Ferguson plot analysis of chaperone ClpB from moderate halophile

Ferguson plot analysis of chaperone ClpB from moderate halophile | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Ferguson plot analysis of chaperone ClpB from moderate halophile Teruo Akuta, Yui Tomioka, Tomoto Ura, Masataka Nakagawa, Tsutomu Arakawa This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4993116/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Jan, 2025 Read the published version in The Protein Journal → Version 1 posted 14 You are reading this latest preprint version Abstract Ferguson plot is a simple method to determine the molecular weight of native proteins and their complexes. We here tested the validity of Ferguson plot based on agarose native gel electrophoresis using multimeric chaperone protein, ClpB, derived from moderate halophile that forms a native hexamer. Ferguson plot showed a single band with a molecular weight of 1,500 kDa, about twice the size of the native hexamer, which may be consistent with the structure of other chaperons that form a double ring comprising a dimer of two hexamer assembly units, i.e, dodecamer. In fact, dynamic light scattering experiment showed two peaks, which appears to correspond to the hexamer and dodecamer structures. Ferguson plot ClpB chaperone assembly Figures Figure 1 Figure 2 Figure 3 Figure 4 1 Introduction Ferguson plot is a simple empirical method to determine the molecular weight of proteins based on electrophoretic mobility on gel electrophoresis [ 1 , 2 ], not requiring such a sophisticated instrument as analytical ultracentrifuge [ 3 , 4 ], light scattering [ 5 – 9 ] and mass photometry [ 10 – 12 ]. When combined with native gel electrophoresis, Ferguson plot can ideally provide the molecular weight of native proteins and their complexes. We have been investigating its applicability and validity [ 12 – 15 ] and here further extended such a study using a multimeric ClpB protein derived from halophilic marine bacterium Chromohalobacter salexigens DSM 3043, which favors high salt environments [ 16 – 17 ]. The ClpB belongs in a major molecular chaperone HSP100 group and forms oligomers, e.g., hexamers or dodecamers. ClpB has been characterized as a chaperone molecule to solubilize and refold denatured and aggregated proteins in cooperation with HSP70 system [ 18 ]. We have used in the previous paper a recombinant ClpB as a model oligomeric protein to compare sieving properties of agarose and polyacrylamide gels, but did not go into detail of its oligomeric state [ 15 ]. This paper reports Ferguson plot analysis of the ClpB preparations in the native state. In order to cover the monomeric state (100 kDa) and the putative functional hexameric state (600 kDa) of the ClpB, we constructed a calibration line of Ferguson plot using several proteins, including bovine serum albumin (68 k Da), ferritin (475 kDa) and tyroglobulin (669 kDa). The assembly of the ClpB was also examined by dynamic light scattering (DLS), which can determines hydrodynamic size of the proteins and their complex. 2 Materials and Methods 2.1 Materials The ClpB protein used in this study was a kind gift of Dr. Tokunaga at Kagoshima University and derived from the GenomeNet, KEGG, Chromohalobacter salexigens DSM 3043 ClpB, Csal_0499. The protein was expressed with N-terminal His-tag (pI 5.11, MW 100 kDa) in recombinant E. coli , not in the native halophilic organism favoring high salt environments, according to a previously reported method [ 17 ]. Bovine serum albumin (BSA, standard grade) was a gift from Proliant Biologicals (Ankeny, IA, USA) and composed of monomer and several oligomers. The monomer BSA (pI 5.4, MW 66.5 kDa) was purified by TSK gel UltraSW Aggregate column (TOSOH, Tokyo, Japan) as described previously [ 13 ]. ferritin (pI 4.4, MW 474 kDa) and thyroglobulin (pI 4.5, MW 669 kDa) was purchased from Cytiva (Tokyo, Japan). According to Cytiva, the exact iron content of this holoferritin product is unclear. However, after the product was treated with thioglycolate and subjected to a size exclusion mini spin column to remove bound irons [ 19 ], no difference in electrophoretic mobility was observed between the apoferritin and holoferritin on a native gel. This suggests that the holoferritin used here may not contain sufficient iron to alter its charge state. UltraPure agarose was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Research grade 2-( N -morpholino)ethanesulfonic acid (MES) was purchased from DOJINDO Laboratories (Kumamoto, Japan). L-Histidine (His) was purchased from Nacalai Tesque (Kyoto, Japan). SuperSep Ace 10–20% gradient gel, and Quick CBB (Coomassie brilliant blue) PLUS were purchased from FUJIFILM Wako Pure Chemical (Tokyo, Japan). A Loading Buffer (6×) was purchased from TaKaRa Bio (Kusatsu, Japan). 2.2 Agarose native gel electrophoresis Agarose native gel electrophoresis in horizontal mode was performed using the Mupid-2 Mini-Gel System (Mupid, Tokyo, Japan) as previously reported [ 12 – 15 , 20 – 25 ]. Agarose was dissolved at 3, 5, 6 and 7% in hot 0.1 M His/0.1 M MES buffer at pH 6.1 and cast onto a flat bed with a comb in the center position. As indicated in the result section, the upper limit of agarose concentration is determined by handling difficulty, e.g., fragility and viscosity: namely, UltraPure agarose above 7% was too viscous, when melted, to pour on to the gel tray. After loading the sample, electrophoresis was run at room temperature or on ice for 50–120 min under a constant voltage of 100 V. 2.3 SDS-polyacrylamide gel electrophoresis SDS-PAGE (10–20% gradient gel) was performed with running buffer (0.1% SDS, 25 mM Tris/192 mM Glycine, pH 8.3). Samples were prepared by mixing with 4× NuPAGE LDS sample buffer (Thermo Fisher Scientific) with or without DTT (Dithiothreitol) and run for 50 min under a constant current of 30 mA. 2.4 Gel Image analysis Agarose or polyacrylamide gels stained with CBB were photographed by image scanner (EPSON GT-X980, Suwa, Japan). The band’s movement distance was measured using the freely available software Image J and a graph was generated using GraphPad Prism 8J (GraphPad Software, Boston, MA). We are primarily concerned with the electrophoretic mobility of the bands for Ferguson plot analysis so that no effort was made to quantify the band intensity. The presented data are representative of at least three experiments. 2.5 Dynamic Light Scattering (DLS) analysis DLS experiments were performed using a Zetasizer Nano ZS light scattering photometer (Malvern Instruments, Worcestershire, UK) equipped with a 4 mW-He–Ne ion laser (λ = 633 nm) at a detection angle of 173° [ 26 ]. BSA, ferritin, and thyroglobulin were measured at 1 mg/mL, and ClpB was measured at 0.5 mg/mL in 0.1 M His/0.1 M MES buffer (pH 6.1). Due to high polydispersity, ClpB was also measured at the same protein concentration in 4 mM HEPES buffer (pH 7.3) containing 15 mM KCl and 1 mM MgCl 2 . DLS measurements were conducted in a disposable cuvette with a 1 cm path length at 25°C. The viscosity of the solutions was approximated using water (η = 0.87 cP). All reported values are the average of four measurements 3 Results Figure 1 A shows SDS-PAGE of purified ClpB used for this study. Under reducing conditions, it showed a single band. Under non-reducing conditions, a main band with identical mobility to the band under reducing condition was observed, suggesting that no intra-molecular or inter-molecular disulfide bonds were formed. ClpB has two cysteine residues in its 860 amino acids, which, according to UniProt, are predicted to have free sulfhydryl (SH) groups. The intra-molecular disulfide bonds, if formed, can make the protein-SDS complex more compact, leading to a faster mobility on SDS-PAGE. It is consistent with the SH groups far apart in the structure, which prevents from forming intra-molecular disulfide bonds even when denatured by SDS. The partially buried SH groups would also prevent from forming inter-molecular disulfide bonds. However, under non-reducing conditions, a few faint high molecular weight bands were observed due to inter-molecular disulfide bonds. It is most likely due to denaturation of ClpB by SDS, exposing the free SH to inter-molecular disulfide bond formation. Figure 1 B shows agarose native gel electrophoresis of ClpB done at pH 6.1, at which oxidation of the SH is unlikely to occur: note that no SDS is present and hence ClpB is in the native state. The ClpB showed a single band, indicating that the sample is homogeneous in the native state under the conditions of the agarose native gel electrophoresis. Figure 1 B also shows native gel profiles as a function of agarose concentration. The mobility was retarded at higher gel concentration. The mobility changes were used to construct Ferguson plot in Fig. 1 C. Figure 1 B also shows agarose native gel electrophoresis profiles of BSA and ferritin as a function of agarose concentration. This figure was assembled from the original figures to compare the results of different proteins side-by-side at an identical agarose concentration, i.e., 3, 5, 6 and 7%. Upon visual inspection, the gel concentration dependence of electrophoretic mobility is much stronger for ClpB and ferritin than BSA. For example, BSA moved slower than the other proteins at 3%, but faster at 7% in both gel types (see panel B). Figure 1 C shows Ferguson plots of three proteins, whose relative mobilities were calculated against the mobility of the tracking dye (bromophenol blue). It appears that the plots can be approximately fit with a linear line for three proteins. In both cases, the slope increased in the order of monomer BSA < ferritin < ClpB, indicating that the hydrodynamic effective size increases in this order. This in turn suggests that the molecular weight of the native protein increases also in this order and hence that the molecular weight of ClpB is greater than ferritin. The relationship between the known molecular weights of standard proteins and their Ferguson plot slopes were plotted in Fig. 2 . Figure 2 showed the agarose native gel electrophoresis profiles of three standard proteins at 1, 3 and 5% agarose concentration, whose molecular weights are listed in the table. The Ferguson plot slopes of these three standard proteins were plotted against their molecular weight and appears to have a linear relationship. The slope of the ClpB was much steeper than these standard proteins and shown in Fig. 2 C, from which the molecular weight of ClpB was determined to be ~ 1,500 kDa. A similar molecular weight was obtained based on the calibration with two standard proteins, BSA and ferritin, in Fig. S1 . DLS experiments were carried out to confirm the assembly state of ClpB obtained by the Ferguson plot analysis. Figure 3 A illustrates the hydrodynamic diameters of control proteins measured by DLS: their autocorrelation functions are plotted in Fig. S2 A. In the 0.1 M His/0.1 M MES buffer (pH 6.1), BSA, ferritin and thyroglobulin exhibited single-peak distributions with intensities exceeding 90% and their hydrodynamic diameters were 10.58 ± 0.12 nm, 18.77 ± 0.39 nm, and 29.67 ± 2.17 nm, respectively, increasing with their molecular weight. The ClpB was polydisperse in this His/MES buffer, complicating accurate fitting. Nevertheless, autocorrelation function results, shown in Fig. S2A suggest that the ClpB is larger than BSA, ferritin, and thyroglobulin. We then performed DLS experiments in 4 mM HEPES buffer (pH 7.3) containing 15 mM KCl and 1 mM MgCl₂, where ClpB remains stable. The autocorrelation function, shown in Fig. S2B, resembled that in the His/MES buffer but allowed for accurate fitting. In this HEPES buffer, the ClpB displayed a two-peak distribution with hydrodynamic diameters of 18.22 ± 0.99 nm (intensity: 39.93 ± 2.52%) and 107.20 ± 10.47 nm (intensity: 60.08 ± 2.52%) (Fig. 3 A and B). Although the molecular weight of the ClpB monomer is approximately 100 kDa, the observed hydrodynamic radius of Peak 1 is close to that of ferritin (Mw: 440 kDa) and the radius of Peak 2 is much larger than that of thyroglobulin (Mw: 669 kDa), indicating that ClpB more likely forms hexamers and dodecamers and exists in an equilibrium state. However, the hydrodynamic radius of Peak 2 appears to be significantly larger than that expected for dodecamers, suggesting that the structure of Peak 2 is more asymmetric than Peak 1. Nevertheless, these results align with agarose native electrophoresis findings (Figs. 2 and 3 ) and suggest that ClpB forms oligomers. 4 Discussion Ferguson plot is in principle based on the electrophoretic mobility of proteins, which is determined by not only the molecular weight but also the shape [ 1 , 2 ]. At the same molecular weight, the elongated particles would move in the gel during electrophoresis more slowly than the compact particles and hence give a larger molecular weight. Traditional more rigorous technologies, such as sedimentation equilibrium [ 3 , 4 ] and light scattering [ 5 – 9 ], can provide a correct molecular weight. However, these technologies require expensive instruments and knowledge of solution thermodynamics. Recently, a much simpler mass photometry that measures the molecular weight of a single particle was added to those technologies, but also requires expensive instrument [ 10 – 12 ]. Another issue with mass photometry is the concentration of macromolecules, which must be diluted so that the light scattering of each single particle can be determined. These technologies also require measurements on purified samples. On the contrary, Ferguson plot has none of these limitations. As long as the target band can be identified, this technology can be used for impure samples and with minute quantities. It appears that Ferguson plot gave a molecular weight of the halophilic ClpB that was inconsistent with the native hexameric assembly determined for the same protein [ 17 ]. In the above paper [ 17 ], the hexamer structure was inferred by glutaraldehyde cross-linking of the purified ClpB protein followed by SDS-gel electrophoresis, which showed a ladder of protein bands composed of monomers to hexamers: namely, not only the hexamers but also monomers and smaller oligomers were observed. This indicates most likely that the ClpB is forming a hexamer, which generates a ladder of cross-linked oligomers due to cross-linking efficiency. It is also possible that there may be larger structures, which could not be cross-linked efficiently by glutaraldehyde. Why does then Ferguson plot not show the hexamer molecular weight? One possibility may be that the cross-linking experiment underestimates the size of the ClpB in solution due to the possibility that the cross-linking efficiency falls to a large extent over the hexamer size as described above. Of course there are other possibilities. Ferguson plot cannot be used for ClpB or any proteins, if their electrophoretic mobility is affected by agarose gels, for example, if the protein structure or charged state changes as a function of agarose gel concentration. Namely, they may lose charges or alter structures at high gel concentration, which would cause slower mobility in gel. Alternatively, ClpB binds to agarose gel that may be dependent on agarose concentration. These effects may be combined with errors in determination of electrophoretic mobility on agarose native gel electrophoresis, which had led to the molecular weights different from the hexameric assembly. There exist other possibilities, e.g., deviation from the linear relationship between the log mobility and gel concentration [ 26 – 30 ] or the effects of gel matrix on macromolecular assembly [ 31 – 34 ]. Another more likely possibility is dimerization of the hexameric assembly unit of the halophilic ClpB. Structural analysis of the human mitochondrial homologue (i.e., non-halophilic) SKD3/CLPB using cryo-electron microscopy revealed a dodecamer structure composed of double hexameric rings, as depicted in Fig. 4 [ 35 ]. Mass photometry done at low protein concentrations identified three distinct peaks corresponding to monomers, hexamers, and dodecamers. At protein concentrations of 91 nM (~ 0.01 mg/mL), the proportion of monomers was dominant, whereas the proportions of hexamers and dodecamers increased at 240 nM (~ 0.024 mg/mL). Negative-staining electron microscopy indicated the formation of large oligomers with significant size heterogeneity [ 36 ]. Analytical ultracentrifugation experiments with E. coli ClpB chaperone at 1 mg/mL [ 37 ], a much higher concentration than the above mass photometry analysis, showed an equilibrium between monomers, hexamers, and dodecamers that was dependent on salt concentration. At 20 and 50 mM salt the dodecamer predominated, and the monomer content increased with rising salt concentration, suggesting that the electrostatic interactions, which can be neutralized by salt, are involved in the chaperone assembly. Agarose native gel electrophoresis of halophilic Chromohalobacter salexigens ClpB, conducted in this study at concentrations ~ 10-fold higher than those used in mass photometry, suggested an increased proportion of oligomers, potentially exceeding dodecamers. Additionally, no salt was added to the electrophoresis system. In the yeast ( Saccharomyces cerevisiae ) homologue HSP104, cryo-electron microscopy reported a hexamer [ 38 ], though a dodecamer cylindrical model has also been proposed [ 39 ]. Atomic force microscopy (AFM) analysis of T. thermophilus ClpB revealed a primary hexameric ring structure, with larger, unstable oligomers also observed [ 40 ]. Our DLS analysis of ClpB also detected two peaks, indicating the presence of two different oligomers in the solution. Although hydrodynamic size of the ClpB could not be calculated in His/MES buffer, which is the same condition used in agarose electrophoresis, the autocorrelation functions suggests that the ClpB without salt may exist in a larger assembly state than in HEPES buffer with salt (Fig. S2B). Thus, depending on the organism and analytical method, the hexamer to dodecamer size may be detected. Similarly, the well-studied chaperone protein E. coli GroEL [ 41 ] exhibited a 14-mer structure composed of two stacked heptameric rings as depicted in Fig. 4 . It should be mentioned that the Ferguson plot and DLS analysis used here are rather qualitative and thus, for studying chaperons, specifically ClpB, it would be necessary to apply other techniques to determine its absolute molecular weight. Such techniques include sedimentation equilibrium [ 3 , 4 ], static light scattering [ 5 – 9 ] and mass photometry [ 10 – 12 ]. These technologies can determine the molecular weight of the particles, regardless of their structures, whether compact or extended, although these techniques also have following limitations. Sedimentation analysis cannot be done at high protein concentration due to increasing non-ideality at higher protein concentration and mass photometry analysis requires a dilute protein solution. Static light scattering analysis is always hampered by large particles, e.g., dusts or contaminating particles, which dominate light scattering. Compared to these techniques, agarose native gel electrophoresis can be done at any protein concentration, as long as bands can be observed and identified. For low concentration samples, more sensitive staining method can be used. Lastly, it is puzzling that ClpB showed a single band on agarose native gel electrophoresis, but two peaks on DLS analysis. One possibility is poor size resolution of 1% UltraPure agarose, resulting in inability to separate the hexamers from the dodecamers. However, even a higher agarose concentration was unable to separate them and provide their respective molecular weight information. Second possibility is the effects of gel structure on assembly state of the ClpB [ 31 – 34 ]. Namely, the small cell confinement of the gel structure enforces the protein-protein interactions and stabilizes the molecular assembly, here dodecamer structures. Thus, it would be of great interest to test more porous or alternatively higher resolution (small cell confinement) gels to evaluate the effects of gel structure on macromolecular assembly. Nevertheless, it may be safe to conclude that the halophilic ClpB forms oligomers in different size in solution and the dodecamer structure is stabilized under confined environments of gel matrix. 5 Conclusion We have tested Ferguson plot for determination of the molecular weight of halophilic chaperone, ClpB, in the native state. The results showed an approximate size of dodecamer, which appears to be in part consistent with the DLS analysis, which showed two peaks corresponding to a hexamer and a dodecamer. Thus, it can be concluded that Ferguson plot, combined with the agarose native gel electrophoresis, can be used to qualitatively determine the oligomerization of proteins. Declarations Acknowledgements We thank Prof. Masao Tokunaga for his help in providing the ClpB samples and manuscript preparation. Author contribution Teruo Akuta: Methodology, Visualization, Writing, Supervision Yui Tomioka: Visualization, Formal analysis Tomoto Ura: Visualization, Formal Analysis Masataka Nakagawa: Visualization Tsutomu Arakawa: Conceptualization, Writing - review & editing, Supervision Funding No external funding was received. Data availability Data are available upon request. Conflict of interests This work was supported by Kyokuto Pharmaceutical Industrial Co., Ltd. Employees T.A. (Teruo Akuta), Y.T., and M.N. are affiliated with Kyokuto Pharmaceuticals. T.U. is affiliated with the University of Tsukuba and the National Institutes for Quantum Science and Technology. 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Science 357 (6348):273-279. https://doi.org/ 10.1126/science.aan1052 Walter GM, Smith MC, Wisén S, Basrur V, Elenitoba-Johnson KS, Duennwald ML, Kumar A, Gestwicki JE (2011) Ordered assembly of heat shock proteins, Hsp26, Hsp70, Hsp90, and Hsp104, on expanded polyglutamine fragments revealed by chemical probes. J Biol Chem 286(47):40486-40493. https://doi.org/ 10.1074/jbc.M111.284448 Uchihashi T, Watanabe YH, Nakazaki Y, Yamasaki T, Watanabe H, Maruno T, Ishii K, Uchiyama S, Song C, Murata K, Iino R, Ando T (2018) Dynamic structural states of ClpB involved in its disaggregation function. Nat Commun 9(1):2147. https://doi.org/10.1038/s41467-018-04587-w Xu Z, Horwich AL, Sigler PB (1997) The crystal structure of the asymmetric GroEL-GroES-(ADP)7 chaperonin complex. Nature 388(6644):741-750. https://doi.org/10.1038/41944 Additional Declarations No competing interests reported. Supplementary Files SupplementaralFigures.pdf Supplemental data Fig. S1. Slope from Ferguson plot using ferritin and BSA as molecular weight marker Fig. S2. Autocorrelation functions from DLS measurement A. Autocorrelation functions of each protein in 0.1 M His/0.1 M MES buffer (pH 6.1) corresponding to agarose native electrophoresis. The results for BSA (▲), ferritin (■), and thyroglobulin (▼) were fittable, and the calculated hydrodynamic diameters are shown in Fig. 3A. ClpB (●) could not be fitted well due to its high polydispersity. B. Autocorrelation functions of ClpB in 0.1 M His/0.1 M MES buffer (pH 6.1)(●) and in 4 mM HEPES buffer (pH 7.3) containing 15 mM KCl and1 mM MgCl 2 , (○). The results in HEPES buffer were fittable and are shown in Fig. 3A. Cite Share Download PDF Status: Published Journal Publication published 05 Jan, 2025 Read the published version in The Protein Journal → Version 1 posted Editorial decision: Revision requested 18 Oct, 2024 Reviews received at journal 07 Oct, 2024 Reviewers agreed at journal 16 Sep, 2024 Reviewers agreed at journal 14 Sep, 2024 Reviews received at journal 14 Sep, 2024 Reviewers agreed at journal 11 Sep, 2024 Reviewers agreed at journal 09 Sep, 2024 Reviews received at journal 09 Sep, 2024 Reviewers agreed at journal 09 Sep, 2024 Reviewers agreed at journal 09 Sep, 2024 Reviewers invited by journal 09 Sep, 2024 Editor assigned by journal 31 Aug, 2024 Submission checks completed at journal 31 Aug, 2024 First submitted to journal 28 Aug, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4993116","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":357289034,"identity":"4d46695c-a407-4f0b-b9a1-a8822c3f989f","order_by":0,"name":"Teruo Akuta","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Teruo","middleName":"","lastName":"Akuta","suffix":""},{"id":357289035,"identity":"2c844fad-89ad-4332-acb8-597adabaa849","order_by":1,"name":"Yui Tomioka","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yui","middleName":"","lastName":"Tomioka","suffix":""},{"id":357289036,"identity":"1b2f9f5f-7e77-452c-990d-5e00274ba507","order_by":2,"name":"Tomoto Ura","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Tomoto","middleName":"","lastName":"Ura","suffix":""},{"id":357289037,"identity":"e5cb1138-8883-40cc-aa8d-a345615a56b6","order_by":3,"name":"Masataka Nakagawa","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Masataka","middleName":"","lastName":"Nakagawa","suffix":""},{"id":357289038,"identity":"10aca173-84f9-42ed-a5b9-6d4a1fe27dab","order_by":4,"name":"Tsutomu Arakawa","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwElEQVRIiWNgGAWjYJACxgYgwc8OJB8Qo5wHpkWy5wADQwJJWgxuJBCpxZ797MOPM2oOyzPcfPzwQQKDnZxuAyFbeNKNJTccO2zYODvN2CCBIdnY7ABBh6UxSD5gu83YLJ1gJpHAcCBxG0Et/M+Yfz74d9u+TfL4NyK1SKSxSW5su53YI8FDrC03nrFZzuz7nzyDJ6fYIMGACL+w96cx3+z5lma7//jxjQ8+VNjJEdSCBgxIUz4KRsEoGAWjAAcAAEM+Qo1c2NuRAAAAAElFTkSuQmCC","orcid":"","institution":"Alliance Protein Laboratories (United States)","correspondingAuthor":true,"prefix":"","firstName":"Tsutomu","middleName":"","lastName":"Arakawa","suffix":""}],"badges":[],"createdAt":"2024-08-28 18:54:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4993116/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4993116/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10930-024-10245-6","type":"published","date":"2025-01-05T15:57:29+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":66189583,"identity":"a4767967-10d7-4260-99be-f78e77bc88d4","added_by":"auto","created_at":"2024-10-08 13:57:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":410765,"visible":true,"origin":"","legend":"\u003cp\u003eAgarose native gel electrophoresis of BSA, ferritin and ClpB as a function of gel concentration and Ferguson plot\u003c/p\u003e\n\u003cp\u003eA. Reducing and non-reducing SDS-PAGE\u003c/p\u003e\n\u003cp\u003eB. Agarose native gel electrophoresis of BSA, ferrtin and ClpB at 3, 5, 6 and 7% agarose.\u003c/p\u003e\n\u003cp\u003eC. Ferguson plots of BSA, ferritin and ClpB.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4993116/v1/b640b2d53d7549f569abc036.png"},{"id":66191539,"identity":"aff1272e-c963-46e1-8ce8-a7dcd98252e3","added_by":"auto","created_at":"2024-10-08 14:13:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":161989,"visible":true,"origin":"","legend":"\u003cp\u003eFerguson plot analysis\u003c/p\u003e\n\u003cp\u003eA. Molecular weight, isoelectric point (acidic or basic at pH 6.1) and state of association.\u003c/p\u003e\n\u003cp\u003eB. Agarose native gel electrophoresis of thyroglobulin (Thyr), ferritin (Ferr) and BSA.\u003c/p\u003e\n\u003cp\u003eC. Slope from Ferguson plot.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4993116/v1/fea6f103e6d5e224a40c0bda.png"},{"id":66189875,"identity":"fac7b5cd-e55b-48ed-830f-fb525afaa1cf","added_by":"auto","created_at":"2024-10-08 14:05:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":76306,"visible":true,"origin":"","legend":"\u003cp\u003eDLS analysis\u003c/p\u003e\n\u003cp\u003eA. Summary of DLS analysis\u003c/p\u003e\n\u003cp\u003eB. Particle size of ClpB\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4993116/v1/2fd5fadb719f02596aabe21f.png"},{"id":66189582,"identity":"59191260-7c60-4379-acf2-6aef988b8bcd","added_by":"auto","created_at":"2024-10-08 13:57:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":57284,"visible":true,"origin":"","legend":"\u003cp\u003eModel of ClpB structure in native state\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4993116/v1/45945c8d70f7a5fa0debcafd.png"},{"id":73093295,"identity":"1d4c6a03-a50b-4175-81b4-fc2a9602a012","added_by":"auto","created_at":"2025-01-06 16:13:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1133585,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4993116/v1/9ac8e10a-a0d5-42c6-95cb-a4783d2b76a5.pdf"},{"id":66189585,"identity":"7ab7e1d0-a457-4db1-b8b4-095faad8ebdf","added_by":"auto","created_at":"2024-10-08 13:57:48","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":92602,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental data\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. S1. \u003c/strong\u003eSlope from Ferguson plot using ferritin and BSA as molecular weight marker\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eFig. S2.\u003c/strong\u003e Autocorrelation functions from DLS measurement\u003c/p\u003e\n\u003cp\u003eA. Autocorrelation functions of each protein in 0.1 M His/0.1 M MES buffer (pH 6.1) corresponding to agarose native electrophoresis. The results for BSA (▲), ferritin (■), and thyroglobulin (▼) were fittable, and the calculated hydrodynamic diameters are shown in Fig. 3A. ClpB (●) could not be fitted well due to its high polydispersity.\u003c/p\u003e\n\u003cp\u003eB. Autocorrelation functions of ClpB in 0.1 M His/0.1 M MES buffer (pH 6.1)(●) and in 4 mM HEPES buffer (pH 7.3) containing 15 mM KCl and1 mM MgCl\u003csub\u003e2\u003c/sub\u003e, (○). The results in HEPES buffer were fittable and are shown in Fig. 3A.\u003c/p\u003e","description":"","filename":"SupplementaralFigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4993116/v1/9d00284fc11a0aafa1426455.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Ferguson plot analysis of chaperone ClpB from moderate halophile","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eFerguson plot is a simple empirical method to determine the molecular weight of proteins based on electrophoretic mobility on gel electrophoresis [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], not requiring such a sophisticated instrument as analytical ultracentrifuge [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], light scattering [\u003cspan additionalcitationids=\"CR6 CR7 CR8\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] and mass photometry [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. When combined with native gel electrophoresis, Ferguson plot can ideally provide the molecular weight of native proteins and their complexes. We have been investigating its applicability and validity [\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] and here further extended such a study using a multimeric ClpB protein derived from halophilic marine bacterium \u003cem\u003eChromohalobacter salexigens\u003c/em\u003e DSM 3043, which favors high salt environments [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The ClpB belongs in a major molecular chaperone HSP100 group and forms oligomers, e.g., hexamers or dodecamers. ClpB has been characterized as a chaperone molecule to solubilize and refold denatured and aggregated proteins in cooperation with HSP70 system [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe have used in the previous paper a recombinant ClpB as a model oligomeric protein to compare sieving properties of agarose and polyacrylamide gels, but did not go into detail of its oligomeric state [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. This paper reports Ferguson plot analysis of the ClpB preparations in the native state. In order to cover the monomeric state (100 kDa) and the putative functional hexameric state (600 kDa) of the ClpB, we constructed a calibration line of Ferguson plot using several proteins, including bovine serum albumin (68 k Da), ferritin (475 kDa) and tyroglobulin (669 kDa). The assembly of the ClpB was also examined by dynamic light scattering (DLS), which can determines hydrodynamic size of the proteins and their complex.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials\u003c/h2\u003e \u003cp\u003eThe ClpB protein used in this study was a kind gift of Dr. Tokunaga at Kagoshima University and derived from the GenomeNet, KEGG, \u003cem\u003eChromohalobacter salexigens\u003c/em\u003e DSM 3043 ClpB, Csal_0499. The protein was expressed with N-terminal His-tag (pI 5.11, MW 100 kDa) in recombinant \u003cem\u003eE. coli\u003c/em\u003e, not in the native halophilic organism favoring high salt environments, according to a previously reported method [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Bovine serum albumin (BSA, standard grade) was a gift from Proliant Biologicals (Ankeny, IA, USA) and composed of monomer and several oligomers. The monomer BSA (pI 5.4, MW 66.5 kDa) was purified by TSK gel UltraSW Aggregate column (TOSOH, Tokyo, Japan) as described previously [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. ferritin (pI 4.4, MW 474 kDa) and thyroglobulin (pI 4.5, MW 669 kDa) was purchased from Cytiva (Tokyo, Japan). According to Cytiva, the exact iron content of this holoferritin product is unclear. However, after the product was treated with thioglycolate and subjected to a size exclusion mini spin column to remove bound irons [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], no difference in electrophoretic mobility was observed between the apoferritin and holoferritin on a native gel. This suggests that the holoferritin used here may not contain sufficient iron to alter its charge state. UltraPure agarose was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Research grade 2-(\u003cem\u003eN\u003c/em\u003e-morpholino)ethanesulfonic acid (MES) was purchased from DOJINDO Laboratories (Kumamoto, Japan). L-Histidine (His) was purchased from Nacalai Tesque (Kyoto, Japan). SuperSep Ace 10\u0026ndash;20% gradient gel, and Quick CBB (Coomassie brilliant blue) PLUS were purchased from FUJIFILM Wako Pure Chemical (Tokyo, Japan). A Loading Buffer (6\u0026times;) was purchased from TaKaRa Bio (Kusatsu, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.2 Agarose native gel electrophoresis\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eAgarose native gel electrophoresis in horizontal mode was performed using the Mupid-2 Mini-Gel System (Mupid, Tokyo, Japan) as previously reported [\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan additionalcitationids=\"CR21 CR22 CR23 CR24\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Agarose was dissolved at 3, 5, 6 and 7% in hot 0.1 M His/0.1 M MES buffer at pH 6.1 and cast onto a flat bed with a comb in the center position. As indicated in the result section, the upper limit of agarose concentration is determined by handling difficulty, e.g., fragility and viscosity: namely, UltraPure agarose above 7% was too viscous, when melted, to pour on to the gel tray. After loading the sample, electrophoresis was run at room temperature or on ice for 50\u0026ndash;120 min under a constant voltage of 100 V.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.3 SDS-polyacrylamide gel electrophoresis\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eSDS-PAGE (10\u0026ndash;20% gradient gel) was performed with running buffer (0.1% SDS, 25 mM Tris/192 mM Glycine, pH 8.3). Samples were prepared by mixing with 4\u0026times; NuPAGE LDS sample buffer (Thermo Fisher Scientific) with or without DTT (Dithiothreitol) and run for 50 min under a constant current of 30 mA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Gel Image analysis\u003c/h2\u003e \u003cp\u003eAgarose or polyacrylamide gels stained with CBB were photographed by image scanner (EPSON GT-X980, Suwa, Japan). The band\u0026rsquo;s movement distance was measured using the freely available software Image J and a graph was generated using GraphPad Prism 8J (GraphPad Software, Boston, MA). We are primarily concerned with the electrophoretic mobility of the bands for Ferguson plot analysis so that no effort was made to quantify the band intensity. The presented data are representative of at least three experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Dynamic Light Scattering (DLS) analysis\u003c/h2\u003e \u003cp\u003eDLS experiments were performed using a Zetasizer Nano ZS light scattering photometer (Malvern Instruments, Worcestershire, UK) equipped with a 4 mW-He\u0026ndash;Ne ion laser (λ\u0026thinsp;=\u0026thinsp;633 nm) at a detection angle of 173\u0026deg; [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. BSA, ferritin, and thyroglobulin were measured at 1 mg/mL, and ClpB was measured at 0.5 mg/mL in 0.1 M His/0.1 M MES buffer (pH 6.1). Due to high polydispersity, ClpB was also measured at the same protein concentration in 4 mM HEPES buffer (pH 7.3) containing 15 mM KCl and 1 mM MgCl\u003csub\u003e2\u003c/sub\u003e. DLS measurements were conducted in a disposable cuvette with a 1 cm path length at 25\u0026deg;C. The viscosity of the solutions was approximated using water (η\u0026thinsp;=\u0026thinsp;0.87 cP). All reported values are the average of four measurements\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA shows SDS-PAGE of purified ClpB used for this study. Under reducing conditions, it showed a single band. Under non-reducing conditions, a main band with identical mobility to the band under reducing condition was observed, suggesting that no intra-molecular or inter-molecular disulfide bonds were formed. ClpB has two cysteine residues in its 860 amino acids, which, according to UniProt, are predicted to have free sulfhydryl (SH) groups. The intra-molecular disulfide bonds, if formed, can make the protein-SDS complex more compact, leading to a faster mobility on SDS-PAGE. It is consistent with the SH groups far apart in the structure, which prevents from forming intra-molecular disulfide bonds even when denatured by SDS. The partially buried SH groups would also prevent from forming inter-molecular disulfide bonds. However, under non-reducing conditions, a few faint high molecular weight bands were observed due to inter-molecular disulfide bonds. It is most likely due to denaturation of ClpB by SDS, exposing the free SH to inter-molecular disulfide bond formation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB shows agarose native gel electrophoresis of ClpB done at pH 6.1, at which oxidation of the SH is unlikely to occur: note that no SDS is present and hence ClpB is in the native state. The ClpB showed a single band, indicating that the sample is homogeneous in the native state under the conditions of the agarose native gel electrophoresis. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB also shows native gel profiles as a function of agarose concentration. The mobility was retarded at higher gel concentration. The mobility changes were used to construct Ferguson plot in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB also shows agarose native gel electrophoresis profiles of BSA and ferritin as a function of agarose concentration. This figure was assembled from the original figures to compare the results of different proteins side-by-side at an identical agarose concentration, i.e., 3, 5, 6 and 7%. Upon visual inspection, the gel concentration dependence of electrophoretic mobility is much stronger for ClpB and ferritin than BSA. For example, BSA moved slower than the other proteins at 3%, but faster at 7% in both gel types (see panel B). Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC shows Ferguson plots of three proteins, whose relative mobilities were calculated against the mobility of the tracking dye (bromophenol blue). It appears that the plots can be approximately fit with a linear line for three proteins. In both cases, the slope increased in the order of monomer BSA\u0026thinsp;\u0026lt;\u0026thinsp;ferritin\u0026thinsp;\u0026lt;\u0026thinsp;ClpB, indicating that the hydrodynamic effective size increases in this order. This in turn suggests that the molecular weight of the native protein increases also in this order and hence that the molecular weight of ClpB is greater than ferritin.\u003c/p\u003e \u003cp\u003eThe relationship between the known molecular weights of standard proteins and their Ferguson plot slopes were plotted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e showed the agarose native gel electrophoresis profiles of three standard proteins at 1, 3 and 5% agarose concentration, whose molecular weights are listed in the table. The Ferguson plot slopes of these three standard proteins were plotted against their molecular weight and appears to have a linear relationship. The slope of the ClpB was much steeper than these standard proteins and shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, from which the molecular weight of ClpB was determined to be ~\u0026thinsp;1,500 kDa. A similar molecular weight was obtained based on the calibration with two standard proteins, BSA and ferritin, in Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDLS experiments were carried out to confirm the assembly state of ClpB obtained by the Ferguson plot analysis. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA illustrates the hydrodynamic diameters of control proteins measured by DLS: their autocorrelation functions are plotted in Fig.\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA. In the 0.1 M His/0.1 M MES buffer (pH 6.1), BSA, ferritin and thyroglobulin exhibited single-peak distributions with intensities exceeding 90% and their hydrodynamic diameters were 10.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 nm, 18.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39 nm, and 29.67\u0026thinsp;\u0026plusmn;\u0026thinsp;2.17 nm, respectively, increasing with their molecular weight. The ClpB was polydisperse in this His/MES buffer, complicating accurate fitting. Nevertheless, autocorrelation function results, shown in Fig. S2A suggest that the ClpB is larger than BSA, ferritin, and thyroglobulin.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe then performed DLS experiments in 4 mM HEPES buffer (pH 7.3) containing 15 mM KCl and 1 mM MgCl₂, where ClpB remains stable. The autocorrelation function, shown in Fig. S2B, resembled that in the His/MES buffer but allowed for accurate fitting. In this HEPES buffer, the ClpB displayed a two-peak distribution with hydrodynamic diameters of 18.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.99 nm (intensity: 39.93\u0026thinsp;\u0026plusmn;\u0026thinsp;2.52%) and 107.20\u0026thinsp;\u0026plusmn;\u0026thinsp;10.47 nm (intensity: 60.08\u0026thinsp;\u0026plusmn;\u0026thinsp;2.52%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and B). Although the molecular weight of the ClpB monomer is approximately 100 kDa, the observed hydrodynamic radius of Peak 1 is close to that of ferritin (Mw: 440 kDa) and the radius of Peak 2 is much larger than that of thyroglobulin (Mw: 669 kDa), indicating that ClpB more likely forms hexamers and dodecamers and exists in an equilibrium state. However, the hydrodynamic radius of Peak 2 appears to be significantly larger than that expected for dodecamers, suggesting that the structure of Peak 2 is more asymmetric than Peak 1. Nevertheless, these results align with agarose native electrophoresis findings (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e) and suggest that ClpB forms oligomers.\u003c/p\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eFerguson plot is in principle based on the electrophoretic mobility of proteins, which is determined by not only the molecular weight but also the shape [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. At the same molecular weight, the elongated particles would move in the gel during electrophoresis more slowly than the compact particles and hence give a larger molecular weight. Traditional more rigorous technologies, such as sedimentation equilibrium [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] and light scattering [\u003cspan additionalcitationids=\"CR6 CR7 CR8\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], can provide a correct molecular weight. However, these technologies require expensive instruments and knowledge of solution thermodynamics. Recently, a much simpler mass photometry that measures the molecular weight of a single particle was added to those technologies, but also requires expensive instrument [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Another issue with mass photometry is the concentration of macromolecules, which must be diluted so that the light scattering of each single particle can be determined. These technologies also require measurements on purified samples. On the contrary, Ferguson plot has none of these limitations. As long as the target band can be identified, this technology can be used for impure samples and with minute quantities.\u003c/p\u003e \u003cp\u003eIt appears that Ferguson plot gave a molecular weight of the halophilic ClpB that was inconsistent with the native hexameric assembly determined for the same protein [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In the above paper [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], the hexamer structure was inferred by glutaraldehyde cross-linking of the purified ClpB protein followed by SDS-gel electrophoresis, which showed a ladder of protein bands composed of monomers to hexamers: namely, not only the hexamers but also monomers and smaller oligomers were observed. This indicates most likely that the ClpB is forming a hexamer, which generates a ladder of cross-linked oligomers due to cross-linking efficiency. It is also possible that there may be larger structures, which could not be cross-linked efficiently by glutaraldehyde.\u003c/p\u003e \u003cp\u003eWhy does then Ferguson plot not show the hexamer molecular weight? One possibility may be that the cross-linking experiment underestimates the size of the ClpB in solution due to the possibility that the cross-linking efficiency falls to a large extent over the hexamer size as described above. Of course there are other possibilities. Ferguson plot cannot be used for ClpB or any proteins, if their electrophoretic mobility is affected by agarose gels, for example, if the protein structure or charged state changes as a function of agarose gel concentration. Namely, they may lose charges or alter structures at high gel concentration, which would cause slower mobility in gel. Alternatively, ClpB binds to agarose gel that may be dependent on agarose concentration. These effects may be combined with errors in determination of electrophoretic mobility on agarose native gel electrophoresis, which had led to the molecular weights different from the hexameric assembly. There exist other possibilities, e.g., deviation from the linear relationship between the log mobility and gel concentration [\u003cspan additionalcitationids=\"CR27 CR28 CR29\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] or the effects of gel matrix on macromolecular assembly [\u003cspan additionalcitationids=\"CR32 CR33\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAnother more likely possibility is dimerization of the hexameric assembly unit of the halophilic ClpB. Structural analysis of the human mitochondrial homologue (i.e., non-halophilic) SKD3/CLPB using cryo-electron microscopy revealed a dodecamer structure composed of double hexameric rings, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003e [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Mass photometry done at low protein concentrations identified three distinct peaks corresponding to monomers, hexamers, and dodecamers. At protein concentrations of 91 nM (~\u0026thinsp;0.01 mg/mL), the proportion of monomers was dominant, whereas the proportions of hexamers and dodecamers increased at 240 nM (~\u0026thinsp;0.024 mg/mL). Negative-staining electron microscopy indicated the formation of large oligomers with significant size heterogeneity [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Analytical ultracentrifugation experiments with \u003cem\u003eE. coli\u003c/em\u003e ClpB chaperone at 1 mg/mL [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], a much higher concentration than the above mass photometry analysis, showed an equilibrium between monomers, hexamers, and dodecamers that was dependent on salt concentration. At 20 and 50 mM salt the dodecamer predominated, and the monomer content increased with rising salt concentration, suggesting that the electrostatic interactions, which can be neutralized by salt, are involved in the chaperone assembly. Agarose native gel electrophoresis of halophilic \u003cem\u003eChromohalobacter salexigens\u003c/em\u003e ClpB, conducted in this study at concentrations\u0026thinsp;~\u0026thinsp;10-fold higher than those used in mass photometry, suggested an increased proportion of oligomers, potentially exceeding dodecamers. Additionally, no salt was added to the electrophoresis system. In the yeast (\u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e) homologue HSP104, cryo-electron microscopy reported a hexamer [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], though a dodecamer cylindrical model has also been proposed [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Atomic force microscopy (AFM) analysis of \u003cem\u003eT. thermophilus\u003c/em\u003e ClpB revealed a primary hexameric ring structure, with larger, unstable oligomers also observed [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Our DLS analysis of ClpB also detected two peaks, indicating the presence of two different oligomers in the solution. Although hydrodynamic size of the ClpB could not be calculated in His/MES buffer, which is the same condition used in agarose electrophoresis, the autocorrelation functions suggests that the ClpB without salt may exist in a larger assembly state than in HEPES buffer with salt (Fig. S2B). Thus, depending on the organism and analytical method, the hexamer to dodecamer size may be detected. Similarly, the well-studied chaperone protein \u003cem\u003eE. coli\u003c/em\u003e GroEL [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] exhibited a 14-mer structure composed of two stacked heptameric rings as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIt should be mentioned that the Ferguson plot and DLS analysis used here are rather qualitative and thus, for studying chaperons, specifically ClpB, it would be necessary to apply other techniques to determine its absolute molecular weight. Such techniques include sedimentation equilibrium [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], static light scattering [\u003cspan additionalcitationids=\"CR6 CR7 CR8\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] and mass photometry [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. These technologies can determine the molecular weight of the particles, regardless of their structures, whether compact or extended, although these techniques also have following limitations. Sedimentation analysis cannot be done at high protein concentration due to increasing non-ideality at higher protein concentration and mass photometry analysis requires a dilute protein solution. Static light scattering analysis is always hampered by large particles, e.g., dusts or contaminating particles, which dominate light scattering. Compared to these techniques, agarose native gel electrophoresis can be done at any protein concentration, as long as bands can be observed and identified. For low concentration samples, more sensitive staining method can be used.\u003c/p\u003e \u003cp\u003eLastly, it is puzzling that ClpB showed a single band on agarose native gel electrophoresis, but two peaks on DLS analysis. One possibility is poor size resolution of 1% UltraPure agarose, resulting in inability to separate the hexamers from the dodecamers. However, even a higher agarose concentration was unable to separate them and provide their respective molecular weight information. Second possibility is the effects of gel structure on assembly state of the ClpB [\u003cspan additionalcitationids=\"CR32 CR33\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Namely, the small cell confinement of the gel structure enforces the protein-protein interactions and stabilizes the molecular assembly, here dodecamer structures. Thus, it would be of great interest to test more porous or alternatively higher resolution (small cell confinement) gels to evaluate the effects of gel structure on macromolecular assembly. Nevertheless, it may be safe to conclude that the halophilic ClpB forms oligomers in different size in solution and the dodecamer structure is stabilized under confined environments of gel matrix.\u003c/p\u003e"},{"header":"5 Conclusion","content":"\u003cp\u003eWe have tested Ferguson plot for determination of the molecular weight of halophilic chaperone, ClpB, in the native state. The results showed an approximate size of dodecamer, which appears to be in part consistent with the DLS analysis, which showed two peaks corresponding to a hexamer and a dodecamer. Thus, it can be concluded that Ferguson plot, combined with the agarose native gel electrophoresis, can be used to qualitatively determine the oligomerization of proteins.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Prof. Masao Tokunaga for his help in providing the ClpB samples and manuscript preparation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTeruo Akuta:\u0026nbsp;Methodology, Visualization,\u0026nbsp;Writing,\u0026nbsp;Supervision\u003c/p\u003e\n\u003cp\u003eYui Tomioka: Visualization, Formal analysis\u003c/p\u003e\n\u003cp\u003eTomoto Ura: Visualization, Formal Analysis\u003c/p\u003e\n\u003cp\u003eMasataka Nakagawa:\u0026nbsp;Visualization\u003c/p\u003e\n\u003cp\u003eTsutomu Arakawa: Conceptualization, Writing - review \u0026amp; editing, Supervision\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eNo external funding was received.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003eData are available upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interests\u003c/strong\u003e This work was supported by Kyokuto Pharmaceutical Industrial Co., Ltd. \u0026nbsp;Employees T.A. (Teruo Akuta), Y.T., and M.N. are affiliated with Kyokuto Pharmaceuticals. T.U. is affiliated with the University of Tsukuba and the National Institutes for Quantum Science and Technology. The authors declare no competing interests. T.A. (Tsutomu Arakawa) previously worked at Alliance Protein Laboratories but currently has no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFerguson KA (2007) The origin of the Ferguson plot. Electrophoresis 28(4): 499-500. https://doi.org/10.1002/elps.200600666\u003c/li\u003e\n\u003cli\u003eFerguson KA (1964) Starch-gel electrophoresis-application to the classification of pituitary proteins and polypeptides. Metabolism 13:SUPPL: 985-1001. \u003cu\u003ehttps://doi.org/10.1016/S0026-0495(64)80018-4\u003c/u\u003e \u003c/li\u003e\n\u003cli\u003ePhilo JS (2006) Is any measurement method optimal for all aggregate sizes and types? 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We here tested the validity of Ferguson plot based on agarose native gel electrophoresis using multimeric chaperone protein, ClpB, derived from moderate halophile that forms a native hexamer. Ferguson plot showed a single band with a molecular weight of 1,500 kDa, about twice the size of the native hexamer, which may be consistent with the structure of other chaperons that form a double ring comprising a dimer of two hexamer assembly units, i.e, dodecamer. In fact, dynamic light scattering experiment showed two peaks, which appears to correspond to the hexamer and dodecamer structures.\u003c/p\u003e","manuscriptTitle":"Ferguson plot analysis of chaperone ClpB from moderate halophile","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-08 13:57:43","doi":"10.21203/rs.3.rs-4993116/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-10-18T05:31:49+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-08T02:32:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"252917132224540576161146667665688077110","date":"2024-09-16T13:38:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"336541057463950840738114894578364687353","date":"2024-09-14T13:53:37+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-14T05:29:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"292821226466688630704723488895461266272","date":"2024-09-11T06:48:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"79417077453621171219800735617878031397","date":"2024-09-10T03:13:44+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-09T14:39:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"329625286009417442165823112886499690030","date":"2024-09-09T09:47:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"315604508327504377266865598368194209060","date":"2024-09-09T07:17:03+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-09-09T05:28:22+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-08-31T14:13:21+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-08-31T14:10:49+00:00","index":"","fulltext":""},{"type":"submitted","content":"The Protein Journal","date":"2024-08-28T18:52:56+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"the-protein-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jopc","sideBox":"Learn more about [The Protein Journal](http://link.springer.com/journal/10930)","snPcode":"10930","submissionUrl":"https://submission.nature.com/new-submission/10930/3","title":"The Protein Journal","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"ae4a58ad-a2d1-47fb-8b03-400f627ba1f2","owner":[],"postedDate":"October 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-01-06T16:01:35+00:00","versionOfRecord":{"articleIdentity":"rs-4993116","link":"https://doi.org/10.1007/s10930-024-10245-6","journal":{"identity":"the-protein-journal","isVorOnly":false,"title":"The Protein Journal"},"publishedOn":"2025-01-05 15:57:29","publishedOnDateReadable":"January 5th, 2025"},"versionCreatedAt":"2024-10-08 13:57:43","video":"","vorDoi":"10.1007/s10930-024-10245-6","vorDoiUrl":"https://doi.org/10.1007/s10930-024-10245-6","workflowStages":[]},"version":"v1","identity":"rs-4993116","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4993116","identity":"rs-4993116","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}