgh Conference on Analytical Chemistry... Res 389, 27 9 Virta, M., J Lampinen and M Karp, 1995, Anal Chem 67, 667 Willardson, B.M., J.F Wilkins, T.A Rand, J.M Schupp, K.K Hill, P Keim and P.J Jackson, 1998, Appl Environ Microbiol 64, 1006 Xu, X., A.L Gerard, B.C Huang, D.C Anderson, D.G Payan and Y Luo, 1998, Nucleic Acids Res 26 , 20 34 Zaccolo, M., F De Giorgi, C.Y Cho, L Feng, T Knapp, P.A Negulescu, S.S Taylor, R.Y Tsien and T Pozzan, 20 00,... events, such as Zn 2 or Ca 2 release and cAMP accumulation, have also been found to be good targets for FRET-based systems An in vitro system 321 Feltus and Daunert for Zn 2 developed by Godwin and Berg (1996) uses a zinc finger peptide as the sensing element Zinc fingers bind zinc tightly and have a great selectivity for Zn(II) over Co(II), Fe(II), and Ni(II) Godwin and Berg (1996) engineered a . Feltus and Daunert H2C-' C~ ~C~ " II 0 N(CH 3 )2 (Q-t 3(:1-t 2) 2 N ~O H O_ O~ N~ II 0 IVD(X3 (OH 3OH 2) 2 N ~ ) O O HN ~ N C CH21 8Doll 1 ,,54 ~/g,~ Figure 2. Structures. Optical Biosensors: Present and Future F.S. Ligler and C.A. Rowe Taitt (editors) 9 20 02 Elsevier Science B.V. All fights reserved CHAPTER. ATP BL oxyluciferin + AMP + PPi + h v FMNI-I2 + R-CHO+ 02 -~ FMN + BL HE0 + RCOOH + h v coelenterazine + 02 + Ca 2+ ~ BL coelenteramide + CO2 + h t, posttranslational formation of an