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Nuclear magnetic resonance approaches for characterizing interactions between the bacterial chaperonin GroEL and unstructured proteins
Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e5, 2013 www.elsevier.com/locate/jbiosc
Nuclear magnetic resonance approaches for characterizing interactions between the bacterial chaperonin GroEL and unstructured proteins Noritaka Nishida,1, 2, y Maho Yagi-Utsumi,2, 3, y Fumihiro Motojima,4 Masasuke Yoshida,4 Ichio Shimada,1 and Koichi Kato2, 3, * Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan,1 Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan,2 Institute for Molecular Science and Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki 444-8787, Japan,3 and Department of Molecular Biosciences, Faculty of Life Sciences, Kyoto Sangyo University, Kita-ku, Kyoto 603-8555, Japan4 Received 27 December 2012; accepted 19 February 2013 Available online xxx
GroELeprotein interactions were characterized by stable isotope-assisted nuclear magnetic resonance (NMR) spectroscopy using chemically denatured bovine rhodanese and an intrinsically disordered protein, a-synuclein, as model ligands. NMR data indicated that proteins tethered to GroEL remain largely unfolded and highly mobile, enabling identification of the interaction hot spots displayed on intrinsically disordered proteins. Ó 2013, The Society for Biotechnology, Japan. All rights reserved. [Key words: GroEL; Nuclear magnetic resonance; Molecular chaperone; Intrinsically disordered protein; Rhodanese; a-Synuclein; Denaturation]
Molecular chaperones are involved in various cellular processes in which proteins undergo folding, unfolding, and/or refolding under physiological and stress conditions (1). It has been proposed that molecular chaperones actively contribute to the suppression of toxic aggregate formation of various amyloidogenic proteins associated with neurodegenerative disorders (2). Furthermore, molecular chaperones have attracted attention due to their potential applicabilities as intelligent nanodevices in bioengineering fields (3). The chaperonin of Escherichia coli GroEL, which is one of the most extensively studied molecular chaperones, forms a large cylindrical complex that assists in the folding of nascent polypeptides and the recovery of proteins after heat shock or other stresses by preventing aggregation (1). GroEL is composed of 14 identical subunits with a molecular mass of 57 kDa that are arranged as two heptameric rings stacked back-to-back (4). The GroEL double-ring complex contains a central cavity with a diameter of approximately 45 A and interacts in an ATP-dependent manner with one or two GroES, a co-chaperone comprising a single heptameric ring of identical 10-kDa subunits (1). Each GroEL subunit is composed of three domains: apical, intermediate, and equatorial domains (4).
* Corresponding author at: Institute for Molecular Science and Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki 444-8787, Japan. Tel.: þ81 564 59 5225; fax: þ81 564 59 5224. E-mail addresses: [email protected], [email protected] (K. Kato). y The first two authors contributed equally to the work. Abbreviations: aSN, a-synuclein; DTT, dithiothreitol; GdmCl, guanidium chloride; HSQC, heteronuclear single-quantum coherence; Rho, rhodanese; SR1, singlering variant of GroEL.
The equatorial domain contains the nucleotide-binding site and mediates intersubunit contacts within and between rings. The apical domain constitutes the rim of the central cavity and provides binding sites for polypeptide substrates and GroES. The intermediate domain acts as a hinge connecting the first two domains. Protein recognition by GroEL is primarily characterized by hydrophobic interactions involving exposed hydrophobic residues of the substrates (5). Based on transferred nuclear Overhauser effect data, it has been shown that an N-terminal peptide derived from bovine rhodanese (Rho), a stringent GroEL substrate, interacted with the apical domain of GroEL in an a-helical conformation (6). On the other hand, a crystallographic study indicated that a phage display-derived peptide was accommodated on the apical domain, adopting a b-hairpin conformation (7). Thus, model peptide studies suggest that both a-helices and b-strands can exhibit GroEL-binding motifs. To elucidate the nature of substrate binding, hydrogen/deuterium exchange monitored by mass spectrometry and nuclear magnetic resonance (NMR) have been applied to proteins bound to GroEL (8,9). These studies showed that a wide range of folding intermediates can interact with GroEL, from ones very early in the folding pathway to late ones that may be native-like. More recently, direct NMR observations have been reported for substrate proteins bound to the single-ring variant of GroEL (SR1) with a molecular mass of 400 kDa (10,11). In these studies, a uniformly 2H- and 15N-labeled substrate (Rho or human dihydrofolate reductase) denatured and then trapped by SR1 was subjected to transverse relaxation-optimized spectroscopy-based NMR measurements. The NMR data suggested that the GroEL-bound substrates include largely disordered regions with higher mobility,
1389-1723/$ e see front matter Ó 2013, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2013.02.012
Please cite this article in press as: Nishida, N., et al., Nuclear magnetic resonance approaches for characterizing interactions between the bacterial chaperonin GroEL and unstructured proteins, J. Biosci. Bioeng., (2013), http://dx.doi.org/10.1016/j.jbiosc.2013.02.012
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which gave observable NMR peaks although spectral assignments could not be made. A detailed understanding of the molecular mechanisms underlying the versatile interactions of GroEL with protein ligands not only gives insights into biological functions of mammalian chaperonins but also facilitates their bioengineering applications in development of bioresponsive nanodevices (12). In view of the situations, we herein report our methodological approaches for further characterization of GroEL-bound states of proteins by direct NMR observation of huge protein complexes with a molecular mass of >800 kDa. We used two model ligands, i.e., chemically denatured Rho and an intrinsically disordered protein, a-synuclein (aSN). The latter protein was identified as the major component of the Lewy bodies of Parkinson’s disease (13). On the basis of NMR data, we discuss the conformational state of these proteins forming a complex with GroEL.
MATERIALS AND METHODS Expression and purification of recombinant proteins GroEL was overexpressed in E. coli BL21(DE3) and purified as described previously (14). Uniformly 15Nlabeled recombinant protein of human aSN was expressed in E. coli BL21(DE3) cells cultivated in M9 minimal medium containing [15N]NH4Cl and purified as described previously (15). Bovine Rho labeled with 13C at the Ca carbon of selected amino acid residues was expressed in E. coli BL21(DE3) cells grown in minimal medium supplemented with amino acids, vitamins, minerals, and 4 g of D-glucose according to the protocol described in the literature (14). To the medium, 13Caenriched amino acid was added instead of the unlabeled corresponding amino acid. The cells were grown to an OD600 of 0.6 at 37 C, induced with 0.4 mM isopropyl-b-D-thiogalactopyranoside, and incubated for an additional 12 h at 27 C. Unlabeled Rho was expressed in M2 YT medium containing 50 mg/mL of ampicillin. Rho was purified as described previously (16).
J. BIOSCI. BIOENG., Circular dichroism (CD) measurements CD spectra were measured at room temperature on Jasco J-725 apparatus using a 1.0-mm path length quartz cell.
RESULTS AND DISCUSSION Conformational state of Rho bound to GroEL It has been established that protein secondary structures can be identified on the basis of chemical shift information of the backbone NMR signals (18). In particular, secondary Ca chemical shifts offer a useful tool for the determination of the secondary structures. In general, Ca atoms involved in a-helices and b-sheets exhibit downfield and upfield shifts, respectively, with respect to those located in a random coil. We attempted to estimate the secondary structures of Rho in the GroEL-bound state on the basis of secondary Ca chemical shift data. For this purpose, we prepared recombinant Rho with 13C labeling at the Ca carbons of selected 11 Tyr, 16 Lys, 25 Leu, or 15 Phe residues. These amino acid residues number 67 in total and are spread over the whole Rho molecule, which comprises 296 amino acid residues (Fig. S1). Fig. 1 exemplifies the 13C NMR spectra of [13Ca]Tyr Rho in the native, urea-denatured, and GroEL-bound states. In the spectrum of native [13Ca]Tyr Rho, 7 and 4 signals (estimated based on the integral ratio) exhibited downfield and upfield shifts, respectively, with respect to the highly degenerate Ca signals observed in the presence of 8 M urea. [13Ca]Tyr Rho, once unfolded in 6 M GdmCl and complexed with GroEL, exhibited remarkable line-broadening but still gave observable Ca signals despite the huge size of the complex
Preparation of GroELeRho complex Rho was lyophilized in 10 mM sodium phosphate (pH 7.3) containing 100 mM KCl and 10 mM MgCl2, dissolved in 6 M guanidium chloride (GdmCl) and 10 mM dithiothreitol (DTT) to a protein concentration of 2.2 mg/mL, and incubated at room temperature for 30 min. To minimize denaturation of GroEL due to locally high concentration of GdmCl, the solution containing denatured Rho was gradually diluted to 25-fold with 10 mM sodium phosphate buffer (pH 7.3) containing 100 mM KCl, 10 mM MgCl2, 10 mM DTT, and 1 mg/mL of GroEL. The mixture was dialyzed against 10 mM sodium phosphate (pH 7.3) containing 100 mM KCl, 10 mM MgCl2, and 10 mM DTT. Formation of the GroELeRho complex was checked by gel filtration using a Superose 12 column (Amersham Pharmacia Biotech). Rho was released from GroEL as a native monomer after incubation with GroES and ATP according to the protocol described in the literature (17), confirming a 1:1 stoichiometry of the GroELeRho complex. NMR measurements For 13C NMR measurements, the proteins were concentrated to a final volume of 2 mL in 10 mM sodium phosphate (pH 7.3) containing 100 mM KCl, 3 mM NaN3, and 10% (v/v) 2H2O and transferred to the 10-mm NMR sample tube (Shigemi). The 13C spectrum of denatured Rho was measured by adding 8 M urea to the sample solution. One-dimensional 13C NMR spectra were recorded at 25 C using a Bruker AMX-400 spectrometer equipped with a probe head specialized for 13C observation with 10-mm bore size using a WALTZ proton decoupling pulse train. For each spectrum, 40,000e80,000 transients of free induction decay after 30 pulse with a repetition period of 1 s were accumulated with 16 K data points and a spectral width of 24,000 Hz. Prior to Fourier transformation, the accumulated free induction decays were multiplied by an exponential window function with line-broadening factors of 30 Hz and 2 Hz to observe peaks originating from the GroEL-bound and -unbound forms of Rho, respectively. 13C chemical shift values are given in parts per million (ppm) calibrated with internal reference 1,4-dioxane at 67.8 ppm. Spectrum of the 13Clabeled Rho in complex with GroEL was presented after subtraction of that of the unlabeled labeled counterpart in order to cancel out the signal from natural isotope abundance of 13C. For 1He15N heteronuclear single-quantum coherence (HSQC) measurements, isotopically labeled aSN was dissolved to a concentration of 50 mM in 10 mM sodium phosphate buffer (pH 7.0) containing 100 mM NaCl and 10% (v/v) 2H2O in the presence or absence of 25 mM GroEL. HSQC measurements were performed at 10 C on a JEOL ECA-920 spectrometer. The spectra were recorded at a 1H observation frequency of 920 MHz with 256 (t1) 2048 (t2) complex points and 64 scans per t1 increment. The spectral width was 2800 Hz for the 15N dimension and 13,830 Hz for the 1H dimension. 1H chemical shifts were referenced to external 2,2-dimetyl-2silapentane-5-sulfonic acid, while 15N chemical shifts were indirectly referenced to 2,2-dimetyl-2-silapentane-5-sulfonic acid using the absolute frequency ratio.
FIG. 1. Ca regions of 13C NMR spectra of [13Ca]Tyr Rho in native (top), urea-denatured (middle), and GroEL-bound (bottom) states. The spectrum of GroEL-bound unlabeled Rho recorded under the same condition has already been subtracted. Asterisk indicates the signal from internal 1,4-dioxane.
Please cite this article in press as: Nishida, N., et al., Nuclear magnetic resonance approaches for characterizing interactions between the bacterial chaperonin GroEL and unstructured proteins, J. Biosci. Bioeng., (2013), http://dx.doi.org/10.1016/j.jbiosc.2013.02.012
VOL. xx, 2013 (833 kDa). The Ca chemical shifts of the observable signals were quite similar to those of the unfolded form. The extensively overlapping peaks corresponding to those observed for the ureadenatured form indicate that those Tyr residues are involved in unfolded segments that are highly mobile in the complex with GroEL. By gel filtration, it was confirmed that Rho was released in
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the native monomer from the complex after incubation in the presence of 5 mM ATP/Mg and GroES (data not shown). Similar experiments were conducted using the Ca resonances from Lys, Leu, and Phe of Rho as spectroscopic probes. In either case, Rho bound to GroEL exhibited highly degenerate resonances with chemical shift values corresponding to those of a random coil
FIG. 2. Summary of NMR spectral change of uniformly 15N-labeled aSN upon interaction with GroEL. (A) Superposition of 1He15N HSQC spectra of uniformly 15N-labeled aSN in the absence (black) and presence (red) of GroEL. (B) Plots of the intensity ratios of the backbone amide peaks of aSN upon interaction with GroEL. Asterisk indicates the amino acid residue that did not exhibit an observable peak in the spectrum, while ‘P’ represents proline residue. (C) The amino acid sequence of aSN indicating the residues that exhibited attenuation in peak intensity (Ibound/Ifree <0.5) with a single underline. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article in press as: Nishida, N., et al., Nuclear magnetic resonance approaches for characterizing interactions between the bacterial chaperonin GroEL and unstructured proteins, J. Biosci. Bioeng., (2013), http://dx.doi.org/10.1016/j.jbiosc.2013.02.012
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(Fig. S2). The present results are consistent with the results of inspection of transverse relaxation-optimized spectroscopy-based spectral data obtained using uniformly 15N-labeled Rho bound to SR1 (11). On the basis of all these data, we conclude that Rho bound to GroEL is largely unstructured and highly mobile. GroEL-binding site of aSN Although the NMR data thus far reported, including the above results, have demonstrated that NMR peaks originating from the protein tethered to GroEL could be directly observed, site-specific assignments of those signals have been extremely difficult (10,11). The observable signals overlapped severely, and moreover, the substrate proteins tended to aggregate at high concentrations even in the presence of GroEL, interfering with the long-term NMR measurements needed for sequential assignments. For more detailed analyses, it is necessary to employ an appropriate model substrate that can interact with GroEL. It has recently been shown that GroEL is capable of suppressing fibrization of amyloidogenic proteins such as b2 microglobulin (19). This prompted us to examine a possible interaction between GroEL and aSN, an intrinsically disordered protein for which we have established spectral assignments (15). Fig. 2A compares 1He15N HSQC spectra of uniformly 15N-labeled aSN in the absence and presence of GroEL. Intriguingly, aSN showed significant spectral changes demonstrating its interaction with GroEL. Noteworthy was the limited number of perturbed peaks with an attenuation of peak intensity, indicating that major parts of GroEL-bound aSN were still unstructured and mobile. These results were in marked contrast with the previously reported HSQC data of uniformly 2H- and 15N-labeled Rho bound to SR1, in which only about 25 cross-peaks could be resolved (11). This situation enabled to identify the GroEL-interacting site of aSN by inspecting attenuation profile of peak intensity. GroEL induced significant attenuation in the intensity of the HSQC peaks originating from the amino acid residues located in N-terminal segments, namely Val3, Met5, Lys6, Leu8, Ser9, Lys10, Lys12, Glu13, Ala17, Glu35, Val37, Leu38, Tyr39, and Val40 (Fig. 2B). This observation indicates that the two discontinuous N-terminal segments are involved in the interaction with GroEL while the remaining regions are still structurally disordered with high mobility. Interestingly, these N-terminal segments of aSN are involved in its interactions with anionic membranes (20) and polyphenol-type compounds such as exifone, which inhibit the formation of toxic aSN aggregates (21). Although the N-terminal segment of aSN has been reported to assume an a-helical conformation in hydrophobic membrane-mimicking environments (20,22,23), CD data showed that GroEL caused virtually no secondary structure transition of aSN (Fig. S3). It has recently reported that aSN interacts with ganglioside-embedding small bicelles without a-helix formation (24). Concluding remarks The present study demonstrates that proteins tethered to GroEL remain largely unfolded and highly mobile. Furthermore, our NMR approach enables identification of the GroEL-philic sites on intrinsically disordered proteins. Elevation of transverse relaxation rate of an intrinsically disordered proteins interacting with a larger protein are often interpreted in terms of the transient formation of intermolecular contacts (25,26). Significant line-broadening of the GroEL-bound form of Rho observed in the present study as well as in the previous report (11) might suggest a dynamic complex in which multiple sites on Rho transiently interacted with GroEL in comparison with aSN, which interacted with GroEL through the rather specific segments. It has recently been revealed that encapsulated denatured protein is loosely tethered to the GroEL/GroES interface and subsequently released either into the cage or out of the cage (27). In this context, a systematic NMR study would be intriguing for
J. BIOSCI. BIOENG., identifying interaction hot spots for GroEL displayed on various intrinsically disordered proteins. This line of investigation would not only provide information on the sequence preferences of GroEL-binding segments of substrates but also offer clues to the substrate recognition mechanisms of mammalian chaperonins associated with amyloidogenic proteins of pathological interest. Supplementary materials related to this article can be found at http://dx.doi.org/10.1016/j.jbiosc.2013.02.012. ACKNOWLEDGMENTS We wish to acknowledge Dr. Kunihiro Kuwajima (National Institute of Natural Sciences) and Dr. Michel Goedert (Medical Research Council Laboratory of Melecular Biology) for kindly providing recombinant protein expression systems. We thank Ms. Tomoko Kunihara and Ms. Yukiko Isono (National Institute of Natural Sciences) for their help with the preparation of recombinant proteins. The analyses in this study were performed, in part, using equipments in the Instrument Center at Institute for Molecular Science. This work was supported in part by the Nanotechnology Platform Project and Grants-in-Aid for Scientific Research on Innovative Areas (20107004, 23107728) and for Research Activity Start-up (23870043) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. References 1. Hartl, F. U.: Molecular chaperones in cellular protein folding, Nature, 381, 571e579 (1996). 2. Ben-Zvi, A. P. and Goloubinoff, P.: Review: mechanisms of disaggregation and refolding of stable protein aggregates by molecular chaperones, J. Struct. Biol., 135, 84e93 (2001). 3. Kinbara, K. and Aida, T.: Toward intelligent molecular machines: directed motions of biological and artificial molecules and assemblies, Chem. Rev., 105, 1377e1400 (2005). 4. Braig, K., Otwinowski, Z., Hegde, R., Boisvert, D. C., Joachimiak, A., Horwich, A. L., and Sigler, P. B.: The crystal structure of the bacterial chaperonin GroEL at 2.8 A, Nature, 371, 578e586 (1994). 5. Chatellier, J., Buckle, A. M., and Fersht, A. R.: GroEL recognises sequential and non-sequential linear structural motifs compatible with extended b-strands and a-helices, J. Mol. Biol., 292, 163e172 (1999). 6. Kobayashi, N., Freund, S. M., Chatellier, J., Zahn, R., and Fersht, A. R.: NMR analysis of the binding of a rhodanese peptide to a minichaperone in solution, J. Mol. Biol., 292, 181e190 (1999). 7. Chen, L. and Sigler, P. B.: The crystal structure of a GroEL/peptide complex: plasticity as a basis for substrate diversity, Cell, 99, 757e768 (1999). 8. Zahn, R., Spitzfaden, C., Ottiger, M., Wüthrich, K., and Plückthun, A.: Destabilization of the complete protein secondary structure on binding to the chaperone GroEL, Nature, 368, 261e265 (1994). 9. Robinson, C. V., Gross, M., Eyles, S. J., Ewbank, J. J., Mayhew, M., Hartl, F. U., Dobson, C. M., and Radford, S. E.: Conformation of GroEL-bound alpha-lactalbumin probed by mass spectrometry, Nature, 372, 646e651 (1994). 10. Horst, R., Bertelsen, E. B., Fiaux, J., Wider, G., Horwich, A. L., and Wüthrich, K.: Direct NMR observation of a substrate protein bound to the chaperonin GroEL, Proc. Natl. Acad. Sci. USA, 102, 12748e12753 (2005). 11. Koculi, E., Horst, R., Horwich, A. L., and Wüthrich, K.: Nuclear magnetic resonance spectroscopy with the stringent substrate rhodanese bound to the single-ring variant SR1 of the E. coli chaperonin GroEL, Protein Sci., 20, 1380e1386 (2011). 12. Ishii, D., Kinbara, K., Ishida, Y., Ishii, N., Okochi, M., Yohda, M., and Aida, T.: Chaperonin-mediated stabilization and ATP-triggered release of semiconductor nanoparticles, Nature, 423, 628e632 (2003). 13. Shults, C. W.: Lewy bodies, Proc. Natl. Acad. Sci. USA, 103, 1661e1668 (2006). 14. Nishida, N., Motojima, F., Idota, M., Fujikawa, H., Yoshida, M., Shimada, I., and Kato, K.: Probing dynamics and conformational change of the GroELGroES complex by 13C NMR spectroscopy, J. Biochem., 140, 591e598 (2006). 15. Sasakawa, H., Sakata, E., Yamaguchi, Y., Masuda, M., Mori, T., Kurimoto, E., Iguchi, T., Hisanaga, S., Iwatsubo, T., Hasegawa, M., and Kato, K.: Ultra-high field NMR studies of antibody binding and site-specific phosphorylation of asynuclein, Biochem. Biophys. Res. Commun., 363, 795e799 (2007). 16. Miller, D. M., Kurzban, G. P., Mendoza, J. A., Chirgwin, J. M., Hardies, S. C., and Horowitz, P. M.: Recombinant bovine rhodanese: purification and comparison with bovine liver rhodanese, Biochim. Biophys. Acta, 1121, 286e292 (1992).
Please cite this article in press as: Nishida, N., et al., Nuclear magnetic resonance approaches for characterizing interactions between the bacterial chaperonin GroEL and unstructured proteins, J. Biosci. Bioeng., (2013), http://dx.doi.org/10.1016/j.jbiosc.2013.02.012
VOL. xx, 2013 17. Mendoza, J. A., Rogers, E., Lorimer, G. H., and Horowitz, P. M.: Chaperonins facilitate the in vitro folding of monomeric mitochondrial rhodanese, J. Biol. Chem., 266, 13044e13049 (1991). 18. Wishart, D. S. and Sykes, B. D.: The 13C chemical-shift index: a simple method for the identification of protein secondary structure using 13C chemical-shift data, J. Biomol. NMR, 4, 171e180 (1994). 19. Chen, J., Yagi, H., Sormanni, P., Vendruscolo, M., Makabe, K., Nakamura, T., Goto, Y., and Kuwajima, K.: Fibrillogenic propensity of the GroEL apical domain: a Janus-faced minichaperone, FEBS Lett., 586, 1120e1127 (2012). 20. Bodner, C. R., Maltsev, A. S., Dobson, C. M., and Bax, A.: Differential phospholipid binding of a-synuclein variants implicated in Parkinson’s disease revealed by solution, NMR Spectrosc. Biochem., 49, 862e871 (2010). 21. Yamaguchi, Y., Masuda, M., Sasakawa, H., Nonaka, T., Hanashima, S., Hisanaga, S., Kato, K., and Hasegawa, M.: Characterization of inhibitor-bound a-synuclein dimer: role of a-synuclein N-terminal region in dimerization and inhibitor binding, J. Mol. Biol., 395, 445e456 (2010). 22. Ulmer, T. S., Bax, A., Cole, N. B., and Nussbaum, R. L.: Structure and dynamics of micelle-bound human a-synuclein, J. Biol. Chem., 280, 9595e9603 (2005).
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23. Bartels, T., Ahlstrom, L. S., Leftin, A., Kamp, F., Haass, C., Brown, M. F., and Beyer, K.: The N-terminus of the intrinsically disordered protein a-synuclein triggers membrane binding and helix folding, Biophys. J., 99, 2116e2124 (2010). 24. Yamaguchi, T., Uno, T., Uekusa, Y., Yagi-Utsumi, M., and Kato, K.: Ganglioside-embedding small bicelles for probing membrane-landing processes of intrinsically disordered proteins, Chem. Commun. (Camb), 49, 1235e1237 (2013). 25. Mittag, T., Orlicky, S., Choy, W. Y., Tang, X., Lin, H., Sicheri, F., Kay, L. E., Tyers, M., and Forman-Kay, J. D.: Dynamic equilibrium engagement of a polyvalent ligand with a single-site receptor, Proc. Natl. Acad. Sci. USA, 105, 17772e17777 (2008). 26. Fawzi, N. L., Ying, J., Ghirlando, R., Torchia, D. A., and Clore, G. M.: Atomicresolution dynamics on the surface of amyloid-b protofibrils probed by solution NMR, Nature, 480, 268e272 (2011). 27. Motojima, F., Motojima-Miyazaki, Y., and Yoshida, M.: Revisiting the contribution of negative charges on the chaperonin cage wall to the acceleration of protein folding, Proc. Natl. Acad. Sci. USA, 109, 15740e15745 (2012).
Please cite this article in press as: Nishida, N., et al., Nuclear magnetic resonance approaches for characterizing interactions between the bacterial chaperonin GroEL and unstructured proteins, J. Biosci. Bioeng., (2013), http://dx.doi.org/10.1016/j.jbiosc.2013.02.012
Nuclear magnetic resonance approaches for characterizing interactions between the bacterial chaperonin GroEL and unstructured proteins Noritaka Nishida,1, 2, y Maho Yagi-Utsumi,2, 3, y Fumihiro Motojima,4 Masasuke Yoshida,4 Ichio Shimada,1 and Koichi Kato2, 3, * Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan,1 Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan,2 Institute for Molecular Science and Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki 444-8787, Japan,3 and Department of Molecular Biosciences, Faculty of Life Sciences, Kyoto Sangyo University, Kita-ku, Kyoto 603-8555, Japan4 Received 27 December 2012; accepted 19 February 2013 Available online xxx
GroELeprotein interactions were characterized by stable isotope-assisted nuclear magnetic resonance (NMR) spectroscopy using chemically denatured bovine rhodanese and an intrinsically disordered protein, a-synuclein, as model ligands. NMR data indicated that proteins tethered to GroEL remain largely unfolded and highly mobile, enabling identification of the interaction hot spots displayed on intrinsically disordered proteins. Ó 2013, The Society for Biotechnology, Japan. All rights reserved. [Key words: GroEL; Nuclear magnetic resonance; Molecular chaperone; Intrinsically disordered protein; Rhodanese; a-Synuclein; Denaturation]
Molecular chaperones are involved in various cellular processes in which proteins undergo folding, unfolding, and/or refolding under physiological and stress conditions (1). It has been proposed that molecular chaperones actively contribute to the suppression of toxic aggregate formation of various amyloidogenic proteins associated with neurodegenerative disorders (2). Furthermore, molecular chaperones have attracted attention due to their potential applicabilities as intelligent nanodevices in bioengineering fields (3). The chaperonin of Escherichia coli GroEL, which is one of the most extensively studied molecular chaperones, forms a large cylindrical complex that assists in the folding of nascent polypeptides and the recovery of proteins after heat shock or other stresses by preventing aggregation (1). GroEL is composed of 14 identical subunits with a molecular mass of 57 kDa that are arranged as two heptameric rings stacked back-to-back (4). The GroEL double-ring complex contains a central cavity with a diameter of approximately 45 A and interacts in an ATP-dependent manner with one or two GroES, a co-chaperone comprising a single heptameric ring of identical 10-kDa subunits (1). Each GroEL subunit is composed of three domains: apical, intermediate, and equatorial domains (4).
* Corresponding author at: Institute for Molecular Science and Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki 444-8787, Japan. Tel.: þ81 564 59 5225; fax: þ81 564 59 5224. E-mail addresses: [email protected], [email protected] (K. Kato). y The first two authors contributed equally to the work. Abbreviations: aSN, a-synuclein; DTT, dithiothreitol; GdmCl, guanidium chloride; HSQC, heteronuclear single-quantum coherence; Rho, rhodanese; SR1, singlering variant of GroEL.
The equatorial domain contains the nucleotide-binding site and mediates intersubunit contacts within and between rings. The apical domain constitutes the rim of the central cavity and provides binding sites for polypeptide substrates and GroES. The intermediate domain acts as a hinge connecting the first two domains. Protein recognition by GroEL is primarily characterized by hydrophobic interactions involving exposed hydrophobic residues of the substrates (5). Based on transferred nuclear Overhauser effect data, it has been shown that an N-terminal peptide derived from bovine rhodanese (Rho), a stringent GroEL substrate, interacted with the apical domain of GroEL in an a-helical conformation (6). On the other hand, a crystallographic study indicated that a phage display-derived peptide was accommodated on the apical domain, adopting a b-hairpin conformation (7). Thus, model peptide studies suggest that both a-helices and b-strands can exhibit GroEL-binding motifs. To elucidate the nature of substrate binding, hydrogen/deuterium exchange monitored by mass spectrometry and nuclear magnetic resonance (NMR) have been applied to proteins bound to GroEL (8,9). These studies showed that a wide range of folding intermediates can interact with GroEL, from ones very early in the folding pathway to late ones that may be native-like. More recently, direct NMR observations have been reported for substrate proteins bound to the single-ring variant of GroEL (SR1) with a molecular mass of 400 kDa (10,11). In these studies, a uniformly 2H- and 15N-labeled substrate (Rho or human dihydrofolate reductase) denatured and then trapped by SR1 was subjected to transverse relaxation-optimized spectroscopy-based NMR measurements. The NMR data suggested that the GroEL-bound substrates include largely disordered regions with higher mobility,
1389-1723/$ e see front matter Ó 2013, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2013.02.012
Please cite this article in press as: Nishida, N., et al., Nuclear magnetic resonance approaches for characterizing interactions between the bacterial chaperonin GroEL and unstructured proteins, J. Biosci. Bioeng., (2013), http://dx.doi.org/10.1016/j.jbiosc.2013.02.012
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which gave observable NMR peaks although spectral assignments could not be made. A detailed understanding of the molecular mechanisms underlying the versatile interactions of GroEL with protein ligands not only gives insights into biological functions of mammalian chaperonins but also facilitates their bioengineering applications in development of bioresponsive nanodevices (12). In view of the situations, we herein report our methodological approaches for further characterization of GroEL-bound states of proteins by direct NMR observation of huge protein complexes with a molecular mass of >800 kDa. We used two model ligands, i.e., chemically denatured Rho and an intrinsically disordered protein, a-synuclein (aSN). The latter protein was identified as the major component of the Lewy bodies of Parkinson’s disease (13). On the basis of NMR data, we discuss the conformational state of these proteins forming a complex with GroEL.
MATERIALS AND METHODS Expression and purification of recombinant proteins GroEL was overexpressed in E. coli BL21(DE3) and purified as described previously (14). Uniformly 15Nlabeled recombinant protein of human aSN was expressed in E. coli BL21(DE3) cells cultivated in M9 minimal medium containing [15N]NH4Cl and purified as described previously (15). Bovine Rho labeled with 13C at the Ca carbon of selected amino acid residues was expressed in E. coli BL21(DE3) cells grown in minimal medium supplemented with amino acids, vitamins, minerals, and 4 g of D-glucose according to the protocol described in the literature (14). To the medium, 13Caenriched amino acid was added instead of the unlabeled corresponding amino acid. The cells were grown to an OD600 of 0.6 at 37 C, induced with 0.4 mM isopropyl-b-D-thiogalactopyranoside, and incubated for an additional 12 h at 27 C. Unlabeled Rho was expressed in M2 YT medium containing 50 mg/mL of ampicillin. Rho was purified as described previously (16).
J. BIOSCI. BIOENG., Circular dichroism (CD) measurements CD spectra were measured at room temperature on Jasco J-725 apparatus using a 1.0-mm path length quartz cell.
RESULTS AND DISCUSSION Conformational state of Rho bound to GroEL It has been established that protein secondary structures can be identified on the basis of chemical shift information of the backbone NMR signals (18). In particular, secondary Ca chemical shifts offer a useful tool for the determination of the secondary structures. In general, Ca atoms involved in a-helices and b-sheets exhibit downfield and upfield shifts, respectively, with respect to those located in a random coil. We attempted to estimate the secondary structures of Rho in the GroEL-bound state on the basis of secondary Ca chemical shift data. For this purpose, we prepared recombinant Rho with 13C labeling at the Ca carbons of selected 11 Tyr, 16 Lys, 25 Leu, or 15 Phe residues. These amino acid residues number 67 in total and are spread over the whole Rho molecule, which comprises 296 amino acid residues (Fig. S1). Fig. 1 exemplifies the 13C NMR spectra of [13Ca]Tyr Rho in the native, urea-denatured, and GroEL-bound states. In the spectrum of native [13Ca]Tyr Rho, 7 and 4 signals (estimated based on the integral ratio) exhibited downfield and upfield shifts, respectively, with respect to the highly degenerate Ca signals observed in the presence of 8 M urea. [13Ca]Tyr Rho, once unfolded in 6 M GdmCl and complexed with GroEL, exhibited remarkable line-broadening but still gave observable Ca signals despite the huge size of the complex
Preparation of GroELeRho complex Rho was lyophilized in 10 mM sodium phosphate (pH 7.3) containing 100 mM KCl and 10 mM MgCl2, dissolved in 6 M guanidium chloride (GdmCl) and 10 mM dithiothreitol (DTT) to a protein concentration of 2.2 mg/mL, and incubated at room temperature for 30 min. To minimize denaturation of GroEL due to locally high concentration of GdmCl, the solution containing denatured Rho was gradually diluted to 25-fold with 10 mM sodium phosphate buffer (pH 7.3) containing 100 mM KCl, 10 mM MgCl2, 10 mM DTT, and 1 mg/mL of GroEL. The mixture was dialyzed against 10 mM sodium phosphate (pH 7.3) containing 100 mM KCl, 10 mM MgCl2, and 10 mM DTT. Formation of the GroELeRho complex was checked by gel filtration using a Superose 12 column (Amersham Pharmacia Biotech). Rho was released from GroEL as a native monomer after incubation with GroES and ATP according to the protocol described in the literature (17), confirming a 1:1 stoichiometry of the GroELeRho complex. NMR measurements For 13C NMR measurements, the proteins were concentrated to a final volume of 2 mL in 10 mM sodium phosphate (pH 7.3) containing 100 mM KCl, 3 mM NaN3, and 10% (v/v) 2H2O and transferred to the 10-mm NMR sample tube (Shigemi). The 13C spectrum of denatured Rho was measured by adding 8 M urea to the sample solution. One-dimensional 13C NMR spectra were recorded at 25 C using a Bruker AMX-400 spectrometer equipped with a probe head specialized for 13C observation with 10-mm bore size using a WALTZ proton decoupling pulse train. For each spectrum, 40,000e80,000 transients of free induction decay after 30 pulse with a repetition period of 1 s were accumulated with 16 K data points and a spectral width of 24,000 Hz. Prior to Fourier transformation, the accumulated free induction decays were multiplied by an exponential window function with line-broadening factors of 30 Hz and 2 Hz to observe peaks originating from the GroEL-bound and -unbound forms of Rho, respectively. 13C chemical shift values are given in parts per million (ppm) calibrated with internal reference 1,4-dioxane at 67.8 ppm. Spectrum of the 13Clabeled Rho in complex with GroEL was presented after subtraction of that of the unlabeled labeled counterpart in order to cancel out the signal from natural isotope abundance of 13C. For 1He15N heteronuclear single-quantum coherence (HSQC) measurements, isotopically labeled aSN was dissolved to a concentration of 50 mM in 10 mM sodium phosphate buffer (pH 7.0) containing 100 mM NaCl and 10% (v/v) 2H2O in the presence or absence of 25 mM GroEL. HSQC measurements were performed at 10 C on a JEOL ECA-920 spectrometer. The spectra were recorded at a 1H observation frequency of 920 MHz with 256 (t1) 2048 (t2) complex points and 64 scans per t1 increment. The spectral width was 2800 Hz for the 15N dimension and 13,830 Hz for the 1H dimension. 1H chemical shifts were referenced to external 2,2-dimetyl-2silapentane-5-sulfonic acid, while 15N chemical shifts were indirectly referenced to 2,2-dimetyl-2-silapentane-5-sulfonic acid using the absolute frequency ratio.
FIG. 1. Ca regions of 13C NMR spectra of [13Ca]Tyr Rho in native (top), urea-denatured (middle), and GroEL-bound (bottom) states. The spectrum of GroEL-bound unlabeled Rho recorded under the same condition has already been subtracted. Asterisk indicates the signal from internal 1,4-dioxane.
Please cite this article in press as: Nishida, N., et al., Nuclear magnetic resonance approaches for characterizing interactions between the bacterial chaperonin GroEL and unstructured proteins, J. Biosci. Bioeng., (2013), http://dx.doi.org/10.1016/j.jbiosc.2013.02.012
VOL. xx, 2013 (833 kDa). The Ca chemical shifts of the observable signals were quite similar to those of the unfolded form. The extensively overlapping peaks corresponding to those observed for the ureadenatured form indicate that those Tyr residues are involved in unfolded segments that are highly mobile in the complex with GroEL. By gel filtration, it was confirmed that Rho was released in
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the native monomer from the complex after incubation in the presence of 5 mM ATP/Mg and GroES (data not shown). Similar experiments were conducted using the Ca resonances from Lys, Leu, and Phe of Rho as spectroscopic probes. In either case, Rho bound to GroEL exhibited highly degenerate resonances with chemical shift values corresponding to those of a random coil
FIG. 2. Summary of NMR spectral change of uniformly 15N-labeled aSN upon interaction with GroEL. (A) Superposition of 1He15N HSQC spectra of uniformly 15N-labeled aSN in the absence (black) and presence (red) of GroEL. (B) Plots of the intensity ratios of the backbone amide peaks of aSN upon interaction with GroEL. Asterisk indicates the amino acid residue that did not exhibit an observable peak in the spectrum, while ‘P’ represents proline residue. (C) The amino acid sequence of aSN indicating the residues that exhibited attenuation in peak intensity (Ibound/Ifree <0.5) with a single underline. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article in press as: Nishida, N., et al., Nuclear magnetic resonance approaches for characterizing interactions between the bacterial chaperonin GroEL and unstructured proteins, J. Biosci. Bioeng., (2013), http://dx.doi.org/10.1016/j.jbiosc.2013.02.012
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(Fig. S2). The present results are consistent with the results of inspection of transverse relaxation-optimized spectroscopy-based spectral data obtained using uniformly 15N-labeled Rho bound to SR1 (11). On the basis of all these data, we conclude that Rho bound to GroEL is largely unstructured and highly mobile. GroEL-binding site of aSN Although the NMR data thus far reported, including the above results, have demonstrated that NMR peaks originating from the protein tethered to GroEL could be directly observed, site-specific assignments of those signals have been extremely difficult (10,11). The observable signals overlapped severely, and moreover, the substrate proteins tended to aggregate at high concentrations even in the presence of GroEL, interfering with the long-term NMR measurements needed for sequential assignments. For more detailed analyses, it is necessary to employ an appropriate model substrate that can interact with GroEL. It has recently been shown that GroEL is capable of suppressing fibrization of amyloidogenic proteins such as b2 microglobulin (19). This prompted us to examine a possible interaction between GroEL and aSN, an intrinsically disordered protein for which we have established spectral assignments (15). Fig. 2A compares 1He15N HSQC spectra of uniformly 15N-labeled aSN in the absence and presence of GroEL. Intriguingly, aSN showed significant spectral changes demonstrating its interaction with GroEL. Noteworthy was the limited number of perturbed peaks with an attenuation of peak intensity, indicating that major parts of GroEL-bound aSN were still unstructured and mobile. These results were in marked contrast with the previously reported HSQC data of uniformly 2H- and 15N-labeled Rho bound to SR1, in which only about 25 cross-peaks could be resolved (11). This situation enabled to identify the GroEL-interacting site of aSN by inspecting attenuation profile of peak intensity. GroEL induced significant attenuation in the intensity of the HSQC peaks originating from the amino acid residues located in N-terminal segments, namely Val3, Met5, Lys6, Leu8, Ser9, Lys10, Lys12, Glu13, Ala17, Glu35, Val37, Leu38, Tyr39, and Val40 (Fig. 2B). This observation indicates that the two discontinuous N-terminal segments are involved in the interaction with GroEL while the remaining regions are still structurally disordered with high mobility. Interestingly, these N-terminal segments of aSN are involved in its interactions with anionic membranes (20) and polyphenol-type compounds such as exifone, which inhibit the formation of toxic aSN aggregates (21). Although the N-terminal segment of aSN has been reported to assume an a-helical conformation in hydrophobic membrane-mimicking environments (20,22,23), CD data showed that GroEL caused virtually no secondary structure transition of aSN (Fig. S3). It has recently reported that aSN interacts with ganglioside-embedding small bicelles without a-helix formation (24). Concluding remarks The present study demonstrates that proteins tethered to GroEL remain largely unfolded and highly mobile. Furthermore, our NMR approach enables identification of the GroEL-philic sites on intrinsically disordered proteins. Elevation of transverse relaxation rate of an intrinsically disordered proteins interacting with a larger protein are often interpreted in terms of the transient formation of intermolecular contacts (25,26). Significant line-broadening of the GroEL-bound form of Rho observed in the present study as well as in the previous report (11) might suggest a dynamic complex in which multiple sites on Rho transiently interacted with GroEL in comparison with aSN, which interacted with GroEL through the rather specific segments. It has recently been revealed that encapsulated denatured protein is loosely tethered to the GroEL/GroES interface and subsequently released either into the cage or out of the cage (27). In this context, a systematic NMR study would be intriguing for
J. BIOSCI. BIOENG., identifying interaction hot spots for GroEL displayed on various intrinsically disordered proteins. This line of investigation would not only provide information on the sequence preferences of GroEL-binding segments of substrates but also offer clues to the substrate recognition mechanisms of mammalian chaperonins associated with amyloidogenic proteins of pathological interest. Supplementary materials related to this article can be found at http://dx.doi.org/10.1016/j.jbiosc.2013.02.012. ACKNOWLEDGMENTS We wish to acknowledge Dr. Kunihiro Kuwajima (National Institute of Natural Sciences) and Dr. Michel Goedert (Medical Research Council Laboratory of Melecular Biology) for kindly providing recombinant protein expression systems. We thank Ms. Tomoko Kunihara and Ms. Yukiko Isono (National Institute of Natural Sciences) for their help with the preparation of recombinant proteins. The analyses in this study were performed, in part, using equipments in the Instrument Center at Institute for Molecular Science. This work was supported in part by the Nanotechnology Platform Project and Grants-in-Aid for Scientific Research on Innovative Areas (20107004, 23107728) and for Research Activity Start-up (23870043) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. References 1. Hartl, F. U.: Molecular chaperones in cellular protein folding, Nature, 381, 571e579 (1996). 2. Ben-Zvi, A. P. and Goloubinoff, P.: Review: mechanisms of disaggregation and refolding of stable protein aggregates by molecular chaperones, J. Struct. Biol., 135, 84e93 (2001). 3. Kinbara, K. and Aida, T.: Toward intelligent molecular machines: directed motions of biological and artificial molecules and assemblies, Chem. Rev., 105, 1377e1400 (2005). 4. Braig, K., Otwinowski, Z., Hegde, R., Boisvert, D. C., Joachimiak, A., Horwich, A. L., and Sigler, P. B.: The crystal structure of the bacterial chaperonin GroEL at 2.8 A, Nature, 371, 578e586 (1994). 5. Chatellier, J., Buckle, A. M., and Fersht, A. R.: GroEL recognises sequential and non-sequential linear structural motifs compatible with extended b-strands and a-helices, J. Mol. Biol., 292, 163e172 (1999). 6. Kobayashi, N., Freund, S. M., Chatellier, J., Zahn, R., and Fersht, A. R.: NMR analysis of the binding of a rhodanese peptide to a minichaperone in solution, J. Mol. Biol., 292, 181e190 (1999). 7. Chen, L. and Sigler, P. B.: The crystal structure of a GroEL/peptide complex: plasticity as a basis for substrate diversity, Cell, 99, 757e768 (1999). 8. Zahn, R., Spitzfaden, C., Ottiger, M., Wüthrich, K., and Plückthun, A.: Destabilization of the complete protein secondary structure on binding to the chaperone GroEL, Nature, 368, 261e265 (1994). 9. Robinson, C. V., Gross, M., Eyles, S. J., Ewbank, J. J., Mayhew, M., Hartl, F. U., Dobson, C. M., and Radford, S. E.: Conformation of GroEL-bound alpha-lactalbumin probed by mass spectrometry, Nature, 372, 646e651 (1994). 10. Horst, R., Bertelsen, E. B., Fiaux, J., Wider, G., Horwich, A. L., and Wüthrich, K.: Direct NMR observation of a substrate protein bound to the chaperonin GroEL, Proc. Natl. Acad. Sci. USA, 102, 12748e12753 (2005). 11. Koculi, E., Horst, R., Horwich, A. L., and Wüthrich, K.: Nuclear magnetic resonance spectroscopy with the stringent substrate rhodanese bound to the single-ring variant SR1 of the E. coli chaperonin GroEL, Protein Sci., 20, 1380e1386 (2011). 12. Ishii, D., Kinbara, K., Ishida, Y., Ishii, N., Okochi, M., Yohda, M., and Aida, T.: Chaperonin-mediated stabilization and ATP-triggered release of semiconductor nanoparticles, Nature, 423, 628e632 (2003). 13. Shults, C. W.: Lewy bodies, Proc. Natl. Acad. Sci. USA, 103, 1661e1668 (2006). 14. Nishida, N., Motojima, F., Idota, M., Fujikawa, H., Yoshida, M., Shimada, I., and Kato, K.: Probing dynamics and conformational change of the GroELGroES complex by 13C NMR spectroscopy, J. Biochem., 140, 591e598 (2006). 15. Sasakawa, H., Sakata, E., Yamaguchi, Y., Masuda, M., Mori, T., Kurimoto, E., Iguchi, T., Hisanaga, S., Iwatsubo, T., Hasegawa, M., and Kato, K.: Ultra-high field NMR studies of antibody binding and site-specific phosphorylation of asynuclein, Biochem. Biophys. Res. Commun., 363, 795e799 (2007). 16. Miller, D. M., Kurzban, G. P., Mendoza, J. A., Chirgwin, J. M., Hardies, S. C., and Horowitz, P. M.: Recombinant bovine rhodanese: purification and comparison with bovine liver rhodanese, Biochim. Biophys. Acta, 1121, 286e292 (1992).
Please cite this article in press as: Nishida, N., et al., Nuclear magnetic resonance approaches for characterizing interactions between the bacterial chaperonin GroEL and unstructured proteins, J. Biosci. Bioeng., (2013), http://dx.doi.org/10.1016/j.jbiosc.2013.02.012
VOL. xx, 2013 17. Mendoza, J. A., Rogers, E., Lorimer, G. H., and Horowitz, P. M.: Chaperonins facilitate the in vitro folding of monomeric mitochondrial rhodanese, J. Biol. Chem., 266, 13044e13049 (1991). 18. Wishart, D. S. and Sykes, B. D.: The 13C chemical-shift index: a simple method for the identification of protein secondary structure using 13C chemical-shift data, J. Biomol. NMR, 4, 171e180 (1994). 19. Chen, J., Yagi, H., Sormanni, P., Vendruscolo, M., Makabe, K., Nakamura, T., Goto, Y., and Kuwajima, K.: Fibrillogenic propensity of the GroEL apical domain: a Janus-faced minichaperone, FEBS Lett., 586, 1120e1127 (2012). 20. Bodner, C. R., Maltsev, A. S., Dobson, C. M., and Bax, A.: Differential phospholipid binding of a-synuclein variants implicated in Parkinson’s disease revealed by solution, NMR Spectrosc. Biochem., 49, 862e871 (2010). 21. Yamaguchi, Y., Masuda, M., Sasakawa, H., Nonaka, T., Hanashima, S., Hisanaga, S., Kato, K., and Hasegawa, M.: Characterization of inhibitor-bound a-synuclein dimer: role of a-synuclein N-terminal region in dimerization and inhibitor binding, J. Mol. Biol., 395, 445e456 (2010). 22. Ulmer, T. S., Bax, A., Cole, N. B., and Nussbaum, R. L.: Structure and dynamics of micelle-bound human a-synuclein, J. Biol. Chem., 280, 9595e9603 (2005).
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23. Bartels, T., Ahlstrom, L. S., Leftin, A., Kamp, F., Haass, C., Brown, M. F., and Beyer, K.: The N-terminus of the intrinsically disordered protein a-synuclein triggers membrane binding and helix folding, Biophys. J., 99, 2116e2124 (2010). 24. Yamaguchi, T., Uno, T., Uekusa, Y., Yagi-Utsumi, M., and Kato, K.: Ganglioside-embedding small bicelles for probing membrane-landing processes of intrinsically disordered proteins, Chem. Commun. (Camb), 49, 1235e1237 (2013). 25. Mittag, T., Orlicky, S., Choy, W. Y., Tang, X., Lin, H., Sicheri, F., Kay, L. E., Tyers, M., and Forman-Kay, J. D.: Dynamic equilibrium engagement of a polyvalent ligand with a single-site receptor, Proc. Natl. Acad. Sci. USA, 105, 17772e17777 (2008). 26. Fawzi, N. L., Ying, J., Ghirlando, R., Torchia, D. A., and Clore, G. M.: Atomicresolution dynamics on the surface of amyloid-b protofibrils probed by solution NMR, Nature, 480, 268e272 (2011). 27. Motojima, F., Motojima-Miyazaki, Y., and Yoshida, M.: Revisiting the contribution of negative charges on the chaperonin cage wall to the acceleration of protein folding, Proc. Natl. Acad. Sci. USA, 109, 15740e15745 (2012).
Please cite this article in press as: Nishida, N., et al., Nuclear magnetic resonance approaches for characterizing interactions between the bacterial chaperonin GroEL and unstructured proteins, J. Biosci. Bioeng., (2013), http://dx.doi.org/10.1016/j.jbiosc.2013.02.012