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Unfolding study of native bacteriorhodopsin under acidic condition
ARTICLE IN PRESS Ultramicroscopy 109 (2009) 948–951
Contents lists available at ScienceDirect
Ultramicroscopy journal homepage: www.elsevier.com/locate/ultramic
Unfolding study of native bacteriorhodopsin under acidic condition Takashi Kodama a,b, Tatsuya Koyanagi a, Hiroshi Sekiguchi c, Atsushi Ikai c, Hiroyuki Ohtani a, a b c
Department of Biomolecular Engineering, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan Department of Mechanical Engineering, Stanford University, Stanford, CA 94305, USA Department of Life Science, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan
a r t i c l e in f o
PACS: 87.14.eq 87.64.Dz 87.80.Nj Keywords: Atomic force Microcopy Force curve measurement Single molecule bacteriorhodopsin Purple membrane Blue membrane
a b s t r a c t In this study, we measured the structural properties of ‘‘blue membrane’’, which is specific formation of a purple membrane (PM) upon acid titration or removal of cations, by the force curve measurement mode of an atomic force microscopy. The PM fragments were immobilized on a glass substrate and force curve measurements were carried out on the fragments under neutral (pH 7.2) and acidic (pH 2.4) condition. The results revealed that peak positions of the unfolding spectra obtained under acidic condition were shifted to the shorter extension region than those at neutral pH and that the relative position of only the first peak was changed by about 5 nm. These results suggest the possibility that the specific secondary structure is formed at the site from its C-terminus to helices F in acidified PM. & 2009 Elsevier B.V. All rights reserved.
1. Introduction Purple membrane (PM) is the lipid biomembrane of Halobacterium halobium, and it contains bacteriorhodopsin (bR) trimer [1]. The retinal chromophore of bR absorbs visible light and it can utilize the energy to transport protons against an electrochemical potential across the cell membrane via a sequential reaction through various intermediate state [2]. The bR has unique spectroscopic properties. For example, the absorption spectra are dramatically varied depending on the aqueous condition, such as pH and ionic strength [3,4]. Therefore, relation between its structural and spectroscopic properties has been studied by many researchers. Among them, ‘‘blue membrane (BM)’’ is a special formation of the PM upon acid titration or removal of cations [4–9]. Under acidic condition, the maximum absorption wavelength is shifted to 568–604 nm, and bR forms the BM (see Fig. 1a). While the peak shift is due to the change in the surface charge [6], the details have not been clarified. However, recent improvement of an atomic force microcopy (AFM) enables us to carry out the direct observation of biological samples under physiological condition [10,11]. Up to now, the topographic images of the bR trimer have been reported, and the unfolding pathways of a single bR molecule have been measured under various experimental conditions [12–16]. Therefore, in this study, we carried out the single molecule unfolding study of bR under
Corresponding author. Fax: +81 45 924 5816.
E-mail address: [email protected] (H. Ohtani). 0304-3991/$ - see front matter & 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ultramic.2009.03.006
neutral and acidic condition and comparison of two cases was made.
2. Experimental 2.1. AFM measurement The high-resolution topographic imaging and force curve measurements were carried out by Nanoscope 3a system (Veeco Instruments, NY) and Smena liquid head (NT-MDT, Russia) [17], respectively. The precise value of the spring constant was obtained with thermal fluctuation method [18] after all experiments. The z calibration of piezo elements was performed with laser interferometric method [19] for the force curve measurements. 2.2. Sample preparation The PM was purified by the method reported by Oesterhelt et al. [1], and the membrane fragments were immobilized on a substrate surface by the following two methods. First, after dilution of the sample with buffer solution, the fragments were adsorbed on a freshly cleaved mica surface. Tris–HCl buffer with 300 mM KCl and 300 mM sodium citrate buffer were used for the adjustment of the pH of the liquid phase to 7.6 and 3.4, respectively. Second, the fragments were also immobilized on a glass surface according to the following procedure [17]. The glass surface was modified with 3-(diethoxymethylsilyl)propylamine
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Fig. 1. Spectroscopic and structural information of bacteriorhodopsin molecules. (a) Absorption spectra of purple membranes under different aqueous condition. The solid and dotted line represent the absorption spectra of the light-adapted and the dark-adapted purple membrane at neutral pH, respectively. The broken line corresponds to that of purple membrane under acidic condition (blue membrane). (b) Schematic of a native bR molecule.
Fig. 2. The topographic images of purple membranes observed by an AFM. The panels (a) and (b) are the high-resolution topographic images measured on a fleshly cleaved mica surface at pH 7.6 and 3.4, respectively. On the other hand, the panels (c) and (d) shows the purple membranes immobilized on a glass surface at pH 7.2 and 2.4, respectively. These images were acquired by the tapping mode measurement.
by silane coupling method, and disuccinimidyl suberate (DSS) was reacted with –NH2 groups of the substrate surface. After washing the surface well, the substrate was immersed into the PM stock solution overnight.
2.3. Experimental condition In the high-resolution topographic imaging, we used Tris–HCl buffer with 300 mM KCl (pH 7.6) and 300 mM sodium citrate (pH 3.4) buffer for the experiments under neutral and acidic
condition, respectively. All topographic images were acquired by tapping measurement mode of the AFM system. On the other hand, all force curve measurements were carried out in liquid, and the pH of the liquid phase was adjusted to pH 7.2 and 2.4 with phosphate-buffered saline and citrate buffer with 150 mM NaCl, respectively. The ionic strength was kept constant in all the experiments. After visualization of membrane fragments on the substrate, force curve measurements were carried out on the membrane surface. The velocity of an AFM probe was 3.5 mm/s. All force scans were analyzed after the conversion of the data to force–extension curves. All experiments were performed for many
ARTICLE IN PRESS 950
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times, and only the force–extension curves exhibiting a full length above 60 nm were selected and analyzed in 10% of all retraction curve we detected an adhesion peak.
3. Results and discussion Figs. 2a and b are the topographic images of PM fragments measured under neutral and acidic condition, respectively. The figures show that the regular bR trimer structure was visualized under neutral condition. These results are consistent with the previous studies reported elsewhere [12–16]. On the other hand, the single bR molecules have not been observed under acidic condition. The membrane fragments could not be made flat on mica surface because of their strong aggregation. Furthermore, direction of the PM fragment cannot be controlled by using this sample preparation procedure. Thus, PM fragments were immobilized on a glass substrate by chemical bonding between the silanized glass surface and the N-terminus of bR molecules [17]. Figs. 2c and d are the topographic images of PMs on the glass substrate obtained at neutral and acidic pH, respectively. The results show that single fragments are flatly expanded on the substrate under both conditions. The chemical immobilization prevents the PM fragments from their aggregation under acidic condition. Furthermore, it is considered that the cytoplasmic surface of the PM fragments is made up because the NH2 residues of bR molecules in the PM fragments is chemically attached to the substrate surface. Force curve measurements were carried out with the sample under each liquid phase, and single molecule unfolding spectroscopy attempted to be performed. Figs. 3a and b are the representative force–extension curves of single bR molecules observed at pH 7.2 and 2.4, respectively. In both cases, large adhesion force was observed in lower extension region (o20 nm). This force originates from the interaction between the tip and lipid bilayer surface. At neutral pH, typical well-pronounced three peaks were measured at about 28, 47 and 63 nm. Hereafter, these peaks are called the first, the second and the third peak, respectively. Those exactly correspond to the previous studies reported elsewhere [12,14]. The analysis with the worm-like chain model [20,21] revealed that these peaks originate from unfolding of the helices E and D, B and C, and A, respectively. Further, it has been reported that unfolding of the helices G and F is included in low extension region below 20 nm [14]. In this study, this peak could not be observed because of the large adhesion force below o20 nm. On the other hand, at acidic pH, typical three peaks were also observed, but it is clearly shown in the representative force–extension curves (see Fig. 3b) that the relative positions of these peaks were different. The positions of first and second peak are at 22.5 and 41.5 nm, respectively. They are shifted to the lower extension region than those at neutral pH. Furthermore, two types of results were obtained for the third peak. One is observed at about 55 nm, which is also shifted to lower extension region (see solid line with solid squares in Fig. 3b), and the other is measured at 65 nm, which is almost same observed at neutral pH (see dotted line with open circles in Fig. 3b). The averaged (N ¼ 6) first, second and third peak positions in an unfolding spectrum of acidified bR were 21.87, 41.45 and 57.41 nm, respectively. These averaged values correspond to each peak position of the representative unfolding spectra shown in Fig. 3b except for the third peak position because two types of curves are mixed as we described above. In order to make the comparison of two cases, each representative curve was overlapped with the data obtained at neutral pH (see Fig. 4). Further, the peak-to-peak distance between them was estimated in the figure. In Fig. 4a, it is clearly shown that all peaks of the acidified bR were shifted by about
Fig. 3. The representative unfolding spectra of bR molecules. (a) The solid line with solid squares and dotted line with open circles are force–extension curves of bRs measured at pH 7.2. (b) The solid line, thin line, solid line with solid squares and dotted line with open circles are force–extension curves measured at pH 2.4.
6 nm. However, estimation of the relative distance between these peaks suggests that only the position of the first peak is changed and it influences the position of the other peaks. As we said above, each peak in the force–extension curve originates from the unfolding process of bR subunit. Therefore, its structural change can be qualitatively discussed by the comparison of the peak-topeak distance change. It is considered that the 3-dimensional structure of acidified bR molecules at the site from the C-terminus to Ser158 (loop from the C-terminus to helices G, helices G, loop between helices F and G, helices F and loop between helices E and F) is different from that of neutral bR. However, the amino acid sequence is the same in both conditions. Thus, it is concluded that the unique secondary structure is formed at this site of an acidified bR. This specific structure is probably stable against the extension force and is kept during the unfolding process. This is the reason why only the position of first peak is shifted to lower extension regions. On the other hand, Fig. 4b shows that the first peak is also shifted by about 6 nm at acidic pH but that the third peak is appeared at 65 nm, which corresponds to the full extension of bR. In this case, the specific acidified bR structure may be disrupted simultaneously with the unfolding process of helices A. Previous study has revealed that such a unique structural change was not observed at pH 4.2 [14]. Therefore, it is probably a unique structural property of acidified bR in BM. Side chain pKa of glutamic and aspartic acid residues are below o4.5.
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stable in the lipid bilayer. From these previous studies, it is considered that the 3D structure formed from the C-terminus to helices F is unstable compared with the other subunits and that conformation of the parts is changeable by external perturbation. The present experimental results are compatible with these previous studies. Therefore, it is concluded qualitatively that structural change occurred from its C-terminus to helices F induces the unique spectroscopic property alternation of an acidified bR.
4. Conclusion In this study, we carried out the single molecule unfolding study of an acidified bR molecule, which is formed of bR under acidic condition. The results revealed that only the first peak in the unfolding spectrum of an acidified bR is shifted to lower extension region compared with the spectra measured at neutral pH. The results suggest that stable secondary structure is formed at this part of an acidified bR. It is concluded that the unique spectroscopic property of an acidified bR molecule originates from the structural alternation of the site from the C-terminus to helices F.
Acknowledgements The authors express their sincere gratitude to Professor Masamichi Fujihira, Tokyo Institute of Technology (T.I.T.), for his encouragement and fruitful discussions through out this work. The authors greatly acknowledge the support from the JSPS postdoctoral fellowship for research abroad (Engineering, no. 13). This study was supported in part by a Grant-in-Aid for Creative Scientific Research (no. 19GS0418) from the Japan Society for Promotion of Science. Fig. 4. The comparison of representative unfolding spectra measured at different pH. The dotted lines with open circles and the solid lines with solid squares in these figures represent the force–extension curves measured at pH 7.2 and 2.4, respectively (see Fig. 3). Common unfolding spectrum measured at pH 7.2 is shown as a reference in both panels, and two types of representative spectrum measured at pH 2.4 are shown in panels (a) and (b).
Therefore, the ionization state of these residues is changed at this condition (pH 2.4). Those residues are concentrated in the strands from the C-terminus to helices F. It is well known that the amino acid residues tend to form a stable secondary structure when those are protonated [22]. For example, four glutamic acid residues are concentrated in the strands from its C-terminus to S226. If the strand forms alpha helices, the first peak position would be shifted to lower extension region by approx. 5 nm (22 (0.360.15)). This value corresponds to the experimental results. Further, it has been reported that cation binding to the specific site, such as Ala196, induced conformational alteration of the F–G interhelical loop and the change of spectroscopic property originates from the induced structural change [7–9]. Two acidic amino acid residues, Glu194 and Glu204, are located around the site. Thus, it is also considered that the statistical structural alternation may be occurred at the site. Furthermore, it has been reported that short polypeptide strands corresponding to bR helices F and G does not form stable transmembrane alpha helices in reconstituted phospholipid vesicles while any other strands A–E forms it spontaneously [16,23]. In the report, the author suggested that external force from any other strands make them
References [1] D. Oesterhelt, W. Stoeckenius, Methods Enzymol. 31 (1974) 667. [2] K. Gerwert, G. Souvignier, B. Hess, Proc. Natl. Acad. Sci. USA 87 (1990) 9774. [3] P.C. Mowery, R.H. Lozier, Q. Chae, Y.-W. Tseng, M. Taylor, W. Stoeckenius, Biochemistry 18 (1979) 4100. [4] D. Oesterhelt, W. Stoeckenius, Nat. New Biol. 233 (1971) 149. [5] H. Ohtani, T. Kobayashi, J.-I. Iwai, A. Ikegami, Biochemistry 25 (1986) 3356. [6] I. Szundi, W. Stoeckenius, Biophys. J. 56 (1989) 369. [7] N.Y. Zhang, M.A. El-Sayed, Biochemistry 32 (1993) 14173. [8] S. Tuzi, A. Naito, H. Saitoˆ, Biochemistry 33 (1994) 15046. [9] S. Tuzi, S. Yamaguchi, M. Tanio, H. Konishi, S. Inoue, A. Naito, R. Needleman, J.K. Lanyi, H. Saitoˆ, Biophys. J. 76 (1999) 1523. [10] G. Binnig, C.F. Quate, Ch. Gerber, Phys. Rev. Lett. 56 (1986) 930. [11] P.K. Hansma, V.B. Elings, O. Marti, C.E. Bracker, Science 242 (1988) 209. [12] F. Oesterhelt, D. Oesterhelt, M. Pfeiffer, A. Engel, H.E. Gaub, D.J. Mu¨ller, Science 288 (2000) 143. [13] D.J. Mu¨ller, J.B. Heymann, F. Oesterhelt, C. Mo¨ller, H. Gaub, G. Bu¨ldt, A. Engel, Biochim. Biophys. Acta Bioenergetics 1460 (2000) 27. [14] D.J. Mu¨ller, M. Kessler, F. Oesterhelt, C. Mo¨ller, D. Oesterhelt, H. Gaub, Biophys. J. 83 (2002) 3578. [15] H. Janovjak, M. Kessler, D. Oesterhelt, H. Gaub, D.J. Mu¨ller, EBMO J. 22 (2003) 5220. [16] M. Kessler, H.E. Gaub, Structure 14 (2006) 521. [17] T. Kodama, H. Ohtani, H. Arakawa, A. Ikai, Appl. Phys. Lett. 86 (2005) 043901. [18] J.L. Hutter, J. Bechhoefer, Rev. Sci. Instrum. 84 (1993) 1868. [19] M. Jaschke, H.-J. Butt, Rev. Sci. Instrum. 66 (1995) 1258. [20] J.F. Marko, E.D. Siggia, Macromolecules 28 (1995) 8759. [21] M. Rief, M. Gautel, F. Oesterhelt, J.M. Fernandez, H.E. Gaub, Science 276 (1997) 1109. [22] A. Idiris, M.T. Alam, A. Ikai, Protein Eng. 13 (2000) 763. [23] J.F. Hunt, P. Rath, K.J. Rothschild, D.M. Engelman, Biochemistry 36 (1997) 15177.
Contents lists available at ScienceDirect
Ultramicroscopy journal homepage: www.elsevier.com/locate/ultramic
Unfolding study of native bacteriorhodopsin under acidic condition Takashi Kodama a,b, Tatsuya Koyanagi a, Hiroshi Sekiguchi c, Atsushi Ikai c, Hiroyuki Ohtani a, a b c
Department of Biomolecular Engineering, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan Department of Mechanical Engineering, Stanford University, Stanford, CA 94305, USA Department of Life Science, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan
a r t i c l e in f o
PACS: 87.14.eq 87.64.Dz 87.80.Nj Keywords: Atomic force Microcopy Force curve measurement Single molecule bacteriorhodopsin Purple membrane Blue membrane
a b s t r a c t In this study, we measured the structural properties of ‘‘blue membrane’’, which is specific formation of a purple membrane (PM) upon acid titration or removal of cations, by the force curve measurement mode of an atomic force microscopy. The PM fragments were immobilized on a glass substrate and force curve measurements were carried out on the fragments under neutral (pH 7.2) and acidic (pH 2.4) condition. The results revealed that peak positions of the unfolding spectra obtained under acidic condition were shifted to the shorter extension region than those at neutral pH and that the relative position of only the first peak was changed by about 5 nm. These results suggest the possibility that the specific secondary structure is formed at the site from its C-terminus to helices F in acidified PM. & 2009 Elsevier B.V. All rights reserved.
1. Introduction Purple membrane (PM) is the lipid biomembrane of Halobacterium halobium, and it contains bacteriorhodopsin (bR) trimer [1]. The retinal chromophore of bR absorbs visible light and it can utilize the energy to transport protons against an electrochemical potential across the cell membrane via a sequential reaction through various intermediate state [2]. The bR has unique spectroscopic properties. For example, the absorption spectra are dramatically varied depending on the aqueous condition, such as pH and ionic strength [3,4]. Therefore, relation between its structural and spectroscopic properties has been studied by many researchers. Among them, ‘‘blue membrane (BM)’’ is a special formation of the PM upon acid titration or removal of cations [4–9]. Under acidic condition, the maximum absorption wavelength is shifted to 568–604 nm, and bR forms the BM (see Fig. 1a). While the peak shift is due to the change in the surface charge [6], the details have not been clarified. However, recent improvement of an atomic force microcopy (AFM) enables us to carry out the direct observation of biological samples under physiological condition [10,11]. Up to now, the topographic images of the bR trimer have been reported, and the unfolding pathways of a single bR molecule have been measured under various experimental conditions [12–16]. Therefore, in this study, we carried out the single molecule unfolding study of bR under
Corresponding author. Fax: +81 45 924 5816.
E-mail address: [email protected] (H. Ohtani). 0304-3991/$ - see front matter & 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ultramic.2009.03.006
neutral and acidic condition and comparison of two cases was made.
2. Experimental 2.1. AFM measurement The high-resolution topographic imaging and force curve measurements were carried out by Nanoscope 3a system (Veeco Instruments, NY) and Smena liquid head (NT-MDT, Russia) [17], respectively. The precise value of the spring constant was obtained with thermal fluctuation method [18] after all experiments. The z calibration of piezo elements was performed with laser interferometric method [19] for the force curve measurements. 2.2. Sample preparation The PM was purified by the method reported by Oesterhelt et al. [1], and the membrane fragments were immobilized on a substrate surface by the following two methods. First, after dilution of the sample with buffer solution, the fragments were adsorbed on a freshly cleaved mica surface. Tris–HCl buffer with 300 mM KCl and 300 mM sodium citrate buffer were used for the adjustment of the pH of the liquid phase to 7.6 and 3.4, respectively. Second, the fragments were also immobilized on a glass surface according to the following procedure [17]. The glass surface was modified with 3-(diethoxymethylsilyl)propylamine
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Fig. 1. Spectroscopic and structural information of bacteriorhodopsin molecules. (a) Absorption spectra of purple membranes under different aqueous condition. The solid and dotted line represent the absorption spectra of the light-adapted and the dark-adapted purple membrane at neutral pH, respectively. The broken line corresponds to that of purple membrane under acidic condition (blue membrane). (b) Schematic of a native bR molecule.
Fig. 2. The topographic images of purple membranes observed by an AFM. The panels (a) and (b) are the high-resolution topographic images measured on a fleshly cleaved mica surface at pH 7.6 and 3.4, respectively. On the other hand, the panels (c) and (d) shows the purple membranes immobilized on a glass surface at pH 7.2 and 2.4, respectively. These images were acquired by the tapping mode measurement.
by silane coupling method, and disuccinimidyl suberate (DSS) was reacted with –NH2 groups of the substrate surface. After washing the surface well, the substrate was immersed into the PM stock solution overnight.
2.3. Experimental condition In the high-resolution topographic imaging, we used Tris–HCl buffer with 300 mM KCl (pH 7.6) and 300 mM sodium citrate (pH 3.4) buffer for the experiments under neutral and acidic
condition, respectively. All topographic images were acquired by tapping measurement mode of the AFM system. On the other hand, all force curve measurements were carried out in liquid, and the pH of the liquid phase was adjusted to pH 7.2 and 2.4 with phosphate-buffered saline and citrate buffer with 150 mM NaCl, respectively. The ionic strength was kept constant in all the experiments. After visualization of membrane fragments on the substrate, force curve measurements were carried out on the membrane surface. The velocity of an AFM probe was 3.5 mm/s. All force scans were analyzed after the conversion of the data to force–extension curves. All experiments were performed for many
ARTICLE IN PRESS 950
T. Kodama et al. / Ultramicroscopy 109 (2009) 948–951
times, and only the force–extension curves exhibiting a full length above 60 nm were selected and analyzed in 10% of all retraction curve we detected an adhesion peak.
3. Results and discussion Figs. 2a and b are the topographic images of PM fragments measured under neutral and acidic condition, respectively. The figures show that the regular bR trimer structure was visualized under neutral condition. These results are consistent with the previous studies reported elsewhere [12–16]. On the other hand, the single bR molecules have not been observed under acidic condition. The membrane fragments could not be made flat on mica surface because of their strong aggregation. Furthermore, direction of the PM fragment cannot be controlled by using this sample preparation procedure. Thus, PM fragments were immobilized on a glass substrate by chemical bonding between the silanized glass surface and the N-terminus of bR molecules [17]. Figs. 2c and d are the topographic images of PMs on the glass substrate obtained at neutral and acidic pH, respectively. The results show that single fragments are flatly expanded on the substrate under both conditions. The chemical immobilization prevents the PM fragments from their aggregation under acidic condition. Furthermore, it is considered that the cytoplasmic surface of the PM fragments is made up because the NH2 residues of bR molecules in the PM fragments is chemically attached to the substrate surface. Force curve measurements were carried out with the sample under each liquid phase, and single molecule unfolding spectroscopy attempted to be performed. Figs. 3a and b are the representative force–extension curves of single bR molecules observed at pH 7.2 and 2.4, respectively. In both cases, large adhesion force was observed in lower extension region (o20 nm). This force originates from the interaction between the tip and lipid bilayer surface. At neutral pH, typical well-pronounced three peaks were measured at about 28, 47 and 63 nm. Hereafter, these peaks are called the first, the second and the third peak, respectively. Those exactly correspond to the previous studies reported elsewhere [12,14]. The analysis with the worm-like chain model [20,21] revealed that these peaks originate from unfolding of the helices E and D, B and C, and A, respectively. Further, it has been reported that unfolding of the helices G and F is included in low extension region below 20 nm [14]. In this study, this peak could not be observed because of the large adhesion force below o20 nm. On the other hand, at acidic pH, typical three peaks were also observed, but it is clearly shown in the representative force–extension curves (see Fig. 3b) that the relative positions of these peaks were different. The positions of first and second peak are at 22.5 and 41.5 nm, respectively. They are shifted to the lower extension region than those at neutral pH. Furthermore, two types of results were obtained for the third peak. One is observed at about 55 nm, which is also shifted to lower extension region (see solid line with solid squares in Fig. 3b), and the other is measured at 65 nm, which is almost same observed at neutral pH (see dotted line with open circles in Fig. 3b). The averaged (N ¼ 6) first, second and third peak positions in an unfolding spectrum of acidified bR were 21.87, 41.45 and 57.41 nm, respectively. These averaged values correspond to each peak position of the representative unfolding spectra shown in Fig. 3b except for the third peak position because two types of curves are mixed as we described above. In order to make the comparison of two cases, each representative curve was overlapped with the data obtained at neutral pH (see Fig. 4). Further, the peak-to-peak distance between them was estimated in the figure. In Fig. 4a, it is clearly shown that all peaks of the acidified bR were shifted by about
Fig. 3. The representative unfolding spectra of bR molecules. (a) The solid line with solid squares and dotted line with open circles are force–extension curves of bRs measured at pH 7.2. (b) The solid line, thin line, solid line with solid squares and dotted line with open circles are force–extension curves measured at pH 2.4.
6 nm. However, estimation of the relative distance between these peaks suggests that only the position of the first peak is changed and it influences the position of the other peaks. As we said above, each peak in the force–extension curve originates from the unfolding process of bR subunit. Therefore, its structural change can be qualitatively discussed by the comparison of the peak-topeak distance change. It is considered that the 3-dimensional structure of acidified bR molecules at the site from the C-terminus to Ser158 (loop from the C-terminus to helices G, helices G, loop between helices F and G, helices F and loop between helices E and F) is different from that of neutral bR. However, the amino acid sequence is the same in both conditions. Thus, it is concluded that the unique secondary structure is formed at this site of an acidified bR. This specific structure is probably stable against the extension force and is kept during the unfolding process. This is the reason why only the position of first peak is shifted to lower extension regions. On the other hand, Fig. 4b shows that the first peak is also shifted by about 6 nm at acidic pH but that the third peak is appeared at 65 nm, which corresponds to the full extension of bR. In this case, the specific acidified bR structure may be disrupted simultaneously with the unfolding process of helices A. Previous study has revealed that such a unique structural change was not observed at pH 4.2 [14]. Therefore, it is probably a unique structural property of acidified bR in BM. Side chain pKa of glutamic and aspartic acid residues are below o4.5.
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stable in the lipid bilayer. From these previous studies, it is considered that the 3D structure formed from the C-terminus to helices F is unstable compared with the other subunits and that conformation of the parts is changeable by external perturbation. The present experimental results are compatible with these previous studies. Therefore, it is concluded qualitatively that structural change occurred from its C-terminus to helices F induces the unique spectroscopic property alternation of an acidified bR.
4. Conclusion In this study, we carried out the single molecule unfolding study of an acidified bR molecule, which is formed of bR under acidic condition. The results revealed that only the first peak in the unfolding spectrum of an acidified bR is shifted to lower extension region compared with the spectra measured at neutral pH. The results suggest that stable secondary structure is formed at this part of an acidified bR. It is concluded that the unique spectroscopic property of an acidified bR molecule originates from the structural alternation of the site from the C-terminus to helices F.
Acknowledgements The authors express their sincere gratitude to Professor Masamichi Fujihira, Tokyo Institute of Technology (T.I.T.), for his encouragement and fruitful discussions through out this work. The authors greatly acknowledge the support from the JSPS postdoctoral fellowship for research abroad (Engineering, no. 13). This study was supported in part by a Grant-in-Aid for Creative Scientific Research (no. 19GS0418) from the Japan Society for Promotion of Science. Fig. 4. The comparison of representative unfolding spectra measured at different pH. The dotted lines with open circles and the solid lines with solid squares in these figures represent the force–extension curves measured at pH 7.2 and 2.4, respectively (see Fig. 3). Common unfolding spectrum measured at pH 7.2 is shown as a reference in both panels, and two types of representative spectrum measured at pH 2.4 are shown in panels (a) and (b).
Therefore, the ionization state of these residues is changed at this condition (pH 2.4). Those residues are concentrated in the strands from the C-terminus to helices F. It is well known that the amino acid residues tend to form a stable secondary structure when those are protonated [22]. For example, four glutamic acid residues are concentrated in the strands from its C-terminus to S226. If the strand forms alpha helices, the first peak position would be shifted to lower extension region by approx. 5 nm (22 (0.360.15)). This value corresponds to the experimental results. Further, it has been reported that cation binding to the specific site, such as Ala196, induced conformational alteration of the F–G interhelical loop and the change of spectroscopic property originates from the induced structural change [7–9]. Two acidic amino acid residues, Glu194 and Glu204, are located around the site. Thus, it is also considered that the statistical structural alternation may be occurred at the site. Furthermore, it has been reported that short polypeptide strands corresponding to bR helices F and G does not form stable transmembrane alpha helices in reconstituted phospholipid vesicles while any other strands A–E forms it spontaneously [16,23]. In the report, the author suggested that external force from any other strands make them
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