Thermal Unfolding of Proteins Probed at the Single ... - ACS Publications

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Thermal Unfolding of Proteins Probed at the Single Molecule Level Using Nanopores Linda Payet,†,‡ Marlène Martinho,†,⊥ Manuela Pastoriza-Gallego,‡ Jean-Michel Betton,§ Loïc Auvray,∥ Juan Pelta,*,†,‡ and Jérôme Mathé*,† LAMBE, UMR 8587 CNRS-CEA-UEVE, Université d’Évry val d’Essonne, Évry, France LAMBE, UMR 8587 CNRS-CEA-UEVE, Université de Cergy-Pontoise, Cergy-Pontoise Cedex, France § Unité de Biochimie Structurale, CNRS-URA 3528, Institut Pasteur, Paris, France ∥ Laboratoire MSC, CNRS UMR-7057, Université Paris-Diderot, Paris, France † ‡

S Supporting Information *

ABSTRACT: The nanopore technique has great potential to discriminate conformations of proteins. It is a very interesting system to mimic and understand the process of translocation of biomacromolecules through a cellular membrane. In particular, the unfolding and folding of proteins before and after going through the nanopore are not well understood. We study the thermal unfolding of a protein, probed by two protein nanopores: aerolysin and α-hemolysin. At room temperature, the native folded protein does not enter into the pore. When we increase the temperature from 25 to 50 °C, the molecules unfold and the event frequency of current blockade increases. A similar sigmoid function fits the normalized event frequency evolution for both nanopores, thus the unfolding curve does not depend on the structure and the net charge of the nanopore. We performed also a circular dichroism bulk experiment. We obtain the same melting temperature (around 45 °C) using the bulk and single molecule techniques.

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extremely promising to mimic and describe the protein translocation processes that take place in living organisms. Approaches to study protein folding/unfolding are essentially of two kinds: classical bulk experiments, such as circular dichroism (CD) or fluorescence spectroscopy, and singlemolecule sensing, such as atomic force microscopy (AFM), optical tweezers, or fluorescence resonance energy transfer (FRET).26−28 Nanopores retain qualities of both types: the frequency of probing events is sufficiently high to easily yield significant statistics, which is an important feature of bulk measurements and hard to attain with AFM. However, while bulk measurements necessarily give averaging results across the sample, the pore probes macromolecules one-by-one and thus enables, for example, the separate analysis of the dynamics of several coexisting conformations in a sample.20 Also, contrary to the other single-molecule techniques, nanopores do not require labeled molecules, so the preparation of the samples is simpler and the molecules are not chemically modified. While it is well established that unfolding is necessary for some proteins to pass through narrow channels, only two studies describe the unfolding curves of proteins as a function of the denaturing agent concentration and their transport against an external parameter: curves of unfolded maltose binding protein (MBP) and a mutant as a function of

anopores with ionic current measurements are the subject of extensive research in the field of sensing and characterization of single biopolymers.1 This technique consists in measuring continuously an ionic current through a channel induced by an externally applied electric field. When a macromolecule goes through the pore, the current temporarily falls down. The frequency and the duration of the current variations are characteristic of the physical chemistry such as conformation and charge of the diffusing molecule. In particular, studies had exhibited the potential of using nanopores for single-stranded DNA or RNA ultrafast and low cost sequencing 2 and to detect selectively microRNA biomarkers3 useful, for example, in cancer diagnosis.4 The study of proteins by nanopore presents also a great interest and it is promising for applications. The nanopore technique was shown to be suitable to probe the structure of peptides,5−7 to perform binding essays,8−11 to study protein translocation,12−19 and also to follow the unfolding process of proteins in the presence of denaturing agent13,20 or the folding of peptides and proteins by metal ions.21,22 The transport of unfolded protein through a channel plays a role in biological processes: protein import or export, protein synthesis or degradation. Human diseases are related to bad transport or misfolding.23 The understanding of the connection between protein translocation and folding is crucial. After their translocation, polypeptide chains need to be correctly refolded to be functional.24,25 Given the first results obtained, the nanopore technique looks © 2012 American Chemical Society

Received: January 13, 2012 Accepted: April 9, 2012 Published: April 9, 2012 4071

dx.doi.org/10.1021/ac300129e | Anal. Chem. 2012, 84, 4071−4076

Analytical Chemistry

Article

Figure 1. Electrical detection of protein unfolding through an aerolysin pore (left) and α-hemolysin pore (right) as a function of temperature. The electrical recordings were performed at 70 mV in aerolysin and 100 mV in α-hemolysin. The left panels, from top to bottom, represent recordings without MalE219 at 25 °C, with MalE219 at 25, 42, and 50 °C, respectively. The right panels, from top to bottom, represent recordings without MalE219 at 20 °C, with MalE219 at 20, 40, and 50 °C, respectively.

Supporting Information. The recordings were performed at 70 mV for aerolysin and at 100 mV for α-hemolysin. We found that according to the protein nanopore, these voltage bias are optimized to have stable experimental conditions for a long time and at any temperature. By recording the current−voltage curves, we check the stability of the pores (i.e., no gating observed) under the different experimental conditions (data not shown). It is well-known that lipid bilayers and αhemolysin nanopores are stable at high temperature (up to nearly 100 °C).36 We showed that it is also the case for aerolysin at least up to 70 °C. The protein, MalE219, is added in the cis-chamber (see definition in the Supporting Information).

guanidium chloride concentration has been obtained with the evolution of event frequency of current blockades in an αhemolysin13 or an aerolysin pore.29 We use in this study two passive channels, α-hemolysin and aerolysin. The structure of α-hemolysin was solved by Song et al.30 For aerolysin, there is no crystal structure available, yet electron microscopy provides a model of the pore shape31,32 to be compared with α-hemolysin’s one. It shows that as αhemolysin, the aerolysin channel is heptameric and formed a βbarrel that can perforate the membranes. In contrast with αhemolysin, aerolysin has no vestibule domain. The length of both nanopores are similar (around 10 nm), but the diameter of α-hemolysin (1.5−4.6 nm) is larger than the aerolysin one (1−1.7 nm). On the other hand, the net charge of the pores differs widely: +7e for α-hemolysin against −52e for aerolysin. In this paper, we are interested in the study of protein denaturation by temperature. Temperature can easily be combined with nanopore technique in a reversible way without otherwise disturbing the samples, and this fact is important for further observation of the refolding process. We study the thermal unfolding of MalE219, a variant of MBP, using two different protein nanopores, aerolysin and α-hemolysin (α-HL). We confirm that aerolysin, due to its smaller diameter, is a better candidate to detect different conformations of proteins. We also compare the thermodynamic data obtained with the nanopore technique and CD experiments.

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RESULTS AND DISCUSSION We recorded ion current traces for each temperature, ranging from 20 to 70 °C, before and after addition of MalE219. Figure 1 shows typical electrical recordings for aerolysin (left panels from top to bottom, without MalE219 at 25 °C and with MalE219 at 25, 42, and 50 °C) and for α-hemolysin (right panels from top to bottom, without MalE219 at 20 °C and with MalE219 at 20, 40, and 50 °C). For any temperature, we analyzed only traces with open pore current constant within 10% all along the acquisition. Thus, the shown data correspond to membranes and pores stable during the measurements with limited evaporation. We note that the magnitude of the noise is similar at each temperature. We observe that the open pore current, i.e., the baseline of the electrical trace, increases with temperature. This effect has been previously observed with α-hemolysin36−38 and with OmpF and OmpC porins39,40 and has been found consistent with buffer conductivity variation. Before addition of MalE219, no current blockades appear (Figure 1 top left and right). Adding proteins in the cis chamber induces blocking of the ion current (Figure 1, three lower panels left and right) related to the obstruction of the pore by the proteins. Furthermore, by comparing the current traces in Figure 1 with MalE219, it is clearly visible that increasing the

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EXPERIMENTAL SECTION MBP is a recombinant protein of Escherichia coli widely studied as a model of protein folding and translocation.33 We work with one of its destabilized variants, MalE219,17 which allows nanopore experiments in a convenient range of temperature, as we will show. As MBP, the size and the net charge of MalE219 are 370 residues (40 707 Da) and −8e, respectively. These proteins consist of two domains forming an ellipsoidal structure with overall dimensions of 30 Å × 40 Å × 65 Å.34 Electrical recording setup used is similar to the one presented by Bates et al.35 For details on statistical analysis, see the 4072



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temperature from 50 °C increases the event frequency. The increase of the event rate with temperature could be due to the enhancement of thermal motion, which makes the diffusion of MalE219 in any conformation to the pore more frequent. In fact, we will show below that the increase of the event rate with temperature is mainly related to thermal unfolding of the protein, the same way that we showed previously that the event frequency increases with denaturing agent concentration for different proteins.13,29 We also observe that different levels of blockades exist at any temperature. To characterize theses different current blockades, we present in Figure 2 the event density plots for MalE219 with aerolysin at different temperatures. It represents the current blockade (Ib) normalized by the corresponding open pore

current (Io) as a function of the blockade time. The histograms are normalized by the total duration of the recording. Thus their total intensity reflects the event frequency. The blockade events are defined from the electrical recordings systematically as events with a blockade current lower than the two thresholds Th1 and Th2 (see definition in the Supporting Information). From theses graphs, we observe the limit of detection for the short time (around 7 μs) induced by the acquisition rate (106 Hz) and the thresholds used to define an event as explained previously.41 At 25 °C, all the events visible on the trace of Figure 1 are characterized by a short blockade time (lower than 30 μs and average time of around 10 μs). In previous work,42,43 these short blockade times observed for α-hemolysin in the presence of ssDNA have been assigned to collision events (sometimes called bumping or straddling in other papers), i.e., macromolecules approaching the nanopore entrance without translocation. Pastoriza-Gallego et al. also detected collision events for α-hemolysin and aerolysin with unfolded proteins17,44 corresponding to the higher current blockade. In particular, it was shown that the duration of these short events is independent of the polymer length17,42 and applied potential.42 However their frequency decreases with the voltage.17 At 25 °C, we observe only rare collisions, due to the fact that MalE219 is folded in this condition and cannot translocate due to its relative large size compared to the nanopore diameter. When temperature increases, in addition to the collisions, events with longer blockade time and lower blockade current appear. Seeing the relatively low levels of the blockade current, we associate these latter events with thermally unfolded MalE219 fully confined in the nanopore; these events are called transport events in this paper. From 42 °C, we see clearly two major types of events with a normalized current blockade of 0.3 and 0.1, respectively. These two types of events may be associated with different directions for the entrance of the proteins (by N-terminal or C-terminal) or different conformations of the protein in the pore. Translocations with different blockade currents have been observed previously for ssDNA entering the nanopore either by the 3′ or 5′ end.45 For proteins, different blockade currents have been observed for different conformations of different secondary structures.5,20 Pastoriza-Gallego et al. also observed different blockade currents for fully unfolded MalE219 in guanidium in aerolysin.17 Therefore, these events cannot be associated to partially unfolded proteins inserted in the pore. This sensitivity of the protein nanopores may be interesting as tools for conformation detection, and further experiments should help to assign the different blockade currents detected for MalE219 going through aerolysin. In addition, in Figure 2, we see a distortion of the distribution for short events through a higher blockade current. This is due to the low pass filtering as described previously.20,41 Distortions appear for events shorter than 30 μs. Therefore, for high temperatures, the transport events appear with a higher blockade current than for low temperatures (see Figure S-2 in the Supporting Information for a detailed explanation). This makes the distinction between collision and transport events more difficult when temperature increases. For α-hemolysin, the event duration distributions (Figure S-3 in the Supporting Information) are characterized by collision events and one single type of transport event at any temperature with a normalized blockade current of 0.15. We have also observed rare events of duration greater than 100 ms

Figure 2. Two dimensional (Ib,Tt) histograms of events for MalE219 with aerolysin pore at different temperatures. The probability is in logarithmic scale to enhance the different clouds of events. The histograms are normalized by the recording duration. The total intensity of the histograms thus reflects the event frequency. Two types of events are observed: collisions and transport events. The collisions are characterized by short duration (around 10 μs) and high normalized current blockade and are the only events observed at 25 °C. Transport events are seen from 35 °C, they last longer and with lower normalized current blockade, and their duration decreases with increasing temperature to reach the detection limit (around 7 μs). The oval shape area is drawn to point out one type of the two transport events defined for aerolysin. These events are characterized by a normalized blockade current of 0.3 at 42 °C. The arrows show the variation of their durations and normalized blockade currents with temperature. The average time for these events is used to plot Figure 3. 4073



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below 42 °C associated with partially folded proteins blocking the pore entrance. These partially folded conformations have been previously observed for chemically denaturated wild type MalE,13 and we recently determined using NMR that these events are probably due to proteins partially unfolded by their C-terminal end.46 In Figure 3, we represent the average blockade time as a function of temperature for α-hemolysin and for aerolysin. For

Figure 4 shows the variation of the normalized event rate as a function of temperature determined for α-hemolysin and

Figure 4. Thermal unfolding transition curves of MalE219 detected by, respectively, α-hemolysin and aerolysin pores. The curves represent the normalized event rate (equivalent to the proportion of unfolded conformations) as a function of temperature. Below 50 °C, the data are fitted to a sigmoid. Above 50 °C, the frequency decreases due to the detection limit of short duration time events.

Figure 3. Blockade time as a function of temperature for α-hemolysin and the aerolysin pore. The points at 20 and 22 °C for α-hemolysin and aerolysin, respectively, correspond to MalE219 unfolded in the presence of 1 M guanidium chloride. The lines are drawn to guide the eyes.

aerolysin. To calculate the event rate, we consider all the events because it is difficult to distinguish the collision from the transport events at high temperature (see explanation above). In fact, the collision rate does not affect much the variation of the total event rate with temperature. Indeed, the collision rate varies with the intrinsic dynamic effect of temperature, but it is theoretically linear with the diffusion coefficient. We see this small effect for temperatures below 35 °C when there is no transport yet. Therefore, the shape and the magnitude of the variation are mainly due to the increase of the transport rate with temperature. The event rates are normalized to the maximum frequency (2.6 and 28 Hz) obtained, respectively, for the two nanopores. For both nanopores, we discern two regions of event rate variation according to the temperature. Below 50 °C, the plot of the average event rate as a function of temperature shows an increasing sigmoid dependence. From 60 °C, the frequency drops. Interestingly, taking into account the errors on the measurements, we can see the good superposition of the sigmoid for both nanopores. Thus, the normalization annihilates the effect of MalE219 concentration (0.35 and 2.79 μM for α-hemolysin and aerolysin, respectively), the effect of the applied voltage bias on the event frequency, and more importantly the effect of the net charge and the geometry of the nanopores. With bulk measurements such as circular dichroism or tryptophan fluorescence, the measurable parameter (ellipticity or fluorescence) depends linearly on the concentration of the probed molecule. When describing an unfolding transition, this parameter varies as a sigmoid as a function of temperature either if it is a two-state transition or if the different unfolding states have very similar melting temperatures. With the nanopore technique, the event rate is linear with the concentration of molecules capable of going into the pore.13 We therefore associate its variation in the region below 50 °C to the variation of the proportion of unfolded proteins. To confirm that this dependence is related mainly to the unfolding process and not only to the intrinsic dynamics effect of temperature, we determine the event frequency for fully unfolded MalE219 by guanidium going through aerolysin at 22 and 50 °C. We find, respectively, 20 and 24 Hz ± 4, i.e., a variation of only 20%. Besides, we find that the transport frequency for unfolded ssDNA increases exponentially with

α-hemolysin, this time is defined by the longer characteristic time obtained from the time distribution fit. For aerolysin, since several time scales can be extracted from the event duration distributions, we focused on the event with a normalized current of 0.3 which are clearly visible from 42 °C. This time scale does not characterize all the events, but its evolution with temperature clearly shows a decrease of the transport time with temperature. As expected, the dynamics of MalE219 is accelerated with an increase of temperature, but at high temperature (from 60 °C), the blockade time reaches a plateau due to the resolution limit of the technique, as we will see below. This behavior has been observed for ssDNA translocating α-hemolysin for temperature varying from 15 to 40 °C37 and also for ampicillin in OmpF from 5 to 55 °C.47 We also notice that the transport dynamics of MalE219 are systematically slower in aerolysin compared to α-hemolysin. This agrees with our previous investigation where we found that the mean transport time of chemically unfolded protein is higher in aerolysin compared to α-hemolysin for the same applied voltage.17 This difference may be due also to the existence of a more slowing down interaction of the protein with aerolysin than with α-hemolysin. The interaction between the protein and the pore may be of three kinds: electrostatic, hydrophobic, or steric. Electrostatic interactions may influence transport time as Rincor-Restrepo et al. described for ssDNA.48 However, considering the overall charge of the protein, we can expect these interactions to be weak. We also expect very few hydrophobic interactions between the pore and the protein because at any temperature, although the proteins confined in the nanopore are completely unfolded, the pores are not denatured. The main difference in diffusion through the respective pore may be explained by different geometry along the nanopore: smaller diameter was suggested for aerolysin allowing more steric interactions. It is useful to remember this sensitivity difference between aerolysin and α-hemolysin when one has to choose a pore for single-molecule detection, in particular for identification of different conformation states of a macromolecule. 4074



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sigmoid. Thus, the thermal unfolding transition does not depend on the nanopore net charge and geometry. We show that melting temperature determined by the nanopore technique agrees with the one determined by circular dichroism. While CD gives structural information on average for all the states in solution, nanopores allow one to detect protein conformations at the single molecule level. In particular, we observe with aerolysin that we may have different conformations of unfolded MalE219 in the pore. The nanopore technique has great potential to help resolve fundamental questions concerning the interaction of macromolecules with nanopores and their translocation in vivo in addition to be used as chemical or conformation sensors.

temperature and not with a sigmoid dependence (data to be published). This sigmoid dependence agrees with our previous work, where we followed the nanopore technique with the denaturation of proteins by a chemical agent.13,29 For temperature higher than 50 °C, the rate drops. This would have been explained by aggregation of proteins if we would have observed in parallel an increase of the transport time with temperature in this region. In contrast, as we notice above, the event duration decreases to reach a plateau corresponding to the limit of detection (see Figure 2) of the short time events. Therefore more and more events are too fast to be resolved at high temperature inducing a decrease in the observed event rate. Kozhinjampara et al. had also observed this type of frequency decrease at high temperature for translocation of antibiotic through OmpF membrane channels.47 To extend the measurement of the event rate at a higher temperature, one could increase the transport time by increasing the viscosity of the buffer, for example. By comparing the single-molecule nanopore data (for temperature below 50 °C) to CD bulk detection of thermal unfolding, we confirm again that the sigmoidal variation of the event rate is related to the unfolding process. In Figure 5, we

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (J.P.); [email protected] (J.M.). Present Address

⊥ Laboratoire de Bioénergétique et Ingénierie des Protéines, Equipe de Biophysique des Métalloprotéines, CNRS UPR 9036, Marseille, France.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS At the time of the data acquisition Linda Payet and Marlène Martinho were Postdoctoral Fellows of Genopole. This work was supported by grant funding from ANR Blanche “TRANSFOLDPROT” BLAN08-1_339991. We thank F. Gisous Van Der Goot for the vector of recombinant aerolysin.

Figure 5. Thermal unfolding transition curve of MalE219 assessed by circular dichroism. The curve represents the normalized unfolded fraction of MalE219 as a function of temperature. The normalized data were obtained from the measurement of the ellipticity at 222 nm with a scan rate of 0.5 °C min−1 and after baseline subtraction. The data are fitted to a sigmoid.

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REFERENCES

(1) Howorka, S.; Siwy, Z. Chem. Soc. Rev. 2009, 38, 2360−2384. (2) Branton, D.; et al. Nat. Biotechnol. 2008, 26, 1146−1153. (3) Wanunu, M.; Dadosh, T.; Ray, V.; Jin, J.; McReynolds, L.; Drndic, M. Nat. Nanotechnol. 2010, 5, 807−814. (4) Wang, Y.; Zheng, D.; Tan, Q.; Wang, M. X.; Gu, L.-Q. Nat. Nanotechnol. 2011, 6, 668−674. (5) Sutherland, T. C.; Long, Y.-T.; Stefureac, R.-I.; Bediako-Amoa, I.; Kraatz, H.-B.; Lee, J. S. Nano Lett. 2004, 4, 1273−1277. (6) Stefureac, R.; Long, Y.-t.; Kraatz, H.-B.; Howard, P.; Lee, J. S. Biochemistry 2006, 45, 9172−9179. (7) Wang, H.-Y.; Ying, Y.-L.; Li, Y.; Kraatz, H.-B.; Long, Y.-T. Anal. Chem. 2011, 83, 1746−1752. (8) Movileanu, L.; Schmittschmitt, J. P.; Martin Scholtz, J.; Bayley, H. Biophys. J. 2005, 89, 1030−1045. (9) Han, A.; Creus, M.; Schulermann, G.; Linder, V.; Ward, T. R.; de Rooij, N. F.; Staufer, U. Anal. Chem. 2008, 80, 4651−4658. (10) Sexton, L. T.; Horne, L. P.; Sherrill, S. A.; Bishop, G. W.; Baker, L. A.; Martin, C. R. J. Am. Chem. Soc. 2007, 129, 13144−13152. (11) Niedzwiecki, D. J.; Grazul, J.; Movileanu, L. J. Am. Chem. Soc. 2010, 132, 10816−10822. (12) Fologea, D.; Ledden, B.; McNabb, D. S.; Li, J. Appl. Phys. Lett. 2007, 91, 539011−539013. (13) Oukhaled, G.; Mathé, J.; Biance, A. L.; Bacri, L.; Betton, J. M.; Lairez, D.; Pelta, J.; Auvray, L. Phys. Rev. Lett. 2007, 98, 15801−15805. (14) Mohammad, M.; Movileanu, L. Eur. Biophys. J. 2008, 37, 913− 925.

display the CD thermal unfolding curve for MalE219. Although we may have different unfolding conformations, a sigmoid function is the best fit for the data obtained with both methods. We determine the melting temperature of MalE219: 44 °C ± 1 and 45.5 °C ± 0.5, respectively, for the nanopore technique and CD measurements. Thus, both techniques give values that are in good agreement.

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CONCLUSIONS In conclusion, we demonstrate that we can study thermal unfolding of proteins by electrical detection of single-molecule going through nanopores. For both aerolysin and α-hemolysin, as noticed previously, we detected mainly two types of events: (1) short events with high blockade currents corresponding to collisions which appear at any temperature and (2) long events with low blockade current and with duration associated to transport. These latter events arise when temperature increases and are therefore related to the thermally unfolded proteins crossing the nanopore. As expected, transport dynamics are sped up by thermal motion. Also, for both nanopores, the event frequency, linearly related to the proportion of unfolded proteins, varies as a function of temperature as a growing 4075



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(15) Makarov, D. E. Acc. Chem. Res. 2009, 42, 281−289. (16) Firnkes, M.; Pedone, D.; Knezevic, J.; Doblinger, J.; Rant, U. Nano Lett. 2010, 10, 2162−2167. (17) Pastoriza-Gallego, M.; Rabah, L.; Gibrat, G.; Thiebot, B.; van der Goot, F. G.; Auvray, L.; Betton, J.-M.; Pelta, J. J. Am. Chem. Soc. 2011, 133, 2923−2931. (18) Freedman, K. J.; Jürgens, M.; Prabhu, A.; Ahn, C. W.; Jemth, P.; Edel, J. B.; Kim, M. J. Anal. Chem. 2011, 83, 5137−5144. (19) Yusko, E. C.; Johnson, J. M.; Majd, S.; Prangkio, P.; Rollings, R. C.; Li, J.; Yang, J.; Mayer, M. Nat. Nanotechnol. 2011, 6, 253−260. (20) Talaga, D. S.; Li, J. J. Am. Chem. Soc. 2009, 131, 9287−9297. (21) Stefureac, R.; Waldner, L.; Howard, P.; Lee, J. S. Small 2008, 4, 59−63. (22) Baran, C.; Smith, G. S. T.; Bamm, V. V.; Harauz, G.; Lee, J. S. Biochem. Biophys. Res. Commun. 2010, 391, 224−9. (23) Dobson, C. M. Nature 2003, 426, 884−890. (24) Rapoport, T. A. Nature 2007, 450, 663−669. (25) Wickner, W.; Schekman, R. Science 2005, 310, 1452−1456. (26) Borgia, A.; Williams, P. M.; Clarke, J. Annu. Rev. Biochem. 2008, 77, 101−125. (27) Kapanidis, A. N.; Strick, T. Trends Biochem. Sci. 2009, 34, 234− 243. (28) Crampton, N.; Brockwell, D. J. Curr. Opin. Struct. Biol. 2010, 20, 508−517. (29) Merstoff, C.; Cressiot, B.; Pastoriza-Gallego, M.; Oukhaled, A.; Betton, J.-M.; Auvray, L.; Pelta, J. ACS Chem. Biol. 2012, DOI: 10.1021/cb2004737. (30) Song, L.; Hobaugh, M. R.; Shustak, C.; Cheley, S.; Bayley, H.; Gouaux, J. E. Science 1996, 274, 1859−1865. (31) Parker, M. W.; Buckley, J. T.; Postma, J. P. M; Tucker, A. D.; Leonard, K.; Pattus, F.; Tsernoglou, D. Nature 1994, 367, 292−295. (32) Tsitrin, Y.; Morton, C. J.; El Bez, C.; Paumard, P.; Velluz, M.-C.; Adrian, M.; Dubochet, J.; Parker, M. W.; Lanzavecchia, S.; van der Goot, F. G. Nat. Struct. Mol. Biol. 2002, 9, 729−733. (33) Miot, M.; Betton, J.-M. Microb. Cell Fact. 2004, 3, 4. (34) Spurlino, J. C.; Lu, G. Y.; Quiocho, F. A. J. Biol. Chem. 1991, 266, 5202−5219. (35) Bates, M.; Burns, M.; Meller, A. Biophys. J. 2003, 84, 2366− 2372. (36) Kang, X. f.; Gu, L. Q.; Cheley, S.; Bayley, H. Angew. Chem., Int. Ed. 2005, 44, 1495−1499. (37) Meller, A.; Branton, D. Electrophoresis 2002, 23, 2583−2591. (38) Jung, Y.; Bayley, H.; Movileanu, L. J. Am. Chem. Soc. 2006, 128, 15332−15340. (39) Chimerel, C.; Movileanu, L.; Pezeshki, S.; Winterhalter, M.; Kleinekathöfer, U. Eur. Biophys. J. 2008, 38, 121−125. (40) Biró, I.; Pezeshki, S.; Weingart, H.; Winterhalter, M.; Kleinekathöfer, U. Biophys. J. 2010, 98, 1830−1839. (41) Pedone, D.; Firnkes, M.; Rant, U. Anal. Chem. 2009, 81, 9689− 9694. (42) Kasianowicz, J.; Brandin, E.; Branton, D.; Deamer, D. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 13770−13773. (43) Meller, A.; Nivon, L.; Brandin, E.; Golovchenko, J.; Branton, D. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 1079−1084. (44) Pastoriza-Gallego, M.; Gibrat, G.; Thiebot, B.; Betton, J.-M.; Pelta, J. Biochim. Biophys. Acta, Biomembr. 2009, 1788, 1377−1386. (45) Mathé, J.; Aksimentiev, A.; Nelson, D. R.; Schulten, K.; Meller, A. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 12377−12382. (46) Merstorf, C.; Maciejak, O.; Mathé, J.; Pastoriza-Gallego, M.; Thiebot, B.; Clement, M.-J.; Curmi, P. A.; Pelta, J.; Auvray, L.; Savarin, P. Biochemistry, submitted for publication. (47) Mahendran, K.; Chimerel, C.; Mach, T.; Winterhalter, M. Eur. Biophys. J. 2009, 38, 1141−1145. (48) Rincon-Restrepo, M.; Mikhailova, E.; Bayley, H.; Maglia, G. Nano Lett. 2011, 11, 746−750.

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Thermal Unfolding of Proteins Probed at the Single ... - ACS Publications

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