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Fluorescence resonance energy transfer by S-layer coupled fluorescence dyes
Sensors and Actuators B 185 (2013) 553–559
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Fluorescence resonance energy transfer by S-layer coupled fluorescence dyes Ulrike Weinert a,∗ , Katrin Pollmann a , Johannes Raff a,b a b
Helmholtz-Zentrum Dresden-Rossendorf, Helmholtz Institute Freiberg for Resource Technology, Halsbrücker Straße 34, 09599 Freiberg, Germany Helmholtz-Zentrum Dresden-Rossendorf, Institute of Resource Ecology, Bautzner Landstraße 400, 01328 Dresden, Germany
a r t i c l e
i n f o
Article history: Received 27 February 2013 Received in revised form 8 May 2013 Accepted 15 May 2013 Available online 25 May 2013 Keywords: Fluorescence resonance energy transfer Surface layer proteins EDC Chemical modification Sensory layers Detection
a b s t r a c t In this paper two fluorescence dyes were coupled to surface layer (S-layer) proteins of Lysinibacillus sphaericus A12 and Lysinibacillus sphaericus B53 to easily generate a fluorescence resonance energy transfer (FRET). S-layer proteins are structure proteins which self-assemble in aqueous solutions, on surfaces and at interfaces forming 2D-paracrystalline structures with a defined symmetry in nanometer range. These properties and the fact, that a lot of modifiable functional groups are available on their surface, make them a perfect coating and binding matrix for the generation of functionalized surfaces, e.g. needed for a sensor assembly. Here we chemically link two fluorescence dyes, which are able to perform a FRET, to S-layer proteins by carbodiimide-crosslinking chemistry. Fluorescence dyes were coupled to the protein with a yield of around 54 mol%, demonstrating a modification of every second protein monomer if fluorescence dyes are statistical distributed. A FRET could be detected between the two fluorescence dyes when linked to protein polymers whereas no FRET could be detected if fluorescence dyes are linked to protein monomers. This demonstrates, that the polymer structure is essential for FRET and that fluorescence dyes are statisticaly distributed on protein polymers with a close proximity of donor and acceptor dye. Due to the fact that the used S-layer proteins build a unit cell of p4 symmetry, it can be assumed that two fluorescence dyes are linked to one unit cell. In this paper the FRET pair arrangement and its optimization is described in which the FRET efficiency can be increased from 6 to 40%, simply by varying the molar ratio of donor:acceptor. In result a sensory surface can be generated and used for detection of numerous substances in water like pharmaceuticals or heavy metals. © 2013 Elsevier B.V. All rights reserved.
1. Introduction 1.1. S-layer proteins as universal binding matrix S-layer proteins are structure proteins which are present in many bacteria and almost all archaea as outermost cell envelope. They were discovered by Houwink in 1953 when he saw a regular grid structure on freeze-etched bacteria cells of Spirillum serpens [1]. Although the chemical properties of bacterial S-layer proteins are very similar in different organisms their functions are quite diverse. They can serve e.g. as molecular or ion trap, as protecting layer or binding matrix for exoenzymes [2–4]. They cover the whole cell as paracrystalline structures with a defined symmetry and specific lattice parameters. If S-layer proteins are isolated, they maintain self-assembling properties and are able to autocatalytically form polymer lattice structures in aqueous solutions, on surfaces and at interfaces [5].
∗ Corresponding author. Tel.: +49 351 260 2012; fax: +49 351 260 3553. E-mail address: [email protected] (U. Weinert). 0925-4005/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2013.05.051
One further interesting property of S-layer proteins is their high content of COOH and NH2 -groups which are usually around 15 mol% [6]. Those functional groups can be easily modified by various crosslinkers e.g. 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and glutaraldehyde [7] In one example Protein A was successfully linked to S-layer proteins of Clostridium thermohydrosulfuricum L111-69 [8]. In another approach glucose oxidase was linked to S-layer proteins of Bacillus coaguluns E38-66 by glutaraldehyde [9]. In further work glutaraldehyde is used as stabilization reagent for S-layer protein’s polymer structure and amino group blocking. EDC is then used as crosslinking reagent to link various enzymes to S-layer proteins and build sensory layers up to biosensors [10,11]. Thereby activity of enzymes is dependent of the structure of enzymes [7]. In recently published work, fusion proteins composed of Slayers and different fluorescent proteins were produced. Such fusion proteins show sensitivity toward pH-change and could therefore be used as pH-sensor [12,13]. In another paper a bifluorescent fusion S-layer protein was constructed and an energy transfer with an efficiency of about 20% between both fluorescence dyes could be measured by steady state fluorescent measurements. FRET only occurs in this array if proteins are in their polymer form
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[14]. In this study S-layer proteins from isolates of a uranium mining waste pile near Johoanngeorgenstadt (JG) in Saxony, Germany were used, namely Lysinibacillus sphaericus JG-A121 (A12) and L. sphaericus JG-B53 (B53). The reassembled lattice of those S-layer proteins show a p4 symmetry with a unit cell size of about 13 nm. The S-layer protein of A12 was formerly used as sensor molecule for the detection of uranium [15]. The application shows that those S-layer proteins are long term stable in aqueous solutions. In this paper we describe a method to simply modify S-layer proteins by EDC chemistry without using glutaraldehyde for protein crosslinking. In result, stable S-layer proteins which are functionalized with a FRET pair are generated. The latter can be used e.g. in combination with specific binding molecules for sensor technology. 1.2. Fluorescence resonance energy transfer (FRET) based biosensors FRET is a universal tool in biochemistry for the monitoring of e.g. protein folding, protein interactions and structure changes. In all these applications FRET-induced changes in fluorescence is used to monitor these phenomena [16,17]. Challenging by using FRET-based sensors is to detect changes of the fluorescence correctly and to exclude misinterpretation [18–20]. According to Roda, there are five different methods commonly used to detect FRET. He claimed to prefer the determination of lifetime because the results are more reliable compared to all other methods [18]. Domingo and co-workers applied four different methods to detect FRET and calculate its efficiency using the fluorescent proteins CFP and YFP. Their results show that especially in unknown systems a standard for FRET detection is needed and at least two different methods should be used to get reliable results [19]. Or in other words Vogel and co-workers [20] provokingly said, that they contend that the popularity of FRET has risen in concert with degradation in the validity of the interpretation of biological FRET experiments. Their report specially refers to FRET measurements in living cells where protein interactions are studied and a “random FRET” of 5% can be always detected. But nevertheless the above statement applies to all FRET measurements. Therefore, we will concentrate our work not only on construction of a FRET-system on S-layer proteins but also on verifying an energy transfer between two immobilized fluorescence dyes. The aim of our work is to generate an energy transfer between the FRET pair HiLyteFluorTM 488 (H488) and HiLyteFluorTM 555 (H555). We linked both fluorescence dyes to surface layer proteins by carbodiimide chemistry. We examined different coupling strategies which include functionalization of NH2 or COOH groups, modification of S-layer protein monomers and polymers and we compared two S-layer proteins regarding their modification rate and their polymer stability after functionalization. In result we could find a modification maximum of around 0.54 moldyes /molS-Layer which means that two fluorescence dyes are found on one unit cell. Therefor we methodically analyzed fluorescence spectra and time resolved measurements, in order to exclude “random FRET signals”. In a second step we optimized the FRET efficiency of the linked fluorescence dyes by varying the donor to acceptor ratio. An assembly containing S-layer proteins and a protein bound FRET pair is suitable for further the construction of an improved sensory layer.
1 A12 – Lysinibacillus sphaericus JG-A12; B53 – Lysinibacillus sphaericus JG-B53; H488 – HiLyteFluorTM 488; H555 – HiLyteFluorTM 555.
2. Materials and methods 2.1. Functionalization of S-layer proteins with fluorescence dyes For all experiments S-layer proteins from strains L. sphaericus JG-A12 (A12) and L. sphaericus JG-B53 (B53) were used. Functionalization of S-layer proteins was done either by NH2 or COOH groups. As described by [21] A12 contains 103 NH2 groups (8.5 mol%) and 125 COOH groups (10.4 mol%). For B53 84 NH2 groups (9.5 mol%) and 94 COOH (10.7 mol%) are available in their primary structure [22]. Lyophilized S-layer proteins were dissolved either in 50 mM MES at pH 5.6 for a polymer suspension or in 50 mM MES pH 5.6 with additional fresh prepared 3 M Urea to get a monomer solution. Concentration of all samples was adjusted to 10 mg/ml with corresponding buffer solution. Modification of S-layer proteins with fluorescence dyes were performed by EDC chemistry or succimidyl ester chemistry. In both cases an amide bond between fluorescence dyes and S-layer proteins was created. Fluorescence dyes HiLyte FluorTM 488 hydrazide (H488), HiLyte FluorTM 555 (H555) hydrazide, HiLyte FluorTM 488 succimidyl ester (H488SE) and HiLyte FluorTM 555 succimidyl ester (H555SE) were purchased from MoBiTec GmbH (Göttingen) with HPLC purity. For coupling fluorescence dyes to S-layer protein’s NH2 group H488SE and H555SE with a molar ratio of 3:1 were used. S-layer proteins and fluorescence dyes with a succimidyl coupling group were mixed and incubated for two hours at room temperature in dark. The succimidyl ester will covalently bind to the amino group of the S-layer proteins. Fluorescence dyes H488 and H555, which contained a hydrazide group as binding group, were used for chemical modification of Slayer protein’s COOH groups. Modification was performed by EDC. Molar ratio of fluorescence dyes was 3:1 (donor: acceptor). Fluorescence dyes and proteins were mixed in a molar ratio of 2:1 (fluorescence dye: protein). 200 mM EDC dissolved in 50 mM MES pH 5.6 was added to proteins and fluorescence dye mixture to a final EDC concentration of 20 mM, were mixed gently and incubated for two hours at 4 ◦ C in dark. Modified protein polymer solution was centrifuged at 12,400 × g for 1 h at 4 ◦ C and washed twice in 50 mM MES pH 5.6 in order to remove unbound fluorescence dyes. Functionalized protein monomer solution was dialyzed against 10 mM CaCl2 solution overnight in order to further purify the protein. Dialyzed proteins were centrifuged at 12,400 × g for 1 h at 4 ◦ C. An occurring pellet of precipitated fluorescence dyes and denatured proteins was removed to get a supernatant containing only fluorescence labeled protein monomers. All labeled proteins were stored at 4 ◦ C in dark until further investigation. 2.2. Determination of the number of coupled fluorescence dyes In order to determine binding capacity UV/Vis measurements were performed with Nanodrop (Nanodrop2000, Thermo Fisher Scientific, Waltham, Massachusetts, USA). For measurements 10 l of samples were diluted with 10 l of 6 M guanidine hydrochloride solution and incubated for at least one hour. 2 l were used for measurements and all samples were measured at wavelengths 280 nm (protein concentration), 508 nm (H488), 504 nm (H488SE) and 552 nm (H555 and H555SE). Protein concentration was determined in the range of 0–5 mg/ml. Concentration of fluorescence dyes was determined by using Beer-Lambert law. The spectra of fluorescence dyes H555 and H555SE show an additional shoulder at a wavelength of 500 nm which interferes with the absorption of the donor dye. Therefore the absorbance at
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508 nm (A508 ) of H488 and H488SE has to be corrected (Acorr ) as follows: Acorr = A508 − (0.45 × A552 ) whereas A552 is the measured absorbance at a wavelength of 552 nm and 0.45 is an experimental determined correction factor. 2.3. Detection of FRET Steady state and time resolved fluorescence measurements were performed with the system Quantamaster 40-Q25 (Photon Technology International (Canada) Inc., London, Canada). 2.3.1. Steady state fluorescence measurements Samples were diluted 1:10 (v/v) in 10 mM CaCl2 to a final protein concentration of 0.1 mg/ml. Measurements were performed with a slit width of 2 nm. Samples were excited at two different wavelengths, 250 and 450 nm, to determine protein concentration and donor emission (H488 or H488SE) in diluted samples. 2.3.2. Time resolved measurements Samples were used in concentration of 10 mg/ml and slit width was 12 nm. For excitation of the donor, samples were excited at 450 nm and time-resolved emission was measured at 520 nm (donor emission). All samples were measured five times and an average of all 5 spectra was used for data analysis. In order to eliminate runaway values and to smooth data a Savitzky–Golay smoothing with 9 data point was performed with the average spectra [23]. Calculation of lifetime was performed by the following equation: y = y0 + A × exp(−x/) In which A is the amplitude and y0 the offset-correction. Determination of the fluorescence lifetime was achieved by a self-written program within the software of OriginLab Version OriginPro 8.6. OG Sr1. FRET-efficiency EFRET was then determined with the following equation: EFRET =
DA D
DA represents the lifetime of donor dye in presence of the acceptor and D represents lifetime of donor dye in absence of acceptor dye. 2.4. Optimization of FRET For optimization of the energy transfer different molar ratios of donor: acceptor were incubated with S-layer proteins of strain A12 or B53. Molar ratio ranges from 0.5 up to 10.5 (donor: acceptor). Thereby the volume of added fluorescence dyes was still the same but the total amount of added fluorescence dyes differed. In order to exclude false interpretation reference solutions containing only different molar ratios of H488:H555 in 10 mM CaCl2 were also investigated. 3. Results 3.1. Characterization of the FRET pair and its optimization Donor and acceptor dyes were mixed in different ratios and UV/vis-measurements were performed in order to calculate the total amount of donor and acceptor in solution and to verify the former introduced Eq. (1) for the calculation of H488 in solution in presence of the acceptor dye H555.
Fig. 1. Diagram of different molar ratios of donor and acceptor against fluorescence ratio of wavelength 525 and 565 nm, excitation wavelength is 450 nm, also shown is the moving average; Inlet: Emission spectra of donor and acceptor dye when excited at wavelength 450 nm.
Fluorescence measurements of various molar ratios of H488:H555 were performed in order to exclude a “random FRET” signal because of different signal intensities. Samples were excited at a wavelength of 450 nm and emission spectrum was detected. In Fig. 1 the emission ratio 525 to 565 versus the ratio of fluorescence dyes is plotted. Ratio 525/565 rises exponentially with rising ratio H488:H555. H555 shows a little emission at 565 nm when excited at a wavelength of 450 nm (Inlet Fig. 1). If no FRET occurs the plot should show a linear curve. Because of little self-quenching effect this curve is not linear. If FRET occurs a local minimum should have been detected. In addition, lifetime of H488 was also calculated for all used ratios. Results show an average lifetime of H488 of about 4.41 ± 0.09 ns independent of the amount of H555. In conclusion fluorescence and lifetime measurements show definitely no FRET between donor and acceptor dye if both fluorescence dyes are in solution.
3.2. Optimization of the FRET-pair binding to S-layer proteins and its influence on the S-layer stability For modification of S-layer proteins at their NH2 -groups, succimidyl ester activated fluorescence dyes were used. For modification of COOH-groups, hydrazide-modified fluorescence dyes were used for binding. Quantification of the amount of linked fluorophores to S-layer proteins where calculated with the help of Eq. (1). In result a modification rate of S-layer proteins in range of 0.15–0.6 moldye /molprotein could be determined. For an energy transfer a distance of less than 10 nm between donor and acceptor dye is necessary. If S-layer proteins build up unit cells, containing 4 monomers, or even polymer structures A modification rate of about 50 mol% will result in total distance of 6.5 nm between the FRET pair as one unit cell gains 13 nm. Thus a recrystallization of modified protein monomers is essential. Furthermore, functionalized S-layer proteins should be able to recrystallize on surfaces for later sensory applications. For this reason the recrystallization process of functionalized protein monomers was also investigated. In result, no recrystallization could be observed, even after two weeks. Only precipitated and denatured proteins could be detected. It can be thusly assumed that modification of S-layer protein monomers disturb recrystallization process. In comparison to that protein
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Fig. 2. Light microscopic image of modified S-layer protein polymers of strain A12; phase contrast and fluorescence images clearly show the typical polymer tubes.
polymers maintain their polymer structure after functionalization (see Fig. 2). The only exclusion was in case of the modification of S-layer’s NH2 -groups of B53 where nearly 50% loss of protein was detected (Fig. 3). Due to the fact that the reference sample, where S-layer proteins from strain B53 were not modified, have also a loss of about 50%, it is reasonable that the used buffer (50 mM MES, pH 5.5) system destabilizes their polymer structure. If S-layer proteins from strain B53 are cross-linked by EDC, no loss of protein polymer can be observed due to the fact that internal crosslinking stabilizes polymer structure. SDS-PAGE of functionalized S-layer proteins showed also a crosslinking as the proteins did not enter the separating gel and remain in the sample wells (Fig. 4).
sufficient evidence for an energy transfer. Influence of acceptor dye on the emission spectra cannot be excluded specially when an excess in acceptor dyes is available ([24] suppl. material).
3.3. Determination of FRET Results of steady state and time-resolved measurements can be seen in Fig. 5 and Table 1. All spectra were normalized to measured protein concentration. If FRET occurs, an emission peak should be detected in the range of 550–570 nm. Only polymeric proteins with modified COOH-groups show a clear second emission peak with a maximum at 575 nm. In other cases only a small shoulder arises, being no
Fig. 3. Protein concentration of the S-layer proteins of A12 and B53 after modification with fluorescence dyes at their NH2 or COOH groups, dashed line showed the original applied protein concentrations.
Fig. 4. SDS-PAGE: 10% SDS-gel of Protein polymers of strain B53 modified with a FRET pair at their (1) NH2 -group or (2) COOH-group, also shown is a protein ladder (3) with size markers at 200, 150, 120, 100, 85, 70, 60, 50, 40, 30, 25, 20 and 15 kDa; white arrows indicate crosslinked protein dimers and oligomers.
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Fig. 5. Emission spectra of S-layer proteins of A12 modified with a FRET-pair; excitation wavelength is 450 nm; monomer and polymers were modified either at their NH2 or COOH group.
In order to clearly demonstrate the energy transfer and to calculate the FRET-efficiency the lifetime of donor dye was determined in presence and absence of acceptor dye and efficiency was calculated according to Eq. (3) (Table 1). With this method the lifetime can be determined with an average error of 0.2 ns which represents nearly 5% of the total lifetime of the donor dye being 4.5 ns in 10 mM CaCl2 . According to that no energy transfer can be detected in case of modified protein monomers independently from protein or modified functional group. If protein polymers were modified at their COOH-group an energy transfer with an efficiency of 10% (A12) and 25.4% (B53) can be detected. If NH2 -groups of protein polymers from A12 were modified, energy transfer can be determined with an efficiency of 7.5%. Fluorescence spectra are consistent with this observed data (see Fig. 5). Furthermore, emission intensities at a wavelength of 525 nm are consistent with absorption spectra at a wavelength of 508 nm with exception of the samples showing a FRET. This linear behavior indicates that no self-quenching of donor dye occurs if it is linked to the protein. 3.4. Optimization of FRET efficiency
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Fig. 6. Plot of introduced molar ratio of donor and acceptor against experimental determined molar ratio of donor and acceptor linked to the protein polymers of B53 (triangle) and A12 (square); also shown is the 100% agreement (dashed gray line).
The fluorescent dye H488 thusly binds better to the S-layer protein than H555. Same results were obtained by modifying proteins either with donor or acceptor dye only. Modification rate of proteins modified with only the acceptor dye H555 is around 0.3 moldyes /molprotein and for proteins modified with only the donor dye H488 the rate is around 0.5 moldyes /molprotein . This result is possibly explainable by different structure and size of the fluorescence dyes. 3.4.2. Determination of FRET-efficiency Steady state and time resolved fluorescence measurements of all samples with a molar donor: acceptor ratio varying from 0.3 to 10.5 mol: mol were analyzed and compared (see Fig. 8). Data were analyzed by a polynomial fit and the local minimum represents the highest FRET-efficiency. In case of A12 a theoretical molar ratio of 6.5 (D: A) and in case of B53 a molar ratio of 6 (D: A) will result in the highest FRET efficiency. Lifetime measurements were performed in order to determine the FRET-efficiency. An increase of FRET-efficiency from roughly 10% (25.4%) to 40% could be reached by changing the molar ratio
3.4.1. Introducing different donor and acceptor ratios to S-layer proteins Protein polymers were modified with different molar ratios of donor and acceptor ranging from 0.2 to 10.5 in order to increase the energy transfer. Results of modification can be seen in Fig. 6. The diagram clearly shows that a molar ratio of linked donor and acceptor can be directly controlled. The fact of controlling the binding ratio was also proclaimed by Prasuhn et al. who modified Quantum dots with dye-labeled peptides by simple using EDC chemistry [25]. average modification rate is around The 0.54 ± 0.16 moldye /molprotein . Thereby it can be noticed that an increasing modification trend goes along with an increasing donor: acceptor ratio (Fig. 7).
Table 1 FRET-efficiency of FRET-pairs coupled to S-layer proteins in their polymeric or monomeric form of strains A12 and B53. sample
monomeric proteins
polymeric proteins
modification rate NH2 COOH
A12 1.3 0.3
A12 7.2 10.3
B53 4.9 2.4
B53 4.5 25.4
Fig. 7. Modification rate of S-layer protein polymers of A12 and B53 with a FRETpair with different molar ratios of donor and acceptor; gray dotted line shows the average modification rate and gray solid line shows standard deviation.
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Fig. 8. Plot of normalized fluorescence intensities of the molar ratio of donor and acceptor (D: A) against the fluorescence ratio at the wavelength 525 and 565 nm, excitation wavelength is 450 nm; Donor and acceptor were modified at the COOHgroups of protein polymers of A12 (square) or B53 (triangle), also shown are unbound fluorescence dyes in different molar ratios (circles).
within the Förster radius (10 nm). A higher modification rate of fluorescence dyes to the S-layer proteins is not mandatory because higher density of fluorescence dyes will cause self-quenching effects and reduces fluorescence intensity. Several papers claim that a modification rate of one up to three fluorescence dyes on protein in solution will result in the highest yield of fluorescence intensity versus protein concentration [26–28]. Another important aspect is the effect of a hydrophilic environment which can also quench fluorescence intensity. Most commercial available fluorescence dyes prefer a more hydrophobic environment like methanol or dimethyl sulfoxide. Fluorescence dyes linked to proteins and transferred into an aqueous environment, the quantum yield will decrease and they sometimes get quenched. If fluorescence dyes are linked to S-layer proteins, this effect could not be observed. A reason can be seen in the hydrophobic environment of the S-layer protein’s surface which will work as a protecting environment for the fluorescence dyes [26,29,30]. The S-layer proteins, which are used in this work, possess 40% of hydrophobic amino acids [21,22]. The S-layer proteins were chemical functionalized with an FRETpair producing a sensory layer, which can be used for the detection of molecules. One requirement for those molecules is their ability to quench fluorescence dyes in order to reduce the energy transfer. Another possibility for would be a genetically introduced FRET pair into S-layer proteins as it was done by Kainz et al. with a FRET efficiency of about 20% [14]. The chemical modification has several advantages over the genetic one: commercial available fluorescence dyes are more stable than GFP and likewise and show no pH-sensibility and high quantum yields. Furthermore a chemical functionalization will enable more flexibility concerning the molecules which can also work as sensory elements like quantum dots and aptamers. With the help of different crosslinkers it will be possible to sequential link those mentioned elements on the surface at the carboxylic, amino or hydroxyl group of S-layer proteins. And in case of our setup a functional protein layer can be produced within a few hours without special equipment.
5. Conclusion Fig. 9. Lifetime of the donor dye H488 linked to S-layer protein polymers of A12 or B53 at their COOH-group in absence and presence of different molar ratios of the acceptor dye.
from 3 to 4 (donor: acceptor). These results were obtained for both S-layer proteins A12 and B53 (Fig. 9). In summary an optimized protocol for the modification of Slayer proteins of A12 and B53 can be created as follows: Protein polymers are used and a FRET pair is linked to their COOH-group by EDC chemistry. S-layer proteins are then additional internal crosslinked to each other by EDC to gain more stability toward degradation to their monomer forms. Furthermore, a quadruplicate excess of donor dye to acceptor dye is used in order to achieve an energy transfer of approximately 40%. Determination of an energy transfer is confirmed by two measurements using static fluorescence and time resolved fluorescence. 4. Discussion Chemical modification of S-layer proteins with a FRET pair yielded in a modification rate of about 0.54 ± 0.16 moldye /molprotein. This means that two fluorescence dyes are linked to one unit cell which consists of four protein monomers. The distance between donor and acceptor can be estimate as 6.5 nm which is clearly
In this paper S-layer proteins of the two wild strains L. sphaericus JG-A12 and L. sphaericus JG-B53 were chemically modified with a FRET-pair by EDC by their COOH and NH2 groups. We systematically optimized the modification procedure in order to gain a high FRET efficiency. Therefore we also established a procedure for a secure FRET detection without misinterpreting. We were able to reach a FRET efficiency of nearly 40% by a quadruplicate excess of acceptor dye. Thereby a modification rate of 0,54 moldye /molprotein could be reached which means that two fluorescence dyes are linked to one unit cell of the S-layer lattice. The advantage of a chemical modified S-layer protein over a genetically introduced FRET-Pair lies in the higher stability of the fluorescence dyes, the possibility to link other molecules to the S-layer proteins like quantum dots or aptamers and to sequential bind different elements on a surface. The hydrophobic environment of the S-layer protein will protect the fluorescence dyes from quenching effects by aqueous solutions and buffer changes. Most fluorescence dyes are hydrophobic and gain the highest fluorescence yields in organic solutions like methanol. In result those constructed sensory layer can be used for the detection of numerous substances in water like heavy metals and pharmaceuticals. Heavy metals will work as fluorescence quenchers and lead to a disturbance of FRET. As enhancement and improvement of sensitivity S-layer proteins can be further modified with functional molecules like aptamers which will be able to
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Biographies Ulrike Weinert has studied chemical engineering at the Dresden University of Applied Sciences from 2003 to 2008 and finished her studies as diploma engineer. She received a scholarship from the BMBF and worked as a scholarship student at the Helmholtz-Zentrum Dresden-Rossendorf from 2008 to 2009. From 2009 up to know she is a PhD student at the Helmholtz-Zentrum Dresden-Rossendorf at the department of Helmholtz Institute Freiberg for Resource Technology.
Katrin Pollmann, 40 years old (coordinator) studied Biology at the Universities Münster and Osnabrück from 1992 to 1998. In 2002, after graduating from PhD at the Helmholtz-Zentrum für Infektionsforschung in Braunschweig, Dept. Microbiology, she joined the Helmholtz-Zentrum Dresden Rossendorf. After successful evaluation of her Young Investigator’s Group “NanoBio” (Nanostructured biocomposites) in 2011, she established her group « Geobiotechnology » at the newly founded Helmholtz Institute for Resource Technology.
Johannes Raff has studied diploma degree in Microbiology in 1996 (University of Bayreuth) and PhD in Biochemistry in 2002 (Institute of Biochemistry, University of Leipzig). PhD thesis on the interaction of metals with bacterial surface layer proteins and its application. The scientific and application oriented work was awarded in 2002, 2007 and 2011. Since 2007 senior scientist at the Institute of Resource Ecology/Helmholtz Institute Freiberg and coordinator of several projects in the field of radioecology and bionanotechnology. Current focus on the interaction of metals with bioligands and application of bacterial surface layer proteins for the development of new filter materials, (photo)catalysts and sensors.
Contents lists available at SciVerse ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Fluorescence resonance energy transfer by S-layer coupled fluorescence dyes Ulrike Weinert a,∗ , Katrin Pollmann a , Johannes Raff a,b a b
Helmholtz-Zentrum Dresden-Rossendorf, Helmholtz Institute Freiberg for Resource Technology, Halsbrücker Straße 34, 09599 Freiberg, Germany Helmholtz-Zentrum Dresden-Rossendorf, Institute of Resource Ecology, Bautzner Landstraße 400, 01328 Dresden, Germany
a r t i c l e
i n f o
Article history: Received 27 February 2013 Received in revised form 8 May 2013 Accepted 15 May 2013 Available online 25 May 2013 Keywords: Fluorescence resonance energy transfer Surface layer proteins EDC Chemical modification Sensory layers Detection
a b s t r a c t In this paper two fluorescence dyes were coupled to surface layer (S-layer) proteins of Lysinibacillus sphaericus A12 and Lysinibacillus sphaericus B53 to easily generate a fluorescence resonance energy transfer (FRET). S-layer proteins are structure proteins which self-assemble in aqueous solutions, on surfaces and at interfaces forming 2D-paracrystalline structures with a defined symmetry in nanometer range. These properties and the fact, that a lot of modifiable functional groups are available on their surface, make them a perfect coating and binding matrix for the generation of functionalized surfaces, e.g. needed for a sensor assembly. Here we chemically link two fluorescence dyes, which are able to perform a FRET, to S-layer proteins by carbodiimide-crosslinking chemistry. Fluorescence dyes were coupled to the protein with a yield of around 54 mol%, demonstrating a modification of every second protein monomer if fluorescence dyes are statistical distributed. A FRET could be detected between the two fluorescence dyes when linked to protein polymers whereas no FRET could be detected if fluorescence dyes are linked to protein monomers. This demonstrates, that the polymer structure is essential for FRET and that fluorescence dyes are statisticaly distributed on protein polymers with a close proximity of donor and acceptor dye. Due to the fact that the used S-layer proteins build a unit cell of p4 symmetry, it can be assumed that two fluorescence dyes are linked to one unit cell. In this paper the FRET pair arrangement and its optimization is described in which the FRET efficiency can be increased from 6 to 40%, simply by varying the molar ratio of donor:acceptor. In result a sensory surface can be generated and used for detection of numerous substances in water like pharmaceuticals or heavy metals. © 2013 Elsevier B.V. All rights reserved.
1. Introduction 1.1. S-layer proteins as universal binding matrix S-layer proteins are structure proteins which are present in many bacteria and almost all archaea as outermost cell envelope. They were discovered by Houwink in 1953 when he saw a regular grid structure on freeze-etched bacteria cells of Spirillum serpens [1]. Although the chemical properties of bacterial S-layer proteins are very similar in different organisms their functions are quite diverse. They can serve e.g. as molecular or ion trap, as protecting layer or binding matrix for exoenzymes [2–4]. They cover the whole cell as paracrystalline structures with a defined symmetry and specific lattice parameters. If S-layer proteins are isolated, they maintain self-assembling properties and are able to autocatalytically form polymer lattice structures in aqueous solutions, on surfaces and at interfaces [5].
∗ Corresponding author. Tel.: +49 351 260 2012; fax: +49 351 260 3553. E-mail address: [email protected] (U. Weinert). 0925-4005/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2013.05.051
One further interesting property of S-layer proteins is their high content of COOH and NH2 -groups which are usually around 15 mol% [6]. Those functional groups can be easily modified by various crosslinkers e.g. 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and glutaraldehyde [7] In one example Protein A was successfully linked to S-layer proteins of Clostridium thermohydrosulfuricum L111-69 [8]. In another approach glucose oxidase was linked to S-layer proteins of Bacillus coaguluns E38-66 by glutaraldehyde [9]. In further work glutaraldehyde is used as stabilization reagent for S-layer protein’s polymer structure and amino group blocking. EDC is then used as crosslinking reagent to link various enzymes to S-layer proteins and build sensory layers up to biosensors [10,11]. Thereby activity of enzymes is dependent of the structure of enzymes [7]. In recently published work, fusion proteins composed of Slayers and different fluorescent proteins were produced. Such fusion proteins show sensitivity toward pH-change and could therefore be used as pH-sensor [12,13]. In another paper a bifluorescent fusion S-layer protein was constructed and an energy transfer with an efficiency of about 20% between both fluorescence dyes could be measured by steady state fluorescent measurements. FRET only occurs in this array if proteins are in their polymer form
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[14]. In this study S-layer proteins from isolates of a uranium mining waste pile near Johoanngeorgenstadt (JG) in Saxony, Germany were used, namely Lysinibacillus sphaericus JG-A121 (A12) and L. sphaericus JG-B53 (B53). The reassembled lattice of those S-layer proteins show a p4 symmetry with a unit cell size of about 13 nm. The S-layer protein of A12 was formerly used as sensor molecule for the detection of uranium [15]. The application shows that those S-layer proteins are long term stable in aqueous solutions. In this paper we describe a method to simply modify S-layer proteins by EDC chemistry without using glutaraldehyde for protein crosslinking. In result, stable S-layer proteins which are functionalized with a FRET pair are generated. The latter can be used e.g. in combination with specific binding molecules for sensor technology. 1.2. Fluorescence resonance energy transfer (FRET) based biosensors FRET is a universal tool in biochemistry for the monitoring of e.g. protein folding, protein interactions and structure changes. In all these applications FRET-induced changes in fluorescence is used to monitor these phenomena [16,17]. Challenging by using FRET-based sensors is to detect changes of the fluorescence correctly and to exclude misinterpretation [18–20]. According to Roda, there are five different methods commonly used to detect FRET. He claimed to prefer the determination of lifetime because the results are more reliable compared to all other methods [18]. Domingo and co-workers applied four different methods to detect FRET and calculate its efficiency using the fluorescent proteins CFP and YFP. Their results show that especially in unknown systems a standard for FRET detection is needed and at least two different methods should be used to get reliable results [19]. Or in other words Vogel and co-workers [20] provokingly said, that they contend that the popularity of FRET has risen in concert with degradation in the validity of the interpretation of biological FRET experiments. Their report specially refers to FRET measurements in living cells where protein interactions are studied and a “random FRET” of 5% can be always detected. But nevertheless the above statement applies to all FRET measurements. Therefore, we will concentrate our work not only on construction of a FRET-system on S-layer proteins but also on verifying an energy transfer between two immobilized fluorescence dyes. The aim of our work is to generate an energy transfer between the FRET pair HiLyteFluorTM 488 (H488) and HiLyteFluorTM 555 (H555). We linked both fluorescence dyes to surface layer proteins by carbodiimide chemistry. We examined different coupling strategies which include functionalization of NH2 or COOH groups, modification of S-layer protein monomers and polymers and we compared two S-layer proteins regarding their modification rate and their polymer stability after functionalization. In result we could find a modification maximum of around 0.54 moldyes /molS-Layer which means that two fluorescence dyes are found on one unit cell. Therefor we methodically analyzed fluorescence spectra and time resolved measurements, in order to exclude “random FRET signals”. In a second step we optimized the FRET efficiency of the linked fluorescence dyes by varying the donor to acceptor ratio. An assembly containing S-layer proteins and a protein bound FRET pair is suitable for further the construction of an improved sensory layer.
1 A12 – Lysinibacillus sphaericus JG-A12; B53 – Lysinibacillus sphaericus JG-B53; H488 – HiLyteFluorTM 488; H555 – HiLyteFluorTM 555.
2. Materials and methods 2.1. Functionalization of S-layer proteins with fluorescence dyes For all experiments S-layer proteins from strains L. sphaericus JG-A12 (A12) and L. sphaericus JG-B53 (B53) were used. Functionalization of S-layer proteins was done either by NH2 or COOH groups. As described by [21] A12 contains 103 NH2 groups (8.5 mol%) and 125 COOH groups (10.4 mol%). For B53 84 NH2 groups (9.5 mol%) and 94 COOH (10.7 mol%) are available in their primary structure [22]. Lyophilized S-layer proteins were dissolved either in 50 mM MES at pH 5.6 for a polymer suspension or in 50 mM MES pH 5.6 with additional fresh prepared 3 M Urea to get a monomer solution. Concentration of all samples was adjusted to 10 mg/ml with corresponding buffer solution. Modification of S-layer proteins with fluorescence dyes were performed by EDC chemistry or succimidyl ester chemistry. In both cases an amide bond between fluorescence dyes and S-layer proteins was created. Fluorescence dyes HiLyte FluorTM 488 hydrazide (H488), HiLyte FluorTM 555 (H555) hydrazide, HiLyte FluorTM 488 succimidyl ester (H488SE) and HiLyte FluorTM 555 succimidyl ester (H555SE) were purchased from MoBiTec GmbH (Göttingen) with HPLC purity. For coupling fluorescence dyes to S-layer protein’s NH2 group H488SE and H555SE with a molar ratio of 3:1 were used. S-layer proteins and fluorescence dyes with a succimidyl coupling group were mixed and incubated for two hours at room temperature in dark. The succimidyl ester will covalently bind to the amino group of the S-layer proteins. Fluorescence dyes H488 and H555, which contained a hydrazide group as binding group, were used for chemical modification of Slayer protein’s COOH groups. Modification was performed by EDC. Molar ratio of fluorescence dyes was 3:1 (donor: acceptor). Fluorescence dyes and proteins were mixed in a molar ratio of 2:1 (fluorescence dye: protein). 200 mM EDC dissolved in 50 mM MES pH 5.6 was added to proteins and fluorescence dye mixture to a final EDC concentration of 20 mM, were mixed gently and incubated for two hours at 4 ◦ C in dark. Modified protein polymer solution was centrifuged at 12,400 × g for 1 h at 4 ◦ C and washed twice in 50 mM MES pH 5.6 in order to remove unbound fluorescence dyes. Functionalized protein monomer solution was dialyzed against 10 mM CaCl2 solution overnight in order to further purify the protein. Dialyzed proteins were centrifuged at 12,400 × g for 1 h at 4 ◦ C. An occurring pellet of precipitated fluorescence dyes and denatured proteins was removed to get a supernatant containing only fluorescence labeled protein monomers. All labeled proteins were stored at 4 ◦ C in dark until further investigation. 2.2. Determination of the number of coupled fluorescence dyes In order to determine binding capacity UV/Vis measurements were performed with Nanodrop (Nanodrop2000, Thermo Fisher Scientific, Waltham, Massachusetts, USA). For measurements 10 l of samples were diluted with 10 l of 6 M guanidine hydrochloride solution and incubated for at least one hour. 2 l were used for measurements and all samples were measured at wavelengths 280 nm (protein concentration), 508 nm (H488), 504 nm (H488SE) and 552 nm (H555 and H555SE). Protein concentration was determined in the range of 0–5 mg/ml. Concentration of fluorescence dyes was determined by using Beer-Lambert law. The spectra of fluorescence dyes H555 and H555SE show an additional shoulder at a wavelength of 500 nm which interferes with the absorption of the donor dye. Therefore the absorbance at
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508 nm (A508 ) of H488 and H488SE has to be corrected (Acorr ) as follows: Acorr = A508 − (0.45 × A552 ) whereas A552 is the measured absorbance at a wavelength of 552 nm and 0.45 is an experimental determined correction factor. 2.3. Detection of FRET Steady state and time resolved fluorescence measurements were performed with the system Quantamaster 40-Q25 (Photon Technology International (Canada) Inc., London, Canada). 2.3.1. Steady state fluorescence measurements Samples were diluted 1:10 (v/v) in 10 mM CaCl2 to a final protein concentration of 0.1 mg/ml. Measurements were performed with a slit width of 2 nm. Samples were excited at two different wavelengths, 250 and 450 nm, to determine protein concentration and donor emission (H488 or H488SE) in diluted samples. 2.3.2. Time resolved measurements Samples were used in concentration of 10 mg/ml and slit width was 12 nm. For excitation of the donor, samples were excited at 450 nm and time-resolved emission was measured at 520 nm (donor emission). All samples were measured five times and an average of all 5 spectra was used for data analysis. In order to eliminate runaway values and to smooth data a Savitzky–Golay smoothing with 9 data point was performed with the average spectra [23]. Calculation of lifetime was performed by the following equation: y = y0 + A × exp(−x/) In which A is the amplitude and y0 the offset-correction. Determination of the fluorescence lifetime was achieved by a self-written program within the software of OriginLab Version OriginPro 8.6. OG Sr1. FRET-efficiency EFRET was then determined with the following equation: EFRET =
DA D
DA represents the lifetime of donor dye in presence of the acceptor and D represents lifetime of donor dye in absence of acceptor dye. 2.4. Optimization of FRET For optimization of the energy transfer different molar ratios of donor: acceptor were incubated with S-layer proteins of strain A12 or B53. Molar ratio ranges from 0.5 up to 10.5 (donor: acceptor). Thereby the volume of added fluorescence dyes was still the same but the total amount of added fluorescence dyes differed. In order to exclude false interpretation reference solutions containing only different molar ratios of H488:H555 in 10 mM CaCl2 were also investigated. 3. Results 3.1. Characterization of the FRET pair and its optimization Donor and acceptor dyes were mixed in different ratios and UV/vis-measurements were performed in order to calculate the total amount of donor and acceptor in solution and to verify the former introduced Eq. (1) for the calculation of H488 in solution in presence of the acceptor dye H555.
Fig. 1. Diagram of different molar ratios of donor and acceptor against fluorescence ratio of wavelength 525 and 565 nm, excitation wavelength is 450 nm, also shown is the moving average; Inlet: Emission spectra of donor and acceptor dye when excited at wavelength 450 nm.
Fluorescence measurements of various molar ratios of H488:H555 were performed in order to exclude a “random FRET” signal because of different signal intensities. Samples were excited at a wavelength of 450 nm and emission spectrum was detected. In Fig. 1 the emission ratio 525 to 565 versus the ratio of fluorescence dyes is plotted. Ratio 525/565 rises exponentially with rising ratio H488:H555. H555 shows a little emission at 565 nm when excited at a wavelength of 450 nm (Inlet Fig. 1). If no FRET occurs the plot should show a linear curve. Because of little self-quenching effect this curve is not linear. If FRET occurs a local minimum should have been detected. In addition, lifetime of H488 was also calculated for all used ratios. Results show an average lifetime of H488 of about 4.41 ± 0.09 ns independent of the amount of H555. In conclusion fluorescence and lifetime measurements show definitely no FRET between donor and acceptor dye if both fluorescence dyes are in solution.
3.2. Optimization of the FRET-pair binding to S-layer proteins and its influence on the S-layer stability For modification of S-layer proteins at their NH2 -groups, succimidyl ester activated fluorescence dyes were used. For modification of COOH-groups, hydrazide-modified fluorescence dyes were used for binding. Quantification of the amount of linked fluorophores to S-layer proteins where calculated with the help of Eq. (1). In result a modification rate of S-layer proteins in range of 0.15–0.6 moldye /molprotein could be determined. For an energy transfer a distance of less than 10 nm between donor and acceptor dye is necessary. If S-layer proteins build up unit cells, containing 4 monomers, or even polymer structures A modification rate of about 50 mol% will result in total distance of 6.5 nm between the FRET pair as one unit cell gains 13 nm. Thus a recrystallization of modified protein monomers is essential. Furthermore, functionalized S-layer proteins should be able to recrystallize on surfaces for later sensory applications. For this reason the recrystallization process of functionalized protein monomers was also investigated. In result, no recrystallization could be observed, even after two weeks. Only precipitated and denatured proteins could be detected. It can be thusly assumed that modification of S-layer protein monomers disturb recrystallization process. In comparison to that protein
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Fig. 2. Light microscopic image of modified S-layer protein polymers of strain A12; phase contrast and fluorescence images clearly show the typical polymer tubes.
polymers maintain their polymer structure after functionalization (see Fig. 2). The only exclusion was in case of the modification of S-layer’s NH2 -groups of B53 where nearly 50% loss of protein was detected (Fig. 3). Due to the fact that the reference sample, where S-layer proteins from strain B53 were not modified, have also a loss of about 50%, it is reasonable that the used buffer (50 mM MES, pH 5.5) system destabilizes their polymer structure. If S-layer proteins from strain B53 are cross-linked by EDC, no loss of protein polymer can be observed due to the fact that internal crosslinking stabilizes polymer structure. SDS-PAGE of functionalized S-layer proteins showed also a crosslinking as the proteins did not enter the separating gel and remain in the sample wells (Fig. 4).
sufficient evidence for an energy transfer. Influence of acceptor dye on the emission spectra cannot be excluded specially when an excess in acceptor dyes is available ([24] suppl. material).
3.3. Determination of FRET Results of steady state and time-resolved measurements can be seen in Fig. 5 and Table 1. All spectra were normalized to measured protein concentration. If FRET occurs, an emission peak should be detected in the range of 550–570 nm. Only polymeric proteins with modified COOH-groups show a clear second emission peak with a maximum at 575 nm. In other cases only a small shoulder arises, being no
Fig. 3. Protein concentration of the S-layer proteins of A12 and B53 after modification with fluorescence dyes at their NH2 or COOH groups, dashed line showed the original applied protein concentrations.
Fig. 4. SDS-PAGE: 10% SDS-gel of Protein polymers of strain B53 modified with a FRET pair at their (1) NH2 -group or (2) COOH-group, also shown is a protein ladder (3) with size markers at 200, 150, 120, 100, 85, 70, 60, 50, 40, 30, 25, 20 and 15 kDa; white arrows indicate crosslinked protein dimers and oligomers.
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Fig. 5. Emission spectra of S-layer proteins of A12 modified with a FRET-pair; excitation wavelength is 450 nm; monomer and polymers were modified either at their NH2 or COOH group.
In order to clearly demonstrate the energy transfer and to calculate the FRET-efficiency the lifetime of donor dye was determined in presence and absence of acceptor dye and efficiency was calculated according to Eq. (3) (Table 1). With this method the lifetime can be determined with an average error of 0.2 ns which represents nearly 5% of the total lifetime of the donor dye being 4.5 ns in 10 mM CaCl2 . According to that no energy transfer can be detected in case of modified protein monomers independently from protein or modified functional group. If protein polymers were modified at their COOH-group an energy transfer with an efficiency of 10% (A12) and 25.4% (B53) can be detected. If NH2 -groups of protein polymers from A12 were modified, energy transfer can be determined with an efficiency of 7.5%. Fluorescence spectra are consistent with this observed data (see Fig. 5). Furthermore, emission intensities at a wavelength of 525 nm are consistent with absorption spectra at a wavelength of 508 nm with exception of the samples showing a FRET. This linear behavior indicates that no self-quenching of donor dye occurs if it is linked to the protein. 3.4. Optimization of FRET efficiency
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Fig. 6. Plot of introduced molar ratio of donor and acceptor against experimental determined molar ratio of donor and acceptor linked to the protein polymers of B53 (triangle) and A12 (square); also shown is the 100% agreement (dashed gray line).
The fluorescent dye H488 thusly binds better to the S-layer protein than H555. Same results were obtained by modifying proteins either with donor or acceptor dye only. Modification rate of proteins modified with only the acceptor dye H555 is around 0.3 moldyes /molprotein and for proteins modified with only the donor dye H488 the rate is around 0.5 moldyes /molprotein . This result is possibly explainable by different structure and size of the fluorescence dyes. 3.4.2. Determination of FRET-efficiency Steady state and time resolved fluorescence measurements of all samples with a molar donor: acceptor ratio varying from 0.3 to 10.5 mol: mol were analyzed and compared (see Fig. 8). Data were analyzed by a polynomial fit and the local minimum represents the highest FRET-efficiency. In case of A12 a theoretical molar ratio of 6.5 (D: A) and in case of B53 a molar ratio of 6 (D: A) will result in the highest FRET efficiency. Lifetime measurements were performed in order to determine the FRET-efficiency. An increase of FRET-efficiency from roughly 10% (25.4%) to 40% could be reached by changing the molar ratio
3.4.1. Introducing different donor and acceptor ratios to S-layer proteins Protein polymers were modified with different molar ratios of donor and acceptor ranging from 0.2 to 10.5 in order to increase the energy transfer. Results of modification can be seen in Fig. 6. The diagram clearly shows that a molar ratio of linked donor and acceptor can be directly controlled. The fact of controlling the binding ratio was also proclaimed by Prasuhn et al. who modified Quantum dots with dye-labeled peptides by simple using EDC chemistry [25]. average modification rate is around The 0.54 ± 0.16 moldye /molprotein . Thereby it can be noticed that an increasing modification trend goes along with an increasing donor: acceptor ratio (Fig. 7).
Table 1 FRET-efficiency of FRET-pairs coupled to S-layer proteins in their polymeric or monomeric form of strains A12 and B53. sample
monomeric proteins
polymeric proteins
modification rate NH2 COOH
A12 1.3 0.3
A12 7.2 10.3
B53 4.9 2.4
B53 4.5 25.4
Fig. 7. Modification rate of S-layer protein polymers of A12 and B53 with a FRETpair with different molar ratios of donor and acceptor; gray dotted line shows the average modification rate and gray solid line shows standard deviation.
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Fig. 8. Plot of normalized fluorescence intensities of the molar ratio of donor and acceptor (D: A) against the fluorescence ratio at the wavelength 525 and 565 nm, excitation wavelength is 450 nm; Donor and acceptor were modified at the COOHgroups of protein polymers of A12 (square) or B53 (triangle), also shown are unbound fluorescence dyes in different molar ratios (circles).
within the Förster radius (10 nm). A higher modification rate of fluorescence dyes to the S-layer proteins is not mandatory because higher density of fluorescence dyes will cause self-quenching effects and reduces fluorescence intensity. Several papers claim that a modification rate of one up to three fluorescence dyes on protein in solution will result in the highest yield of fluorescence intensity versus protein concentration [26–28]. Another important aspect is the effect of a hydrophilic environment which can also quench fluorescence intensity. Most commercial available fluorescence dyes prefer a more hydrophobic environment like methanol or dimethyl sulfoxide. Fluorescence dyes linked to proteins and transferred into an aqueous environment, the quantum yield will decrease and they sometimes get quenched. If fluorescence dyes are linked to S-layer proteins, this effect could not be observed. A reason can be seen in the hydrophobic environment of the S-layer protein’s surface which will work as a protecting environment for the fluorescence dyes [26,29,30]. The S-layer proteins, which are used in this work, possess 40% of hydrophobic amino acids [21,22]. The S-layer proteins were chemical functionalized with an FRETpair producing a sensory layer, which can be used for the detection of molecules. One requirement for those molecules is their ability to quench fluorescence dyes in order to reduce the energy transfer. Another possibility for would be a genetically introduced FRET pair into S-layer proteins as it was done by Kainz et al. with a FRET efficiency of about 20% [14]. The chemical modification has several advantages over the genetic one: commercial available fluorescence dyes are more stable than GFP and likewise and show no pH-sensibility and high quantum yields. Furthermore a chemical functionalization will enable more flexibility concerning the molecules which can also work as sensory elements like quantum dots and aptamers. With the help of different crosslinkers it will be possible to sequential link those mentioned elements on the surface at the carboxylic, amino or hydroxyl group of S-layer proteins. And in case of our setup a functional protein layer can be produced within a few hours without special equipment.
5. Conclusion Fig. 9. Lifetime of the donor dye H488 linked to S-layer protein polymers of A12 or B53 at their COOH-group in absence and presence of different molar ratios of the acceptor dye.
from 3 to 4 (donor: acceptor). These results were obtained for both S-layer proteins A12 and B53 (Fig. 9). In summary an optimized protocol for the modification of Slayer proteins of A12 and B53 can be created as follows: Protein polymers are used and a FRET pair is linked to their COOH-group by EDC chemistry. S-layer proteins are then additional internal crosslinked to each other by EDC to gain more stability toward degradation to their monomer forms. Furthermore, a quadruplicate excess of donor dye to acceptor dye is used in order to achieve an energy transfer of approximately 40%. Determination of an energy transfer is confirmed by two measurements using static fluorescence and time resolved fluorescence. 4. Discussion Chemical modification of S-layer proteins with a FRET pair yielded in a modification rate of about 0.54 ± 0.16 moldye /molprotein. This means that two fluorescence dyes are linked to one unit cell which consists of four protein monomers. The distance between donor and acceptor can be estimate as 6.5 nm which is clearly
In this paper S-layer proteins of the two wild strains L. sphaericus JG-A12 and L. sphaericus JG-B53 were chemically modified with a FRET-pair by EDC by their COOH and NH2 groups. We systematically optimized the modification procedure in order to gain a high FRET efficiency. Therefore we also established a procedure for a secure FRET detection without misinterpreting. We were able to reach a FRET efficiency of nearly 40% by a quadruplicate excess of acceptor dye. Thereby a modification rate of 0,54 moldye /molprotein could be reached which means that two fluorescence dyes are linked to one unit cell of the S-layer lattice. The advantage of a chemical modified S-layer protein over a genetically introduced FRET-Pair lies in the higher stability of the fluorescence dyes, the possibility to link other molecules to the S-layer proteins like quantum dots or aptamers and to sequential bind different elements on a surface. The hydrophobic environment of the S-layer protein will protect the fluorescence dyes from quenching effects by aqueous solutions and buffer changes. Most fluorescence dyes are hydrophobic and gain the highest fluorescence yields in organic solutions like methanol. In result those constructed sensory layer can be used for the detection of numerous substances in water like heavy metals and pharmaceuticals. Heavy metals will work as fluorescence quenchers and lead to a disturbance of FRET. As enhancement and improvement of sensitivity S-layer proteins can be further modified with functional molecules like aptamers which will be able to
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Biographies Ulrike Weinert has studied chemical engineering at the Dresden University of Applied Sciences from 2003 to 2008 and finished her studies as diploma engineer. She received a scholarship from the BMBF and worked as a scholarship student at the Helmholtz-Zentrum Dresden-Rossendorf from 2008 to 2009. From 2009 up to know she is a PhD student at the Helmholtz-Zentrum Dresden-Rossendorf at the department of Helmholtz Institute Freiberg for Resource Technology.
Katrin Pollmann, 40 years old (coordinator) studied Biology at the Universities Münster and Osnabrück from 1992 to 1998. In 2002, after graduating from PhD at the Helmholtz-Zentrum für Infektionsforschung in Braunschweig, Dept. Microbiology, she joined the Helmholtz-Zentrum Dresden Rossendorf. After successful evaluation of her Young Investigator’s Group “NanoBio” (Nanostructured biocomposites) in 2011, she established her group « Geobiotechnology » at the newly founded Helmholtz Institute for Resource Technology.
Johannes Raff has studied diploma degree in Microbiology in 1996 (University of Bayreuth) and PhD in Biochemistry in 2002 (Institute of Biochemistry, University of Leipzig). PhD thesis on the interaction of metals with bacterial surface layer proteins and its application. The scientific and application oriented work was awarded in 2002, 2007 and 2011. Since 2007 senior scientist at the Institute of Resource Ecology/Helmholtz Institute Freiberg and coordinator of several projects in the field of radioecology and bionanotechnology. Current focus on the interaction of metals with bioligands and application of bacterial surface layer proteins for the development of new filter materials, (photo)catalysts and sensors.