Author response: Single molecule FRET reveals pore size and opening mechanism of a mechano-sensitive ion channel

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract The mechanosensitive channel of large conductance, which serves as a model system for mechanosensitive channels, has previously been crystallized in the closed form, but not in the open form. Ensemble measurements and electrophysiological sieving experiments show that the open-diameter of the channel pore is >25 Å, but the exact size and whether the conformational change follows a helix-tilt or barrel-stave model are unclear. Here we report measurements of the distance changes on liposome-reconstituted MscL transmembrane α-helices, using a ‘virtual sorting’ single-molecule fluorescence energy transfer. We observed directly that the channel opens via the helix-tilt model and the open pore reaches 2.8 nm in diameter. In addition, based on the measurements, we developed a molecular dynamics model of the channel structure in the open state which confirms our direct observations. https://doi.org/10.7554/eLife.01834.001 eLife digest Bacterial cells are full of fluid, and they will burst if they are not able to respond to a build up of pressure. Fortunately, the membrane of a bacterial cell contains channels that can detect the increased mechanical stress on the cell membrane and then open to relieve the pressure. In many bacterial cells, the last defence against the cell exploding is called the mechanosensitive channel of large conductance (MscL). This is made of five proteins, each of which consists of TM1 and TM2 helixes, which are responsible for opening and closing the channel. Two models have been proposed to explain how the channels are opened. In the barrel-stave model, the TM1 helix moves, while the TM2 helix remains stationary. This results in an open pore that is lined with TM1 and TM2 helixes in the same way that wooden staves line a barrel. In the helix-tilt model, both helixes tilt towards the membrane to open the channel. Wang et al. have now used a technique called single-molecule fluorescence resonance energy transfer (FRET) to explore the structure of the open channel in E. coli in order to determine which model is correct. In this technique an individual channel is labeled with two different fluorescent molecules. By illuminating the channel with light of a wavelength that excites the first fluorescent molecule, and measuring the strength of the fluorescence from the second molecule, it is possible to work out the distance between the two molecules. From this, the structure of the channel and how it opens and closes can be explored. Previous attempts to measure the diameters of open channels using fluorescence techniques have suffered from issues caused by the use of large numbers of fluorescent molecules. This has made it necessary to use computational modeling to extract the required data. By looking at a series of individual proteins, Wang et al. overcame these problems and found that the diameter of the fully open pore is 2.8 nm. The result provides strong support for the helix-tilt model. https://doi.org/10.7554/eLife.01834.002 Introduction Mechanosensitive (MS) channels are essential in both eukaryotes and prokaryotes (Perozo, 2006; Árnadóttir and Chalfie, 2010; Haswell et al., 2011). In eukaryotes, they are involved in diverse processes such as embryonic development, touch, pain, hearing, lung growth, and muscle homeostasis (Hamill and Martinac, 2001; Chalfie, 2009; Árnadóttir and Chalfie, 2010). In bacteria, they are ‘safety valves’, opening their pores to release the pressure to protect cells from hypo-osmotic shock (Booth and Blount, 2012). The rise in antibiotic resistance, and the crucial role MS channels play in bacterial adaptation, makes it important to understand the MS channels as potentially new drug targets (Booth and Blount, 2012). When high pressure (∼10 mN/m) causes the bacterial mechanosensitive channel of large conductance (MscL) to open, it forms a large, nonselective pore with a very high conductance (∼3 nS) that is permeable to various ions and small organic osmolytes. In 1998, MscL from Mycobacterium tuberculosis in the closed state was crystallized by Rees and co-workers (Chang et al., 1998). They showed that MscL is a pentamer made up of five identical subunits (Figure 1A,B). Each subunit consists of one cytoplasmic α-helix (the CP domain) and two transmembrane α-helices (the TM1 and TM2 helices), which extend through the cell membrane and are joined by a periplasmic loop (Figure 1B). TM1 and TM2 are primarily responsible for gating; it has been shown that complete deletion of the CP domain does not change the gating parameters substantially (Anishkin et al., 2003). Figure 1 Download asset Open asset Cartoon representation of the structure of MscL in the closed conformation in the (A) top view and (B) side view (PDB ID: 2OAR [Chang et al., 1998; Steinbacher et al., 2007]), and scheme of single molecule FRET setup. MscL is a homo-pentamer consisting of five identical subunits. Each subunit consists of one cytoplasmic α-helix (CP) and two transmembrane α-helices (TM1 and TM2), which extend through the cell membrane and are joined by a periplasmic loop (Chang et al., 1998). (C) Residues measured using smFRET. Three residues on each of the transmembrane helices (M42C, A27C and I25C on TM1; Y75C, Q80C and V82C on TM2) were chosen. Note that no residues on the CP were chosen because the complete deletion of the CP does not change the gating parameters substantially (Anishkin et al., 2003). (D) Labeled MscL proteins were reconstituted into liposomes, which were then immobilized on a coverslip and used for smFRET experiments. (E) The addition of LPC traps the protein in the open conformation (Perozo et al., 2002b). https://doi.org/10.7554/eLife.01834.003 Despite this progress, the open form of MscL has not been crystallized. This leaves two questions unanswered: what is the exact size of the open pore of MscL, and how does the channel open? Several techniques, for example, permeation of organic ions (Cruickshank et al., 1997), Electron paramagnetic resonance (EPR) (Perozo et al., 2002a, 2002b) and ensemble fluorescence resonance energy transfer (FRET) (Corry et al., 2005b, 2010) have attempted to measure the pore size. However, systematic errors likely result in an overestimation of (Cruickshank et al., 1997), an underestimation of (Corry et al., 2005b, 2010), or an insensitivity to the requisite distances (Perozo et al., 2002a). For example, EPR was only able to establish that the open pore is >25 Å (11). Ensemble FRET, which yielded some insightful results, is potentially sensitive to larger distances (∼80–100 Å) (Roy et al., 2008). However, due to multiple labeling, problems with protein clustering, and the need for Monte-Carlo simulations to extract distance information, there was much variability and uncertainty in the results (Corry et al., 2005a, 2005b, 2010). Another important question is how the MscL channels open, that is how the helices rearrange upon channel activation (i.e., from the closed state to the open state). Currently, there exist two predominant models for the opening of MscL: the barrel-stave model and the helix-tilt model (Figure 2; Perozo, 2006). The barrel-stave model (Figure 2C,D) involves motion of the transmembrane helix 1 (TM1) with the transmembrane helix 2 (TM2) remaining stationary; the open pore is lined by both TM1 and TM2 and the helices are fairly vertical (where the membrane is horizontal). This model derives primarily from the number of transmembrane helices and the large size of the open pore. In contrast, the helix-tilt model (Figure 2E,F), which has been proposed more recently (Sukharev et al., 2001a, 2001b; Betanzos et al., 2002), involves motion of TM1 and TM2, with both swinging away from the pore upon channel opening and both helices tilting toward the plane of membrane. Recent evidence from cysteine-crosslinking experiments, EPR experiments, and ensemble FRET experiments, argue in favor of the helix-tilt model (Betanzos et al., 2002; Perozo et al., 2002a; Corry et al., 2010). Figure 2 Download asset Open asset The opening models for MscL. The MscL opens from (A and B) the closed state, to (C and D) the open state via the barrel-stave model or (E and F) the open state via the helix-tilt model. The top figures (A, C, E) are top views and the bottom figures (B, D, F) are the side views. TM1 helices are shown in red while TM2 in blue. In the barrel-stave model (C and D), TM1 swings away from the pore center but TM2 remains stationary upon channel activation, generating an open pore lined by both TM1 and TM2 and the helices are more parallel to the membrane normal than the membrane plane. In the helix-tilt model (E and F), both TM1 and TM2 swing away from the symmetry axis and both helices tilt toward the plane of membrane. https://doi.org/10.7554/eLife.01834.004 In the present work, we focused on the transmembrane helices involved in the opening of MscL from Escherichia coli (EcoMscL), using a single-molecule fluorescence resonance energy transfer (smFRET). MscL channels were reconstituted in liposomes during smFRET measurements and thus the channels were in their quasi-native environment. In addition, although MscL is a pentamer, we utilized photobleaching to virtually sort out the population of molecules with a single donor and a single acceptor, allowing us to make accurate smFRET measurements. It is the first time that smFRET has been applied to liposome-reconstituted membrane proteins with more than three monomers. We measured movement of three residues on TM1 (M42C, A27C, and I25C; Figure 1C) and three residues on TM2 (Y75C, Q80C and V82C; Figure 1C), from which we determined not only the translational movements but also the tilting of each helix. We observed the tilting of the helices in a model-free fashion, arguing strongly in favor of the helix-tilt model. In addition, from the movement of the residue (I25C) right at the gating region, we determined directly that the open pore reaches 2.8 nm in diameter. Lastly, we developed a molecular dynamics model of the channel structure in the open state based on the smFRET results, while using the crystal structure of the protein in the closed state as a reference. The model of the open structure satisfies all the distance constraints measured from smFRET experiments. The developed open structure confirmed that the pore size of the fully open channel is 2.8 nm in diameter, achieved via the helix-tilt opening model. Results FRET efficiencies Purified MscL mutants (Figure 3—figure supplement 1) were labeled with Alexa Fluor 488 (AF488) and Alexa Fluor 568 (AF568) and reconstituted into ∼50 nm liposomes made of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) with 2% 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-biotinyl (BPE) (Figure 1D). The liposomes were then immobilized on a glass coverslip, via biotin-avidin interaction, for smFRET measurements (Figure 1D). To access the open state of the channels, 1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine or lysophosphatidylcholine (LPC) of 25% molar ratio was added (Perozo et al., 2002b, 2002a; Corry et al., 2005b, 2010; Figure 1E) and incubated for >10 min before immobilization. Just before performing smFRET experiments, the fluorescence spectra of the samples (±LPC) were recorded with excitation at 488 nm to confirm that the channels open up with LPC by observing the shift in the FRET peaks. (The channel activity is also determined by observing the opening of the channels upon application of negative pressure [suction] to the patch pipette. The labeled proteins for patch-clamp experiments are from a different aliquot, although the same batch, of the labeled proteins for smFRET experiments). We emphasize that, although smFRET has been applied to study the conformational changes of channels and transporters (Choi et al., 2010; Zhao et al., 2010, 2011; Akyuz et al., 2013), to our knowledge, it is the first time that smFRET has been used with channels reconstituted to liposomes. Via smFRET measurements, we observed fluorescence intensity traces with one or two photobleaching steps (Figure 3—figure supplement 2A,B). This is the expected result because MscL is a homo-pentamer and the labeling of fluorophores is stochastic. The number of photobleaching steps tells the number of fluorophores attached to a channel. Only the traces showing a single photobleaching step in both the donor and acceptor channels, ensuring that only a single donor and/or acceptor fluorophore, were included in the analysis (Figure 3—figure supplement 2A). Donors were, in most cases, photobleached first, resulting in simultaneous dropping of the fluorescence intensities in both donor and acceptor channels (Figure 3—figure supplement 2A,B). Subtraction of the intensities before and after photobleaching gives the intensities of donor (ID) and acceptor (IA), which are used for the calculation of FRET efficiency. Note that the intensities, ID and IA, automatically remove the direct excitation of acceptor (i.e., the leakage of acceptor emission in the donor channel). However, the leakage of donor emission in the acceptor channel is still present. To measure this leakage, MscL channels were labeled with donors-only and the leakage coefficient (ℓ) was measured experimentally: ℓ = IDA/IDD ≈ 0.09, where IDA is the intensity of donor emission in the acceptor channel and IDD is the intensity of donor emission in the donor channel. Furthermore, to determine the actual FRET efficiency, another instrumental correction was made through the correction factor γ, which accounts for the differences in quantum yield and detection efficiency between the donor and the acceptor. It was calculated as the ratio of change in the acceptor intensity, ΔIA, to the change in the donor intensity, ΔID, upon acceptor photobleaching: from the traces where the acceptor photobleached first (Roy et al., 2008), we estimated the value γ = ΔIA/ΔID ≈ 0.89 ± 0.06 (Figure 3—figure supplement 2C). We analyzed a few hundred traces (varying between 134 and 577 traces) with single photobleaching steps in the absence and presence of LPC for each mutant (Figure 1C, Figure 3). Here we focus on the mutant M42C for the sake of illustration. For the single photobleaching steps of M42C, 428 and 577 traces, in the absence and presence of LPC, respectively, were analyzed. The corrected FRET efficiencies were calculated and their distribution was then plotted and fitted with Gaussians via maximum likelihood estimates, shown in Figure 3A,B, while the number of Gaussians was determined according to the corrected Akaike information criterion (AICc) and the Bayesian information criterion (BIC) (Table 1; Akaike, 1974; Schwarz, 1978; Sugiura, 1978). In the absence of LPC, we observed three peaks at E = 0.1, 0.28 and 0.63, respectively (Figure 3A). In the presence of LPC, the third peak showing the highest FRET efficiency diminishes, leaving mainly two Gaussians (E = 0.1 and 0.23, Figure 3B). This transition (i.e., the highest peak decreases and the lowest peak increases) is more obvious when we plotted the difference between the normalized FRET distributions ( ∑​PX=1, where X = + for in the presence of LPC and X = − for in the absence of LPC) under the two conditions, as shown in Figure 3C: after adding 25% LPC, the peak at E ∼0.6 went away but the fraction of the peak at E ∼0.1 built up. Note that the highest peak at E ∼0.6 does not completely disappear in the presence of 25% LPC, which is consistent with (Perozo et al., 2002b). Figure 3 with 3 supplements see all Download asset Open asset Single molecule FRET results for MscL M42C. The distribution of FRET efficiency of M42C in the (A) absence and (B) presence of LPC were plotted and fitted with Gaussians. (C) The difference between the normalized FRET distributions under the two conditions (±LPC), ΔP, emphasizes the diminishing of the third peak at E ∼0.6 after adding LPC. (D) The variance between the normalized FRET distributions under the two conditions (±LPC), ΔP2, decreases as the fraction of BPE in the liposomes is increased from 2% to 16%. (E) Histograms of FRET efficiencies in the absence (top row, −LPC) and presence (bottom row, +LPC) of LPC for the other five residues (I25C, A27C, Y75C, Q80C, and V82C) measured in the current study. https://doi.org/10.7554/eLife.01834.005 Table 1 The best model of the fittings of FRET efficiency distribution were determined by calculating the corrected Akaike information criterion and Bayesian information criterion: AICc=−2⁡ln⁡LM +2k+2k(k+1)N−k−1, BIC=−2⁡ln⁡LM+k⁡ln⁡N where LM is the maximum likelihood by the model, k the number of parameters of the model, N the number of datapoints used in the fit (Akaike, 1974; Schwarz, 1978; Sugiura, 1978) https://doi.org/10.7554/eLife.01834.009 # of fitting peaks− ln LMAICcBICM42C; No LPC 1−1.90.38.4 2−43.4−76.6−56.4 3−56.4−96.4−64.3 4−58.1−93.5−49.5M42C; With LPC 1−190.9−377.9−369.2 2−279.5−549.0−527.3 3−290.7−565.1−530.5 4−292.7−563.0−515.5 The lowest AICc and BIC values give the best fitting model: three peaks (bold values). In the absence of LPC, the existence of three peaks, rather than two peaks, can be explained by considering the effect of tethering on the liposome. As the MscL channel is a homo-pentamer, we initially expected two distances between donor and acceptor in each state (Rn and Rf in Figure 4A) and thus two peaks for the distribution of FRET efficiency, assuming that all the channels are closed in the absence of LPC. However, this assumption is not necessarily true, especially in our situation where liposomes are immobilized and the proteins are responsive to membrane tension. It had been predicted by theories and observed in experiments that immobilization of liposomes (or vesicles) results in significant membrane tension and possibly rupture (Zhdanov et al., 2006; Chung et al., 2009; Serro et al., 2012). In our experiments, the membrane tension is expected to be high, ∼30–40 kBT, due to the strong interaction between BPE and the surface via biotin-neutravidin (Miyamoto and Kollman, 1993; Rico and Moy, 2007). With such strong interaction, giant unilamellar vesicles ruptured spontaneously, as has been observed experimentally (Chung et al., 2009). The consequence is that some of the MscL channels switch to the open conformation upon the immobilization of the liposomes. (However, the fraction of open channels might be different for different mutants even if the membrane tension is similar). Therefore, the FRET histogram for the no-LPC sample includes a mixture of closed and open MscL channels. To test this hypothesis, control experiments were performed by increasing the fraction of BPE in the liposomes, guided by a theoretical prediction (Zhdanov et al., 2006): if the hypothesis was true, the membrane tension in the liposomes due to immobilization would be higher, more channels would open in the absence of LPC, and therefore the difference between the FRET histograms with and without LPC would be smaller. We varied the fractions of BPE in the liposomes from 2% to 16% and, indeed, observed that the difference between the samples with and without LPC decreases (Figure 3D). We quantified the difference by the (unscaled) variance, ΔP2=∑i=1N(Pi+−Pi−)2, where PiX is the probability distribution of FRET efficiency, ∑i=1NPiX=1. We observed that the variance ΔP2 decreased by 98%, from 0.084 to 0.002, when the fraction of BPE in the liposomes increased from 2% to 16%, supporting the hypothesis that the sample without LPC is a mixture of closed and open channels. Figure 4 with 3 supplements see all Download asset Open asset Movement of residues. (A) Each residue in a red of (or diameter D, where D, as is although upon opening would be at the pore center channel activation, the protein changes from to or the residue by = measured from the pore center (B) of MscL from closed state to open state The residue of of the from the pore (C and D) movements of residues on TM1 and TM2 measured via smFRET. the residues away from the pore arguing in favor of the helix-tilt model. based on the crystal structure of MscL in the closed state (Chang et al., and previously open (Cruickshank et al., Perozo et al., 2002a; Corry et al., 2010) that it is likely that, due to of FRET, the peak (E = is an of two peaks to of the open state and Rf of the closed state and Figure The of the protein (i.e., gives and where is the protein diameter. M42C as an the crystal structure gives the diameter of the protein in the closed state the = resulting in nm. The expected change in the protein size is from to 4 nm (Cruickshank et al., Perozo et al., 2002a; Corry et al., 2010), with the most report of 2.8 nm from ensemble FRET measurements (Corry et al., 2010). the expected = + is which gives the same as (The expected is nm when into the expected from the In the calculation the of for the were However, the side of the residues and the attached fluorescent will an on the order of 2 resulting in that the for and to is even Furthermore, it has been observed that of MscL are of in such as used in the current study et al., 2011). This would to the peaks of FRET in the absence of LPC. Therefore, to be consistent and we use the highest FRET efficiency and for the calculation of distance changes the other we that all mutants give measurements (i.e., with the model that M42C is as shown in Figure 3—figure supplement FRET between MscL channels on the same had been a in ensemble FRET experiments. To the likelihood of and to the we applied two we used labeled channels with for in liposomes. As a we had molar ratio of labeled proteins to than that in the ensemble FRET (Corry et al., 2005b, 2010), the likelihood of We found that adding a mixture of labeled and protein to no more than one fluorescent channel while ensuring of channels into liposomes. In addition, only traces showing a single photobleaching step in both donor and acceptor channels were included in which the FRET between MscL channels in the the likelihood that energy transfer between two channels even in the presence of MscL clustering, the of FRET Another is that we used for peak This was chosen because it does not the data before there are for of and the of size in and the peaks can be with different we then the data and the histograms for the sake of the data is does not change the fitting of The for and is calculated by of et al., 2008). and can be we measured the quantum yield and factor for the fluorophores to each and channel mutant and Figure The quantum of to various MscL mutants are in Table 2; Figure corrected for and It is that the fluorophores used in the current study are of and However, it was expected that this will not the results because they have been used in many smFRET et al., 2002; et al., et al., 2006; et al., et al., the of the are the same while the only difference between the in where the of is we the molecular of the and confirmed that the difference in molecular size is between (Figure supplement Figure with 1 supplement see all Download asset Open asset of (A) and fluorescence spectra of and a used to determine the quantum yield of to MscL (B) of and to MscL mutant corrected for the of the and for the high of the Table 2 of smFRET experiments is the quantum yield of donor (AF488) after to each MscL and are the of donor (AF488) and acceptor (AF568) after is the and are the FRET efficiencies in the closed and open is the change in the distances between donor and acceptor = Figure is the change of the protein diameter = is the translational movement of the measured from the pore = Note that the errors shown in the are the maximum possible errors due to of the The actual errors are expected to be much smaller. The factor was determined by measuring the of the fluorophores (Table 2; Figure Figure supplement The of both donor and acceptor for most residues is and therefore in is to and et al., 2008). we calculated the maximum possible errors in due to of the (Table 2; Figure Figure supplement the actual errors in be much smaller. Another of in in the measurements of which were performed for in + 1 which was not the same for in smFRET experiments in liposomes in although the was the Furthermore, it is possible that the addition of LPC and the conformational change of MscL changes as resulting in errors in and in the distances calculated It is that the of the acceptor is than that of the donor although the acceptor is This be to many as by the = 1 + the coefficient fluorescence and, more the which according to wavelength and The in Table 2 were measured at the where smFRET experiments were to the effect of the size of fluorescent We that the size and of the fluorescent to FRET measurements to the of distances and therefore to To this our is to focus on the movement of of the and then to the model based on the