University of Groningen
Single-molecule fret study on structural dynamics of membrane proteins Aminian Jazi, Atieh
DOI:
10.33612/diss.135802718
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Publication date: 2020
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Aminian Jazi, A. (2020). Single-molecule fret study on structural dynamics of membrane proteins. University of Groningen. https://doi.org/10.33612/diss.135802718
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Chapter 2:
Fluorescence labelling of membrane
transporters and isolated protein domains
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2. Novel approaches for fluorescence labelling of membrane transporters and isolated protein domains
Abstract:
FRET has become a widely used method in combination with single-molecule detection to investigate protein conformational dynamics and heterogeneity. One of the main factors limiting mechanistic insights are poor photophysical properties of dyes and the ne ed for specific attachment strategies of fluorophore to the biomolecule [7]. Site-specific labelling is a crucial step in order to investigate biological systems with smFRET-based single-molecule methods, since it needs to map a relevant reaction coordinate of the system [21]. For proteins, cysteines are artificially introduced at non-conserved and solvent-exposed positions as anchor points for the fluorescent labels. While this strategy has various problems that deal with the molecular biology and preparation of the protein mutants (and their functionality), it is further complicated in multi-subunit proteins such as BetP since one cysteine (in one subunit) appears multiple times in the complex; thus multiple labelling sites are created. Designing an appropriate strategy for site-directed mutagenesis and further conjugation of fluorophores with optimized photophysical properties for FRET will allow to obtain more mechanistic insights. This chapter presents attempts to label homo- and heterotrimeric cysteine constructs of the membrane protein BetP for smFRET studies. Finally, I show labelling of ABC-transporter domains from the amino-acid importer GlnPQ as test systems for characterization of photostabilizer-dye conjugates. With this I established methods to mechanistically study the osmotic stress regulated sodium-coupled betaine symporter BetP. The long-term goals of our studies were monitoring catalytic movements of BetP during substrate transport under up-regulated conditions.
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2.1 Site-specific labelling strategies for smFRET studies of BetP
Atieh Aminian Jazi, Reinhard Krämer, Christine Ziegler & Thorben Cordes(unpublished)
Cysteine labelling of homotrimic BetP: We first describe our studies on improving the labelling of different BetP cysteine variants with high specificity and efficiency for studies of conformational dynamics in BetP. For this purpose, we first selected and characterized the residues of BetP that allow to establish reactive movements of the protein, then we identified non-conserved residues in cytoplasmic and periplasmic side for label attachment. Also, solvent accessibility of the residues within the protein sequence was determined by using the SWISS-MODE server on crystal structures.
Amino acids at positions, which neither affected functionality when mutated nor w ere considered to be involved in conformational changes were replaced by cysteine residues by site -directed mutagenesis. BetP variants were constructed on a cysteine -less BetP (C252T, TM5) as an initial template [26]. Then we demonstrated that the selected mutations had no critical effect on the transport function and activation profile of BetP. For labeling, disulfide bonds are formed in the interaction between sulfhydryl groups of the cysteine residue and commercially available fluorophore-maleimide conjugates. To obtain optimal results we investigated different fluorophore labeling techniques and biochemical experimental approaches to obtain BetP with higher degree of labelling. For this purpose, BetP was first solubilized in DDM detergent and purified via StrepTactin-affinity chromatography (Figure 2.1). We then conducted the FRET labeling (Figure 2.1C). The data shown in Figure 2.1 are the result of a long optimization process. For this we performed the set of experiments in combination with quantitative analysis for varying labeling conditions including variations of buffers, pH values, reducing agents and salts, different incubation times, temperature and distinct molar ratios of protein to fluorophores during labeling. In the first iteration of our final protocol we used 50mM Tris pH7.4 containing 0.05%-0.1% n-dode cyl β-D-maltoside (DDM), during purification of a Strep-Tactin® Macro-Prep column via the gravity flow method. To this a fluorophore mixture (of donor and acceptor-dye) were applied to immobilized BetP. The eluent fraction was subject to SEC-analysis and
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revealed a high amount of non-specific dye-interactions with the column material. The amount of non-specific interaction increased also for hydrophobic fluorescent FRET probes such ATTO555 (Donor) and ATTO647N (Acceptor). Labelling with resin-immobilized BetP turned out to be impractical.
Figure 2.1| overexpression, protein purification and fluorophore labelling of BetP wild type and mutants.A)
Western blot of BetP expression within E. coli cells, DH5αTM-T1R E. coli competent cell were transformed and were cultivated in LB media supplemented with 50 µg/ml carbenicillin. Protein production of BetP was induced through the addition of 200 µg/l Anhydrotetracycline hydrochloride (AHT) at an Optical Density (OD) of 600 of 0.5 and then cells were harvested for analysis after 2 hours. As marker Page Ruler Prestained Protein Ladder (Thermo Scientific Molecular Biology) was used, Lane 1: loaded cells before AHT cell induction. Lane 2: loaded cells after +2 hours AHT induction. B) BetP protein purification SDS-PAGE analysis. Solubilized membrane (10 mg/ml) were loaded onto an SDS-PAGE (12.5 %). Samples collected during purification and were examined by SDS PAGE and Western blot analysis and Each well was loaded with different fractions collected from Strep Tag purification. BetP wild type protein purification (left picture) and L516C_Transmembrane homotrimeric mutant SDS-PAGE (right picture). C)
Size-29
exclusion chromatography profile of labelled Betp_L516C mutant shows a single protein peak at 280 nm (blue)and absorptions at 550 nm and 660 nm(red) were monitored and corresponds to labelled protein with FRET donor fluorophore Alexa 555 and FRET Acceptor ALEXA 647N fluorophore. Protein was injected on a Superose 6 GE 10/300 column with a constant flow rate of 0.5ml/min and protein absorption was monitored at 280 nm. Green box shows selected labelled protein fraction for further smFRET analysis. D) Size-exclusion chromatography profile of labelled Betp_K489C mutant shows a single protein peak at 280 nm (blue)and absorptions at 550 nm and 660 nm(red) were monitored and corresponds to labelled protein with FRET donor fluorophore Alexa 555 and FRET Acceptor ALEXA 647N fluorophore. Green box shows selected labelled protein fraction for further smFRET analysis.
Subsequently, we tried to label the fresh purified protein and removed unbound labels via dialysis methods. For this, we performed series of dialysis experiments to remove the reducing agent dithiothreitol (DTT) used for stabilization of the protein and its cysteins followed by fluorophore incubation of the protein-to-dye ratio of Acceptor and donor 5:4 for ~4 nmol of protein in presence of 0.1mM, 0.5mM and 1M DTT in a deoxygenated reaction buffer. Subsequen tly, the unreacted dyes were washed with sequential dialysis steps to stop cross-reactions of DTT with the maleimide moiety. DTT prevents the interaction of thiol-specific reagents with the cysteine in further steps. The size-exclusion chromatography of labeled BetP protein revealed that the labeling efficiency was increased compared to convenient methods yet indicated protein aggregation. Furthermore, we replaced DTT by TCEP (tris(2-carboxyethyl) phosphine) and also protein immobilization on Strep tag column with desalting method as alternatives for smFRET labeling of BetP.
As a final result of our optimization process, we established a fast and reliable method to label BetP with a minimum incubation time using desalting methods in combination with an optimized fluorophore to protein ratio. This desalting technique aims at removing buffer salt, reducing agent and additional additives directly from purified protein, which was solubilized in detergent or amphipol, in a fast exchange for water. The key for success was to quickly desalt the freely-diffusing protein and to fully remove reducing agent, to allow a start of the sulfhydryl-reaction prior to cysteine-oxidation. Compared to the dialysis method, this approach has the advantage of a much faster reaction, i.e., within a few hours and higher stability of protein. The details of the protocol are provided in the material and methods parts of chapter 3/4.
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After labelling was established for BetP cysteine-mutants, we monitored catalytic movements of BetP during substrate transport. This was done by using structural information on BetP to identify residues that move significantly during the isomerization from inward-facing (IF) to outward-facing (OF) state in the alternating access cycle. As one example we used the d ouble-cysteine mutant S140C/K489C to monitor catalytic activity of BetP via smFRET (Figure 2.2). Postion S140 is located at the cytoplasmic start of TM3, which carries the so-called glycine stretch of BetP. Structure in OF and IF showed that this part of TM3 is displaced by about 6 Å. K489 is located at the beginning of TM11, which is part of the coupling scaffold. This residue is discussed as a lipid interaction side and therefore anchored in the membrane. Structural comparison of IF and OF state suggests that this residue is not moving during alternating access. This mutant was already used in PELDOR measurements and exhibits a WT-type like activation profile. The top view of the BetP crystal structure (Figure 2.2A/B) reveals the challenges of the FRET approach in this particular BetP double-cysteine mutant. The most reduced activity was detected for BetP S140C/C252T/K489C, although normalized data indicated that regulation of the transport for this BetP mutant is still maintained (Figure 2.2D). Because the replacement of K489 in this mutant of BetP seemed to have an impact on the transport activity level, while BetP S140C/C252T alone exhibit only a slight reduced uptake rate compared to the BetP WT. Since the protein is expressed and purified as a homotrimer, the two cysteine residues appear in each subunit. Stochastic labelling with donor and acceptor fluorophore, here Alexa555 and Alexa647, results in FRET histograms that cannot be easily interpreted (Figure 2.2C).
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Figure 2.2| Problems in smFRET investigations of multimeric proteins. Crystal structure of BetP in the double
cysteine context S140C/C252T/K489C. PDB: 4AIN. (A) Side view showing the planar arrangement of BetP with its three monomers (one highlighted in blue). (B) Top view of the crystal structure showing the trimeric structure of BetP. Positions of cysteines and (selected) distances between them are indicated; note that many more distances in particular between neighbouring monomers are relevant and only some selected ones are displayed. (C) 2D ALEX histogram of Alexa555/647-labelled BetPS140C/K489C showing the convolution of FRET interactions and difficulties to use this data for structure analysis. (D) Normalized Uptake measurements of [14C]-labeled glycine betaine as substrate for two BetP-Cysteine mutants S140C/K489C and S516C substrates in E. coli MKH13 in dependence of osmotic stress as explained previously (Ott et al., 2008). uptake rate of two BetP-Cysteine mutants S140C/K489C and S516C in E. coli cells The relative uptake rate of [14C]-labeled betaine in E. coli cells for wild type protein (green circle) is comparable to the one of both mutant proteins (blue: S140C/C252T/K489C; red C252T/S516C), which exhibit 1/3 of total wild type activity.
We then used µs-ALEX (microsecond alternating-laser excitation), where fluorescently-labeled biomolecules diffuse through the excitation volume of a confocal microscope, for smFRET studies of BetP. During their diffusional transit the labelled protein produces fluorescent bursts in two distinct detection channels that are chosen to selectively monitor donor and acceptor emission. In ALEX, green excitation of the sample generates fluorescent signals that allow calculation of apparent FRET via photon streams DD excitation, donor emission) and DA (donor-excitation, acceptor-emission) as E* = DA/(DD+DA). The stoichiometry S is obtained from the ratio of total fluorescence in both channels during green excitation to the total fluorescence during green and red excitation S= (DD+DA)/(DD+DA+AA); AA, acceptor excitation, acceptor emission.
In ALEX brightness ratio S (Y-axis in Figure 2.2C) distinguishes molecular species by their labelling ratio of green to red fluorophores. Low S < 0.2 is indicative of acceptor-only labelled protein,
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while high S > 0.8 corresponds to a donor-only species. Macromolecules containing both dyes are found at S values between these two boundaries (Figure 2.2C, 0.2 > S > 0.8). It is apparent that fluorophore interactions via FRET are highly complex since each protein has six possible labeled positions resulting in a mix of label compositions (heterogeneity of labelled species, Figure 2.2B). In such a scenario the relation of FRET efficiency and interprobe distance R is lost due to the ambiguous interaction of e.g., multiple donor with multiple acceptor fluorophores or signal loss via homo-FRET and energy dissipation. For homotrimeric variants of BetP we had to restrict ourselves to study single cysteine mutants, for which still problems with multiple distinctly labelled species arise. A solution to these problems is detailed in chapter 3.
Protein purification and fluorophore labelling of heterotrimeric BetP:
While problems related to convoluted smFRET distributions with homotrimeric proteins with one cysteine can be solved via photochemical tricks and data sorting (see chapter 3), double-cysteine mutants such as S140C/K489C required a different strategy. More recently, a heterotrimer approach was used as an interprotomeric crosstalk in regulation (Becker et al., 2014). Luckily, for BetP a specific purification strategy was developed by the Krämer lab and conducted in our lab (Figure 2.3 for Heterotrimeric BetP overexpression and purification) that resulted in a heterotrimeric protein complex, which we utilized here to label one protomer specifically for smFRET.
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Figure 2.3| Heterotrimeric BetP overexpression, purification and fluorophore labelling of heterotrimer BetP smFRET of successfully purified and labelled heterotrimeric construct. A) Western blot analysis of Heterotrimeric
BetP Sterp tag purification. Protein production of Heterotrimeric BetP were induced through the addition of 200 µg/l Anhydrotetracycline hydrochloride (AHT) at an Optical Density (OD) of 600 of 0.5 and then cells were harvested for analysis after 2 hours. As marker Page Ruler Prestained Protein Ladder (Thermo Scientific Molecular Biology) was used. B) Western blot analysis of Heterotrimeric BetP His tag protein purifications. C) Western blot analysis of Heterotrimeric BetP FLAG tag protein purification. D) Heterotrimeric BetP protein purification SDS PAGE (12.5 %). analysis. Solubilized membrane (10 mg/ml) were loaded onto an SDS PAGE Samples collected during purification and were examined by SDS-PAGE after Strep tag, His tag and FLAG tag purifications. E) protein structures of Heterotrimeric BetP (S140C/K489C) with label positions of donor and acceptor, PDB=4C7R. F) Corresponding two-dimensional histogram (2D) of Heterotrimeric BetP ( S140C/K489C) with Alexa555/Alexa647 FRET pair with values of S (labelling stoichiometry, dashed lines 0.4 < S < 0.6 donor-acceptor population DA) and Apparent FRET values of
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Biochemical activity and functionality of labelled BetP: Another important point of consideration relates to the biochemical function of the protein after fluorescent labelling, i.e., the labels may interfere with proper folding or function of the transporter. Thus, assays are needed that directly compare the protein activity and its degree of labeling as control experiments to conduct a relevant biophysical study. In Figure 2.2D, the normalized uptake rates of two BetP-Cysteine mutants S140C/K489C and S516C are shown in dependence of osmotic stress. We found a degree of FRET labelling of 76% for homotrimeric mutant BetP S140C-k489C and 40% for homotrimeric mutant L516C (See Figure 2.1 C, D). The relative uptake rate of [ 14C]--labeled glycine betaine for wild type proteins (green circle) was comparable to the one of both mutant proteins (See Figure 2.2 D, blue: S140C/C252T/K489C; red C252T/S516C). (Ultimately, the quality of the final FRET data is not only related to the functionality of the protein, but also to the degree of labeling and the percentage of molecules containing both the donor and the acceptor dye, since only those provide desired FRET information. Especially for smFRET studies, these two requirements, i.e., high labeling efficiency and retained biochemical functionality are challenging hurdles.
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2.2 smFRET studies of substrate-binding proteins with photostabilizer-dye conjugates
Jasper HM van Der Velde, Jens Oelerich, Jingyi Huang, Jochem H Smit, Atieh Aminian Jazi, Silvia Galiani, Kirill Kolmakov, Giorgos Gouridis, Christian Eggeling, Andreas Herrmann, Gerard Roelfes, Thorben Cordes* (published in Nature Communications 2016)
A major limitation of smFRET experiments in general is related to currently available commercial fluorophores. The achievable count-rates and photobleaching properties of commercial fluorophore are restricting both spatial and temporal resolution of smFRET and other experiments. I will here characterize photostabilizer–dye conjugates as FRET donor-acceptor pairs in proteins.While synthetic organic fluorophores have been a major driving force for the recent success of fluorescence-based methods, they intrinsically suffer from transient excursions to dark states (blinking) and irreversible destruction (photobleaching) [8]. Both processes fundamentally limit their applicability and have, for a long time, hampered the development of advanced microscopy techniques with single -molecule sensitivity [9][10] or optical super-resolution <250 nm[11][12]. Lüttke and colleagues [13] introduced covalent binding of triplet-state quenchers and singlet-oxygen scavengers [9] to organic fluorophores as a strategy to reduce the above mentioned effects. Such photostabilizer–dye conjugates with intramolecular triplet-state quenching have ‘self-healing’[14] or ‘self-protecting’[15] properties, preventing photodamage without the use of solution additives. This non-invasive strategy has clear advantages compared with commonly used approaches, where micro- to millimolar concentrations of organic compounds are added to the biochemical buffer system.
We here establish the use of rhodamine- and carbopyronine dyes in conjugation to photostabilizers as FRET dyes. (S)-Nitrophenylalanine, NPA was used as a scaffold for conjugation of a commercially available organic fluorophore (RhodamineB, ATTO647N and KK114), the photostabilizing p-nitrophenyl group [16][17] to a biomolecular target. Using maleimide chemistry, we facilitated the use of NPA-based photostabilizer–dye conjugates for direct labelling of proteins. For this purpose, two different synthesis strategies were
36
developed, accounting for the available quantity of the fluorophore. In the most straightforward case, that is, larger amounts of amine-reactive photostabilizer–dye conjugate are available, the NHS ester of the fluorophore (Figure 2.4, compound 17) can be coupled directly with 2-maleimidoethylamine (Figure 2.4 , compound 19), to yield a maleimide derivative of, for example, NPA–RhodamineB (Figure 2.4, compound 20).
Figure 2.4| Synthesis of reactive photostabilizer–dye conjugates of RhodamineB for direct labelling of primary
amines and thiol residues. The strategy can be extended to other biochemical targets by a varying the linker of molecule. Adapted from van der Velde et al., Nature Communications 2016.
The second strategy is also feasible for small quantities of reactive fluorophore species (<10 mg) that could be due to high prices of commercially available precursors or complicate d synthesis. ATTO647N is a good example of a fluorophore that is often used in demanding fluorescence applications but is not readily available in large amounts. For these cases we optimized the synthesis, to yield a thiol-reactive derivative of ATTO647N containing the photostabilizer NPA (Figure 2.5, compound 25). As shown below both maleimide derivative s can covalently bind to recombinant proteins via solvent-exposed cysteine residues (Figure 2.4, compound 21 and Figure 2.5, compound 26).
To show the benefits of intramolecular photostabilization in fluorescence applications with proteins, we studied the substrate-binding domain 2 (SBD2) of the Lactococcus Lactis type 1
ABC transporter GlnPQ [18]. Using smFRET and alternating laser excitation (ALEX)
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states of the protein were monitored (see Figure 2.6,a and b, open unliganded and closed liganded states of protein). The structural rearrangements of SBD2 on ligand binding cause a change of ∼0.9 nm regarding the distance between two selected amino acids in the protein [18]. As the FRET donor and acceptor fluorophore are attached via male imide chemistry at
these positions in the protein (mutant of SBD2: T369C/S451C), the transfer
efficiency E*reports on the conformational state of the protein, i.e., open or closed. As described previously [18], SBD2 was labelled stochastically using appropriate mixtures of donor and acceptor fluorophores.
Figure 2.5| Simplified synthesis of reactive photo stabilizer–dye conjugates where only small quantities of
fluorophore are available. The resulting NPA–ATTO647N conjugate can be used for direct labelling of thiol residues, for example, in proteins (compound 25). The strategy can be extended to other biochemical targets by a variation of the linker molecule 19.
To understand the effects of intramolecular photostabilization in FRET-based assays, we used different fluorophore combinations: Cy3B or RhodamineB as donor fluorophores and red -emitting dyes such as ATTO647N and KK114 as the acceptor fluorophores. In experiments described further, either the donor (Cy3B or RhodamineB) or the acceptor (ATTO647N or KK114) was stabilized via covalent linkage to NPA. We used smFRET/ALEX, which allows to distinguish the desired proteins labelled with both donor and acceptor (do nor-acceptor) from those labelled with only donor (donor-only) or only acceptor (acceptor-only). Therefore, the
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fluorescence emission of the donor-only (F(DD)), that of the acceptor when excited via FRET (F(DA)) and directly via red excitation light (F(AA)) is determined. In smFRET experiments, individual biomolecules are studied for short time periods of a few milliseconds while diffusing through the excitation volume of a confocal microscope. The challenge of such an experiment is to acquire intense fluorescent bursts during the short observation time under the required excitation intensity of 20-100 kW/cm2.
Figure 2.6| (a) Crystal structures of the SBD2 (T369C/S451C) open (left panel, PDB: 4KR5) and closed state (right panel, PDB:4KQP, after binding of the ligand glutamine shown in red) with label positions of donor (D) and acceptor (A). (b) Corresponding one-dimensional histograms of E*-values for increasing amounts of ligand
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glutamine where an increased E*=0.55±0.1 (dashed line, closed conformation) becomes visible (as opposed to the open conformation, E*=0.36±0.1, dashed line). Excitation intensities of 30 kW cm−2 at 532 nm and 20 kW cm−2 at 640 nm; data evaluated with dual colour burst search and displayed with additional per -bin thresholds of F(DD)+F(DA)+F(AA)>75 and minimal number of counts per bin in ALEX histogram of 3. (c,d,e) 2D histograms of joint pair values of S (labelling stoichiometry) and E* (FRET efficiency, that is, interprobe distance) of Cy3B/ATTO647N in the presence (c) and absence (d) of 2 mM TX and Cy3B/NPA–ATTO647N (e) without ligand glutamine. Excitation intensities of 30 kW cm−2 at 532 nm and 20 kW cm−2 at 640 nm; data evaluated with all photon burst search and displayed with additional per -bin thresholds of F(DD)+F(DA)+F(AA)>100 and minimal number of counts per bin in ALEX histogram of 3. (f,g,h) Histogram of fluorophore brightness values as determined from photon-counting histograms (PCHs) on single-molecule transits of labelled SBD2 diffusing through the observation volume, comparison of donor brightness F(DD) (f), acceptor brightness when excited via FRET, F(DA) (g) and acceptor brightness via direct red excitation F(AA) (h). Excitation intensities of 30 kW cm−2 at 532 nm and 20 kW cm−2 at 640 nm. (i,j,k) Dependence of the mean count rate of F(DD), F(DA) and F(AA) of the different samples for increasing excitation intensity, respectively.
Results of such quality are, however, only available when using 2mM Trolox as a photostabilizer in FRET measurement solution, seen from comparison in Figure 2.6. Here we show data of apo-SBD2 protein (labelled with Cy3B/ATTO647N) in the presence and absence of Trolox compound in buffer solution. In agreement with Kong et al. [10], the high excitation intensities used in our experiments promote acceptor signal fluctuations, that is, blinking and/or bleaching. Cy3B and ATTO647N can hence be seen as a FRET couple where the acceptor photostability is limiting. This appears as a prominent bridge between the donor-only and donor-acceptor population (see Figure 2.6 d), altering both E*/S-values substantially. Closer inspection of the FRET histogram reveals that a significant portion of the molecules show these unwanted photophysical effects. Under these conditions, neither mean E* nor correction factors for accurate FRET determination are directly accessible. Besides the complete loss of information, the overall acquisition time has also increased in the absence of photostabilize r to obtain sufficient statistics from the relevant donor–acceptor species. It should be noted that such photophysical artefacts of the acceptor dye (Figure. 2.6d) are extremely problematic for data interpretation, as they suggest the existence of (non-biological) species in between the donor only and the actual FRET species (see 1D−E* in (Figure. 2.6d) that can only be fitted by the sum of two Gaussians with E*=0.19±0.07 and 0.43±0.1). Furthermore, it remains challenging to separate those two FRET species from D-only molecules when performing smFRET with green excitation in continuous-wave mode.
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Strikingly, the bridge population can be removed by sole photostabilization of the acceptor
fluorophore via scaffolding of ATTO647N to NPA. The ALEX data of apo-SBD2 with Cy3B/NPA–
ATTO647N are shown in Figure 2.6e. Here the bridge between the donor–acceptor and donor-only population is fully removed without the addition of stabilizer to the solution (Figure 2.6d versus Figure 2.6e). The resulting mean E*/S values are changed compared to apo-SBD2 with Cy3B/ATTO647N (2mM Trolox in solution, Figure. 2.6c) accounting for decreased acceptor brightness; mean E* is now found at 0.34±0.09 and mean S at 0.67±0.08 (Figure. 2.6e). These differences are, however, expected considering the results from ATTO647N on DNA scaffold, where a decrease in the overall brightness is observed on conjugation to NPA to ATTO647N fluorophore. It should be mentioned that differences in the donor–acceptor population relative to donor and acceptor only comparing the samples Cy3B/ATTO647N and Cy3B/NPA–ATTO647N are not solely due to photophysics but also due to different labelling ratios of dyes and protein.
Next, we used Cy3B/NPA–ATTO647N to study the biomolecular function of SBD2 (see Figure.
2.6b). Upon addition of the ligand glutamine, the mean E* changes from 0.36±0.1 (open, interprobe distance of ∼4.9 nm) to 0.55±0.1 (closed, with a decreased interprobe distance of ∼4.0 nm). A concentration of 200 μM glutamine saturates the ligand binding and therefore results in a 100% population of the closed state of protein ( Figure. 2.6b). A ligand concentration of 1 μM, which is close to the Kd-value of the protein, consequently results in a mixture of open and closed conformational states of protein ( Figure. 2.6b)
As fluorophore brightness and the resulting photon budget ultimately determine the quality of the final histograms including the necessary measurement time, we quantitatively analysed
Cy3B/ATTO647N and Cy3B/NPA–ATTO647N by means of photon-counting histograms. Data in
Figure 2.5 f,g,h shows the three relevant photon streams used to determine E* and S -values of Cy3B/ATTO647N (donor–acceptor: 0.9>S>0.4; bridge: 0.9>S>0.7) and Cy3B/NPA–ATTO647N (donor–acceptor: 0.9>S>0.4). F(DD) shows that the strongest donor quenching and hence the most efficient FRET is found for the addition of TX to Cy3B/ATTO647N, whereas molecules in
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the bridge (Cy3B/ATTO647N, no TX) show inefficient donor quenching due to a non-functional acceptor (Figure. 2.5 f). Cy3B/NPA–ATTO647N and healthy labelled molecules from the Cy3B/ATTO647N population with no addition of Trolox show a similar performance of the acceptor-based donor quenching. Then F(DA) correlates directly with quenching in F(DD) as seen in Figure 2.5g. Again, the best performance is observed from Cy3B/ATTO647N in the presence of Trolox, while molecules in the bridge show the lowest counts. A striking difference between the bridge and all other conditions is seen in F(AA), that is, direct excitation of the acceptor (Figure. 2.6h). The overall comparison suggest that NPA-based acceptor stabilization is sufficient to remove photophysical artefacts and hence make the smFRET of these pairs useful for biomolecular studies without addition of Trolox. Further optimization of the data quality could, however, be obtained by additional donor stabilization of, for example, Cy3B in this case. This interpretation is supported by excitation intensity-dependent count rates of
Cy3B/ATTO647N and Cy3B/NPA–ATTO647N (Figure. 2.6 i,j,k). NPA-based acceptor
stabilization improves the saturation characteristics in all three channels but the addition of Trolox to the solution (resulting in stabilization of both donor and acceptor fluorophores at the same time) remains superior in terms of achievable count rates.
To study the donor dependence in more detail, we repeated the described experiments using RhodamineB/ATTO647N and NPA–RhodamineB/ATTO647N. Here, the photostability of RhodamineB as donor is the limiting factor for smFRET data quality. For RhodamineB/ATTO647N we find prominent donor blinking in the absence of photostabilize r (see Figure 2.7). The bridge between the donor–acceptor and acceptor-only population can be removed by addition of Trolox in solution (Figure 2.7) or conjugation of RhodamineB to NPA (Figure 2.7). The overall magnitude of the observed effects is lower than for the case of acceptor bleaching/blinking shown in Figure 2.6. Photon-counting histograms and the intensity dependence show a similar trend as before, that is, correlation between F(DD) and F(DA) with the wish to increase donor photostability as much as possible. Our data makes clear that NPA–RhodamineB can be used at significantly higher excitation intensities than
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RhodamineB, as triplet-state population was minimized and would allow for faster and better FRET related data acquisition for protein investigation (Figure 2.7).
Figure 2.7. a/b/c| Two-dimensional histograms (2D) of joint pair values of S (labelling stoichiometry, dashed lines
0.42 < S < 0.75 donor-acceptor population DA) and E* (FRET-efficiency, i.e., interprobe distance) of Cy3B/ATTO647N in the presence (a) and absence (b) of 2mM Trolox, and Cy3B/NPA-ATTO647N (c) without ligand glutamine. Excitation intensities of 30 kW cm-2 at 532 nm and 10 kW cm-2 at 640 nm; data evaluated with all photon burst search and displayed with additional per -bin thresholds of F(DD)+F(DA)+F(AA)>150 and minimal number of counts per bin in ALEX histogram of 3. d/e/f) Histogram of fluorophore brightness values as determined from PCH on single-molecule transits of labelled SBD2 protein diffusing through the observation volume of our confocal microscope: comparison of donor brightness F(DD) (d) acceptor-brightness when excited via FRET, F(DA) (e), and acceptor-brightness via direct red excitation, F(AA) (e). Excitation intensities of 30 kW/cm2 at 532 nm and 20 kW/cm2 at 640 nm. Note the varying stoichiometry ranges used for data evaluation using only bursts for the PCH corresponding to 0.75<S<S.
Our results show that the simple addition of the NPA unit to the acceptor fluorophore is sufficient to gather reliable results from solution-based smFRET experiment (Figure 2.6). Although the addition of a stabilizer on the donor fluorophore can increase the overall
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available photon budget (which should always be maximized), the acceptor strictly requires a solution-based or covalently linked photostabilizer. We consequently suggest to stabilize the acceptor as a minimum and (if possible) also to stabilize the donor to maximize available count rates and hence increasing the data quality. We finally note that all smFRET results of SBD2 with the different pairs of fluorophores shown in this work are in good agreement with biochemical and structural biology data, and with our previous obtained smFRET data, confirming recent hypothesis of an induced-fit type mechanism in GlnPQ [18] In addition, it confirms successful labelling of the protein with the custom-made various NPA derivative, and that the results from the established smFRET assay are indeed independent of the fluorophore pair that is used to monitor the protein conformation. It is noteworthy that spectrally uncorrected apparent FRET is reported here accounting for absolute differences in mean E*-values from varying Förster radius R0 of the fluorophore pairs used or variations in the setup
alignment [18].
Finally, we confirmed these results for a combination of NPA-RhodamineB as donor with the red-emitting dye KK114 [19] as acceptor dye. The results shown in Figure 2.8 are in good agreement with those from Figure 2.7 and show the positive impact of NPA to the photophysical properties of the donor dye. Without addition of ligand, we observed a single donor-acceptor population with an average E* value of 0.59 (Figure 2.8.e/f, upper panel). This value is in agreement with the E* value determined in previous FRET experiments and is indicative of the open protein conformation with 4.9 nm interprobe distance (Figure 2.8a). Absolute differences originated from the varying Förster radius R0 of the fluorophore pairs used here compared to those used previously (Cy3B/ATTO647N and Alexa555/Alexa647)[18]. Upon addition of the ligand (1.1 µM and 200 µM (Figure 2.8f, middle and lower panels) the ALEX-smFRET experiments indicated increasing amounts of a species characterized by E* = 0.72, which indicates the “closed” state (with a decreased interprobe distance of ~4.0 nm). While a ligand concentration of 1.1 µM is close to the Kd-value of the protein,[20] and consequently results in a mix of open and closed states, a concentration of 200 µM saturates ligand binding an therefore results in a 100% population of the closed state. Additionally, it confirms successful labeling of the protein with the custom-made
44
NPA derivative and that the results from the FRET-assay are indeed independent of the fluorophore pair that is used [18].
Figure 2.8| Improving smFRET-ALEX measurements by using NPA-RhodamineB as a donor and KK114 as acceptor: representative study on the protein GlnPQ-SBD2. a) Crystal structures of the SBD2 (T369C/S451C) open (upper
panel) and closed state (lower panel, after binding of the ligand glutamine shown in red) with label positions of donor (D) and acceptor (A). b/c) Histogram of fluorophore brightness values as determined from PCH on single-molecule transits of labeled SBD2 diffusing through the observation volume of our confocal microscope: comparison of donor-brightness F(DD) (b) and acceptor-brightness when excited via FRET, F(DA) (c), when instituting RhodamineB (RhoB, grey) and its NPA-derivative (NPA-RhoB, green) as donor (>8500 transits). Excitation intensities of 30 kW/cm2 at 532 nm and 20 kW/cm2 at 640 nm. d) Dependence of the brightness of F(DD) for increasing green excitation intensity: Comparison of NPA-RhodamineB (NPA-RhoB, green) and RhodamineB (RhoB, black) as a donor on SBD2 (> 1500 transits). e) Two-dimensional histogram (2D) of joint pair values of S (labeling stoichiometry, dashed lines 0.43 < S < 0.6 donor-acceptor population DA) and E* (FRET-efficieny, i.e. interprobe distance) determined from 24180 transits of SBD2 without ligand. Excitation intensities of 30 kW/cm2 at 532 nm and 20 kW/cm2 at 640 nm. f) Corresponding one-dimensional histograms of E* values for increasing amounts of ligand (from upper to lower panels as indicated). An increased population of SBD2 molecules with increased E* = 0.72 values (dashed line, closed conformation) becomes obvious (as opposed to the open conformation, E* = 0.59, dashed line).
0 µM 1.1 µM 200 µM A D 4.9 nm Open D A 4.0 nm Closed
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