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• W2145875360 abstract "Protein aggregation is detrimental to the maintenance of proper protein homeostasis in all cells. To overcome this problem, cells have evolved a network of molecular chaperones to prevent protein aggregation and even reverse existing protein aggregates. The most extensively studied disaggregase systems are ATP-driven macromolecular machines. Recently, we reported an alternative disaggregase system in which the 38-kDa subunit of chloroplast signal recognition particle (cpSRP43) efficiently reverses the aggregation of its substrates, the light-harvesting chlorophyll a/b-binding (LHC) proteins, in the absence of external energy input. To understand the molecular mechanism of this novel activity, here we used biophysical and biochemical methods to characterize the structure and nature of LHC protein aggregates. We show that LHC proteins form micellar, disc-shaped aggregates that are kinetically stable and detergent-resistant. Despite the nonamyloidal nature, the LHC aggregates have a defined global organization, displaying the chaperone recognition motif on its solvent-accessible surface. These findings suggest an attractive mechanism for recognition of the LHC aggregate by cpSRP43 and provide important constraints to define the capability of this chaperone. Protein aggregation is detrimental to the maintenance of proper protein homeostasis in all cells. To overcome this problem, cells have evolved a network of molecular chaperones to prevent protein aggregation and even reverse existing protein aggregates. The most extensively studied disaggregase systems are ATP-driven macromolecular machines. Recently, we reported an alternative disaggregase system in which the 38-kDa subunit of chloroplast signal recognition particle (cpSRP43) efficiently reverses the aggregation of its substrates, the light-harvesting chlorophyll a/b-binding (LHC) proteins, in the absence of external energy input. To understand the molecular mechanism of this novel activity, here we used biophysical and biochemical methods to characterize the structure and nature of LHC protein aggregates. We show that LHC proteins form micellar, disc-shaped aggregates that are kinetically stable and detergent-resistant. Despite the nonamyloidal nature, the LHC aggregates have a defined global organization, displaying the chaperone recognition motif on its solvent-accessible surface. These findings suggest an attractive mechanism for recognition of the LHC aggregate by cpSRP43 and provide important constraints to define the capability of this chaperone. The proper folding of proteins into their native structures is essential for the function and survival of cells. However, environmental stress, molecular crowding, and potential exposure of hydrophobic regions of proteins during their biogenesis (1Hartl F.U. Hayer-Hartl M. Molecular chaperones in the cytosol: from nascent chain to folded protein.Science. 2002; 295: 1852-1858Crossref PubMed Scopus (2786) Google Scholar, 2Balch W.E. Morimoto R.I. Dillin A. Kelly J.W. Adapting proteostasis for disease intervention.Science. 2008; 319: 916-919Crossref PubMed Scopus (1748) Google Scholar, 3Doyle S.M. Wickner S. Hsp104 and ClpB: protein disaggregating machines.Trends Biochem. Sci. 2009; 34: 40-48Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar) pose challenges to protein folding in vivo. In this setting, improper intra- or intermolecular interactions can lead to the aggregation of proteins. Aggregate formation is detrimental to cells as it removes functional proteins (4Weibezahn J. Tessarz P. Schlieker C. Zahn R. Maglica Z. Lee S. Zentgraf H. Weber-Ban E.U. Dougan D.A. Tsai F.T. Mogk A. Bukau B. Thermotolerance requires refolding of aggregated proteins by substrate translocation through the central pore of ClpB.Cell. 2004; 119: 653-665Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar). Moreover, some aggregates, both amorphous ones and those that lead to highly ordered amyloid fibrils, are toxic to cells and have been implicated in a variety of protein folding diseases (5Luheshi L.M. Dobson C.M. Bridging the gap: from protein misfolding to protein misfolding diseases.FEBS Lett. 2009; 583: 2581-2586Crossref PubMed Scopus (86) Google Scholar, 6Broadley S.A. Hartl F.U. The role of molecular chaperones in human misfolding diseases.FEBS Lett. 2009; 583: 2647-2653Crossref PubMed Scopus (122) Google Scholar, 7Powers E.T. Morimoto R.I. Dillin A. Kelly J.W. Balch W.E. Biological and chemical approaches to diseases of proteostasis deficiency.Annu. Rev. Biochem. 2009; 78: 959-991Crossref PubMed Scopus (856) Google Scholar). Cells have evolved elaborate mechanisms to overcome the problems associated with protein aggregation. A specialized class of molecular chaperones, the “disaggregases,” can perform the energetically uphill process of reversing protein aggregation. Thus far, studies of disaggregases have been dominated by the Clp/Hsp100 family of AAA+ 3The abbreviations used are: AAA+ATPases associated with various cellular activitiescpSRPchloroplast signal recognition particleLHClight-harvesting chlorophyll a/b-bindingLHCPLHC proteinTMtransmembraneAβamyloid βTEMtransmission electron microscopyAFMatomic force microscopyThTthioflavin TANS1-anilino-8-naphthalene sulfonateLDAOn-dodecyl-N,N,-dimethylamine-N-oxideDDMn-dodecyl-β-d-maltopyranosideβ-OGn-octyl-β-d-glucopyranosideBNGn-nonyl-β-d-glucopyranosideMTSSL1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl methanethiosulfonateNEMN-ethyl-maleimideGdmHClguanidinium hydrochloride. ATPases (ATPases associated with various cellular activities), such as ClpB in prokaryotes and Hsp104 in yeasts (3Doyle S.M. Wickner S. Hsp104 and ClpB: protein disaggregating machines.Trends Biochem. Sci. 2009; 34: 40-48Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). Both are large hexameric rings (>500 kDa) powered by mechanical forces from ATP hydrolysis and require additional co-chaperones to efficiently disassemble a variety of protein aggregates (8Doyle S.M. Shorter J. Zolkiewski M. Hoskins J.R. Lindquist S. Wickner S. Asymmetric deceleration of ClpB or Hsp104 ATPase activity unleashes protein-remodeling activity.Nat. Struct. Mol. Biol. 2007; 14: 114-122Crossref PubMed Scopus (121) Google Scholar, 9Glover J.R. Lindquist S. Hsp104, Hsp70, and Hsp40: a novel chaperone system that rescues previously aggregated proteins.Cell. 1998; 94: 73-82Abstract Full Text Full Text PDF PubMed Scopus (1098) Google Scholar). The complexity of these disaggregase systems and the promiscuity in their substrate selection have made it difficult to pinpoint their molecular mechanisms of action. Further, AAA+ disaggregase machines were only found in prokaryote and yeast, and no homologues have been identified in higher eukaryotes outside of plastids and mitochondria. It is conceivable that alternative mechanisms of disaggregation, such as the recently described Hsp110–70–40 system (10Duennwald M.L. Echeverria A. Shorter J. Small heat shock proteins potentiate amyloid dissolution by protein disaggregases from yeast and humans.PLoS Biol. 2012; 10: e1001346Crossref PubMed Scopus (140) Google Scholar, 11Rampelt H. Kirstein-Miles J. Nillegoda N.B. Chi K. Scholz S.R. Morimoto R.I. Bukau B. Metazoan Hsp70 machines use Hsp110 to power protein disaggregation.EMBO J. 2012; 31: 4221-4235Crossref PubMed Scopus (217) Google Scholar), could be used in higher eukaryotes. An understanding of alternative disaggregase systems can shed light on novel principles and mechanisms by which cellular chaperones overcome protein aggregates. ATPases associated with various cellular activities chloroplast signal recognition particle light-harvesting chlorophyll a/b-binding LHC protein transmembrane amyloid β transmission electron microscopy atomic force microscopy thioflavin T 1-anilino-8-naphthalene sulfonate n-dodecyl-N,N,-dimethylamine-N-oxide n-dodecyl-β-d-maltopyranoside n-octyl-β-d-glucopyranoside n-nonyl-β-d-glucopyranoside 1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl methanethiosulfonate N-ethyl-maleimide guanidinium hydrochloride. Previously, we identified an efficient disaggregase activity in the chloroplast signal recognition particle 43 subunit (cpSRP43) (12Jaru-Ampornpan P. Shen K. Lam V.Q. Ali M. Doniach S. Jia T.Z. Shan S. ATP-independent reversal of a membrane protein aggregate by a chloroplast SRP subunit.Nat. Struct. Mol. Biol. 2010; 17: 696-702Crossref PubMed Scopus (55) Google Scholar). This provides an example in which a relatively small protein scaffold (38 kDa) can recognize and disrupt large protein aggregates in an ATP-independent mechanism (12Jaru-Ampornpan P. Shen K. Lam V.Q. Ali M. Doniach S. Jia T.Z. Shan S. ATP-independent reversal of a membrane protein aggregate by a chloroplast SRP subunit.Nat. Struct. Mol. Biol. 2010; 17: 696-702Crossref PubMed Scopus (55) Google Scholar), in contrast to the Clp/Hsp100 family of disaggregases. cpSRP43 is part of the protein targeting machinery, the cpSRP, that mediates the delivery of the light-harvesting chlorophyll a/b-binding (LHC) family of proteins to the thylakoid membrane (13Schuenemann D. Gupta S. Persello-Cartieaux F. Klimyuk V.I. Jones J.D. Nussaume L. Hoffman N.E. A novel signal recognition particle targets light-harvesting proteins to the thylakoid membranes.Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 10312-10316Crossref PubMed Scopus (163) Google Scholar, 14Groves M.R. Mant A. Kuhn A. Koch J. Dübel S. Robinson C. Sinning I. Functional characterization of recombinant chloroplast signal recognition particle.J. Biol. Chem. 2001; 276: 27778-27786Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 15Tu C.-J. Schuenemann D. Hoffman N.E. Chloroplast FtsY, chloroplast signal recognition particle, and GTP are required to reconstitute the soluble phase of light-harvesting chlorophyll protein transport into thylakoid membranes.J. Biol. Chem. 1999; 274: 27219-27224Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). The most abundant member of the LHC family, LHCP, comprises ∼30% of the proteins on the thylakoid membrane and is arguably the most abundant membrane protein on earth. The sheer abundance of these proteins and their highly hydrophobic nature demands highly effective chaperones that protect them from aggregation before arrival at the membrane. In the chloroplast stroma, this chaperone function is provided by cpSRP43, which effectively protects LHC proteins from aggregation and can even reverse preformed large LHC protein aggregates (12Jaru-Ampornpan P. Shen K. Lam V.Q. Ali M. Doniach S. Jia T.Z. Shan S. ATP-independent reversal of a membrane protein aggregate by a chloroplast SRP subunit.Nat. Struct. Mol. Biol. 2010; 17: 696-702Crossref PubMed Scopus (55) Google Scholar, 16Falk S. Sinning I. cpSRP43 is a novel chaperone specific for light-harvesting chlorophyll a,b-binding proteins.J. Biol. Chem. 2010; 285: 21655-21661Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). cpSRP43 recognizes a highly conserved 18-amino acid loop between the second and the third transmembrane (TM) domains of LHC proteins, termed L18 (17DeLille J. Peterson E.C. Johnson T. Moore M. Kight A. Henry R. A novel precursor recognition element facilitates posttranslational binding to the signal recognition particle in chloroplasts.Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 1926-1931Crossref PubMed Scopus (74) Google Scholar, 18Tu C.J. Peterson E.C. Henry R. Hoffman N.E. The L18 domain of light-harvesting chlorophyll proteins binds to chloroplast signal recognition particle 43.J. Biol. Chem. 2000; 275: 13187-13190Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). In previous work, we showed that the specific interaction of cpSRP43 with the L18 motif is crucial for the chaperone and disaggregase activity of cpSRP43 (12Jaru-Ampornpan P. Shen K. Lam V.Q. Ali M. Doniach S. Jia T.Z. Shan S. ATP-independent reversal of a membrane protein aggregate by a chloroplast SRP subunit.Nat. Struct. Mol. Biol. 2010; 17: 696-702Crossref PubMed Scopus (55) Google Scholar). This and other observations led us to propose that, in the absence of external energy input, cpSRP43 uses specific binding energy with its substrate proteins to remodel and rescue LHC protein aggregation (12Jaru-Ampornpan P. Shen K. Lam V.Q. Ali M. Doniach S. Jia T.Z. Shan S. ATP-independent reversal of a membrane protein aggregate by a chloroplast SRP subunit.Nat. Struct. Mol. Biol. 2010; 17: 696-702Crossref PubMed Scopus (55) Google Scholar). To gain insights into the molecular mechanism that underlies the novel disaggregase activity of cpSRP43, we need to first understand the nature of the LHC aggregate and identify the structural features that facilitate its disassembly by cpSRP43. To this end, we examined the nature and structure of the LHC aggregate using biophysical and biochemical techniques. We show that LHC proteins form disc-like particles with a relatively amorphous hydrophobic core, but exhibit a defined interior/exterior organization in which the L18 recognition motif is displayed on the solvent-exposed surface. This suggests an attractive mechanism for cpSRP43 to recognize the LHC aggregates and thus initiate their disassembly. LHCP, Lhcb5, and their mutants were purified under denaturing conditions as described (12Jaru-Ampornpan P. Shen K. Lam V.Q. Ali M. Doniach S. Jia T.Z. Shan S. ATP-independent reversal of a membrane protein aggregate by a chloroplast SRP subunit.Nat. Struct. Mol. Biol. 2010; 17: 696-702Crossref PubMed Scopus (55) Google Scholar), except that 6 m GdmHCl was used instead of 8 m urea for Lhcb5. Aβ1–40 and recrystallized thioflavin T (ThT) were generous gifts from Dr. J. W. Kelly. 1-Anilino-8-naphthalene sulfonate (ANS) and bis-ANS were from Sigma and Invitrogen, respectively. n-Dodecyl-N,N,-dimethylamine-N-oxide (LDAO), n-dodecyl-β-d-maltopyranoside (DDM), n-octyl-β-d-glucopyranoside (β-OG), and n-nonyl-β-d-glucopyranoside (BNG) were from Anatrace. Triton X-100 was from Sigma, and SDS was from Bio-Rad. Urea and GdmHCl were molecular biology grade from MP and Sigma, respectively. 1-Oxyl-2,2,5,5-tetramethylpyrroline-3-methyl methanethiosulfonate (MTSSL) was from Toronto Research Chemicals, N-ethyl-maleimide was from Sigma, and N-(1-pyrene)-maleimide was from Invitrogen. Light scattering experiments were performed as described previously (12Jaru-Ampornpan P. Shen K. Lam V.Q. Ali M. Doniach S. Jia T.Z. Shan S. ATP-independent reversal of a membrane protein aggregate by a chloroplast SRP subunit.Nat. Struct. Mol. Biol. 2010; 17: 696-702Crossref PubMed Scopus (55) Google Scholar). For formation of aggregates (see Fig. 3, black), unfolded LHCP in 8 m urea was directly diluted into Buffer D (50 mm KHEPES, pH 7.5, 200 mm NaCl) to the desired final concentration; the final concentration of urea was equalized among different samples. The critical micelle concentration is obtained as the x-intercept from the linear fit of the data (19Hurshman A.R. White J.T. Powers E.T. Kelly J.W. Transthyretin aggregation under partially denaturing conditions is a downhill polymerization.Biochemistry. 2004; 43: 7365-7381Crossref PubMed Scopus (287) Google Scholar). For serial dilution experiments (see Fig. 3, red), the sample at 1 μm LHCP was serially diluted (by 2-fold) into fresh Buffer D and allowed 10–30 min to equilibrate before the measurement. LHCP aggregates were formed by diluting unfolded LHCP in 8 m urea into Buffer D to a final concentration of 2 μm. After incubation at 25 °C for 5 min, the sample was diluted 5-fold and immediately deposited onto a glow-discharged 200-mesh Formvar grid (Ted Pella Inc.). After a 45-s adsorption time, the grid was washed in water and then stained with 1% uranyl acetate for 45 s. Transmission electron microscopy (TEM) images were obtained on a 120-kV Tecnai T12 electron microscope coupled with a CCD camera. The diameters of the particles were measured using ImageJ (20Abramoff M.D. Magelhães P.J. Ram S.J. Image processing with Image J.Biophotonics Int. 2004; 11: 36-42Google Scholar). 1 μm LHCP aggregate in Buffer D was deposited onto a freshly cleaved mica and incubated for 5 min at 25 °C to allow equilibration. The wafer was rinsed with Millipore water and dried under the weak flux of nitrogen. Atomic force microscopy (AFM) images were taken immediately after the sample was prepared. A Digital Instrument Nanoscope IIIA AFM system in tapping mode was used throughout at ambient conditions. A sharp TESP tip (Veeco) was used in the experiment. Typical values for the force constant, resonance frequency, and tip radius were 42 newtons/m, 320 kHz, and 8 nm, respectively. Particle sizes were obtained by calculating the projected area of each particle at half-maximal height onto the surface. This is because the apparent lateral size of surface features is usually overestimated due to the broadening effect of the AFM tip. The cross-sectional area at half the maximum height provides a more realistic distribution of sizes of the particles. All fluorescence experiments were carried out in Buffer D using a FluoroLog 3-22 spectrofluorometer (HORIBA Jobin Yvon). For bis-ANS experiments, 1 mm bis-ANS was added to Buffer D with or without 1 μm LHCP aggregate. The samples were excited at 395 nm and then scanned from 410 to 620 nm, with excitation and emission band passes of 2 and 5 mm, respectively. For ThT experiments, 20 mm recrystallized ThT was added to Buffer D containing no aggregate, aggregates from 1 or 5 μm LHCP, or 15 μm freshly sonicated Aβ1–40 amyloid. The samples were excited at 440 nm and then scanned from 470 to 570 nm, with the excitation and emission band passes of 3 and 7 mm, respectively. For comparison, ThT fluorescence from 1 and 5 μm unfolded LHCP in 8 m urea was measured. For pyrene excimer experiments, DTT-reduced single cysteine mutants of Lhcb5 in 6 m GdmHCl were labeled with a 30-fold molar excess of pyrene maleimide at room temperature in the dark for 2 h. Excess pyrene was removed by gel filtration, and the efficiency of spin labeling (90–100%) was determined by LC-MSDSL 1100 series (Agilent Technologies, Santa Clara, CA). The samples were prepared by diluting pyrene-labeled Lhcb5 pairs into Buffer D for a final concentration of 1.5 μm for each variant. Spectra were obtained from excitation at 317 nm and then scanned from 360 to 560 nm, with excitation and emission band passes of 3 and 6 mm, respectively. The amount of excimer fluorescence, indicated by a red shift to 445 nm, is normalized against the nonexcited fluorescence signal at 376 nm. Statistically, when two variants A and B are mixed, there is a population distribution of homo-pairs (e.g. 25% A-A and 25% B-B) and hetero-pairs (50% A-B). The equation below corrects for the real hetero-pair excimer (FAB) FAB=2×(FAB,app−0.25FA−0.25FB)(Eq. 1) where FAB,app is the apparent ratio of excimer fluorescence (I445/I376) between two pyrene-labeled variants, and FA and FB is the ratio of excimer fluorescence of each individual variants measured separately. Unfolded LHCP was diluted to 10 μm in Buffer D and incubated at 25 °C for 5 min. Aggregation was complete, judged by the absence of LHCP in the supernatant after centrifugation at 13,000 rpm in a microcentrifuge for 30 min. The pellet was dissolved with 50 μl of detergent or chemical denaturants at different concentrations for 30 min at 25 °C. The mixtures were then spun at 13,000 rpm in a microcentrifuge for 30 min, and soluble and pellet fractions were boiled and visualized by SDS-PAGE. For Fig. 2B, the assay was performed as described for amyloid fibrils (21Chernoff Y.O. Uptain S.M. Lindquist S.L. Analysis of prion factors in yeast.Methods Enzymol. 2002; 351: 499-538Crossref PubMed Scopus (96) Google Scholar). Briefly, aggregation of 10 μm LHCP in Buffer D preceded for 5 min at 25 °C. The mixture was then mixed with 2% SDS-PAGE loading buffer and incubated at either 25 °C or 100 °C for 10 min prior to SDS-PAGE. Only the proteins that migrated into the resolving gel (e.g. solubilized portion) were visualized. Spin labeling reactions were performed in 6 m GdmHCl, 50 mm KHEPES, pH 7.5, and 2 mm EDTA. Reduced and degassed single cysteine mutants of Lhcb5 were labeled with a 3–5-fold molar excess of MTSSL at room temperature in the dark for 2–3 h. Excess MTSSL was removed by gel filtration, and the efficiency of spin labeling (80–100%) was determined by electron paramagnetic resonance (EPR) using a 2,2,6,6-tetramethyl peperidine-N-oxyl calibration curve according to manufacturer's instructions (Bruker). EPR spectra were acquired using a 9.4-GHz (X-band) EMX EPR spectrometer (Bruker) equipped with an ER 4119HS cavity at 20–23 °C. To form the aggregate, the individual spin-labeled proteins in GdmHCl were diluted into Buffer D. The concentrations of the aggregate samples were 30–100 μm. Data acquisition was as previously described (22Lam V.Q. Akopian D. Rome M. Henningsen D. Shan S. Lipid activation of the signal recognition particle receptor provides spatial coordination of protein targeting.J. Cell Biol. 2010; 190: 623-635Crossref PubMed Scopus (40) Google Scholar). Cysteine mutants of Lhcb5 in 6 m GdmHCl were reduced with 2.5 mm tris(2-carboxyethyl)phosphine at room temperature for 2 h. Each mutant was diluted into Buffer D to a final concentration of 3.3 μm and incubated on ice for 10 min to form the aggregate followed by the addition of 100 μm N-ethyl-maleimide (NEM). The reaction was quenched with 50 mm DTT at various time points, concentrated under vacuum, and redissolved in 0.2% formic acid, and ∼25 pmol of protein was analyzed on an LC-MSD SL 1100 series (Agilent). The samples were chromatographed on a 2.1 × 150-mm ZORBAX 300SB-C3 column (Agilent) using a gradient consisting of 0.2% formic acid and 0.2% formic acid in acetonitrile (89.8%) and methanol (10%). Intact masses were measured in the single quadrupole and quantified using the software ChemStation software (Agilent). Control experiments where different ratios of unalkylated and alkylated proteins were mixed and subjected to MS analysis show the quantification of ratios of alkylated species to be reliable (see Fig. 6E). The reported accessibilities were calculated as a ratio between the alkylation of each cysteine mutant under aggregation Buffer D versus denaturing 6 m GdmHCl. To characterize the surface features of LHC protein aggregates, we used an established collection of small molecule dyes. Exposure of hydrophobic patches or crevices within aggregates can be probed by extrinsic fluorescent molecular dyes such as ANS and bis-ANS (23Stryer L. The interaction of a naphthalene dye with apomyoglobin and apohemoglobin. A fluorescent probe of non-polar binding sites.J. Mol. Biol. 1965; 13: 482-495Crossref PubMed Scopus (1335) Google Scholar, 24Rosen C.G. Weber G. Dimer formation from 1-anilino-8-naphthalenesulfonate catalyzed by bovine serum albumin. A new fluorescent molecule with exceptional binding properties.Biochemistry. 1969; 8: 3915-3920Crossref PubMed Scopus (199) Google Scholar). We tested whether the aggregates of LHCP, the most abundant member of the LHC protein family, share this feature. Indeed, the fluorescence of both ANS (data not shown) and bis-ANS (Fig. 1A) increased significantly in the presence of 1 μm LHCP aggregate, accompanied by a blue shift of the fluorescence emission spectra. These results strongly suggest that LHCP aggregates contain exposed hydrophobic microdomains that allow the binding of these dyes, consistent with the highly hydrophobic nature of this protein. We next used ThT to probe the structural organization of the LHCP aggregate. ThT is often used as a diagnostic for the formation of amyloid fibrils generated by amyloid-β (Aβ), α-synuclein, and other amyloidogenic proteins (25LeVine H. Quantification of β-sheet amyloid fibril structures with thioflavin T.Methods Enzymol. 1999; 309: 274-284Crossref PubMed Scopus (1200) Google Scholar). Similar to bis-ANS, the fluorescence of ThT exhibited a significant increase in intensity and a blue shift in spectrum in the presence of the LHCP aggregate (Fig. 1B, blue lines). The extent of these fluorescence changes is comparable with that induced by mature amyloid fibrils generated by the Aβ1–40 peptide (Fig. 1B, red versus blue, and Fig. 1C). As microscopy analyses did not indicate fibril formation in the LHCP aggregate (see below), these results suggest that ThT is not highly specific for amyloid fibrils, consistent with recent work observing ThT fluorescence of nonfibrillar aggregates of β-lactoglobulin and transthyretin (19Hurshman A.R. White J.T. Powers E.T. Kelly J.W. Transthyretin aggregation under partially denaturing conditions is a downhill polymerization.Biochemistry. 2004; 43: 7365-7381Crossref PubMed Scopus (287) Google Scholar, 26Carrotta R. Bauer R. Waninge R. Rischel C. Conformational characterization of oligomeric intermediates and aggregates in β-lactoglobulin heat aggregation.Protein Sci. 2001; 10: 1312-1318Crossref PubMed Scopus (124) Google Scholar). Instead, this dye possibly binds to hydrophobic grooves that are often present in amyloid fibrils but can also be generated by other types of aggregates (27Groenning M. Olsen L. van de Weert M. Flink J.M. Frokjaer S. Jørgensen F.S. Study on the binding of thioflavin T to β-sheet-rich and non-β-sheet cavities.J. Struct. Biol. 2007; 158: 358-369Crossref PubMed Scopus (198) Google Scholar). To probe the stability of the LHCP aggregate, we tested its solubility in various detergents, including LDAO, DDM, β-OG, BNG, and Triton X-100. By analyzing the amount of proteins in the soluble and insoluble fractions after medium speed sedimentation (see “Experimental Procedures”), we showed that none of these detergents were able to solubilize the LHCP aggregate at or above their respective concentrations typically used for membrane protein solubilization (Fig. 2A). In addition, we tested the solubility of the LHCP aggregate in SDS using an established protocol for amyloid fibrils (21Chernoff Y.O. Uptain S.M. Lindquist S.L. Analysis of prion factors in yeast.Methods Enzymol. 2002; 351: 499-538Crossref PubMed Scopus (96) Google Scholar). This assay evaluated solubility of the aggregate based on the mobility of the protein in SDS-PAGE after incubation with SDS-containing buffer at room temperature (see “Experimental Procedures”). “SDS-insoluble” amyloid fibrils or oligomeric protein aggregates cannot enter the resolving gel unless boiled (21Chernoff Y.O. Uptain S.M. Lindquist S.L. Analysis of prion factors in yeast.Methods Enzymol. 2002; 351: 499-538Crossref PubMed Scopus (96) Google Scholar). LHCP aggregate showed significant resistance to 2% SDS in this procedure as only 24% of the aggregates could be solubilized and migrated into the gel without boiling (Fig. 2B, right panel). SDS could solubilize large LHCP aggregates only after extensive incubation and boiling of the sample (Fig. 2B, left panel). Taken together, the detergent resistance of the LHC protein aggregate suggests the presence of highly stable packing interactions within the aggregate that must be overcome by cpSRP43. Formation of large LHC aggregates can be monitored based on light scattering at 360 nm (12Jaru-Ampornpan P. Shen K. Lam V.Q. Ali M. Doniach S. Jia T.Z. Shan S. ATP-independent reversal of a membrane protein aggregate by a chloroplast SRP subunit.Nat. Struct. Mol. Biol. 2010; 17: 696-702Crossref PubMed Scopus (55) Google Scholar). The scattering intensity increases linearly with LHCP concentration above ∼100 nm (Fig. 3, black), suggesting that aggregate formation was complete under these conditions. However, the linearity broke down at lower LHCP concentrations (Fig. 3 and inset, black). This was not due to limitations in instrument sensitivity; when preformed LHCP aggregates were diluted, linearity in light scattering intensity was observed at all concentrations and extrapolated through zero (Fig. 3 and inset, red). These observations show that: (i) the LHCP aggregate is kinetically stable and virtually irreversible once it has formed; and (ii) formation of the LHCP aggregate requires a critical protein concentration, reminiscent of the critical micelle concentration during micelle formation. An analogous, “critical aggregate concentration” of 125 nm was obtained for the LHCP aggregates from these data (see “Experimental Procedures”). This micelle-like characteristic begins to suggest a globular morphology of the LHC aggregates. To directly observe the global structure of LHC aggregates, we examined them using TEM and AFM. Negatively stained TEM images revealed LHCP aggregates to be circular particles (Fig. 4, A and B). Analysis of the size of these particles resulted in a distribution that fits well to a Gaussian function, with diameters of 12 ± 2 nm (Fig. 4C). Consistent with the EM images, AFM analysis also showed LHCP aggregates to be disc-shaped particles (Fig. 5, A and B) with mean area of 214 ± 94 nm2 (Fig. 5C) or mean diameter of 16 ± 5 nm, in good agreement with the EM measurements. Strikingly, the heights of the aggregates measured by AFM are “quantized” and peaked at integrals of 0.7–0.8 nm (Fig. 5D and inset). These results suggest that LHC proteins form disc-shaped aggregates with a height of 0.7–0.8 nm, and these discs can further stack upon one another.FIGURE 5AFM analysis of LHCP aggregates. A, large field view of AFM topographic image showing well separated LHCP aggregates. Large clusters are occasionally observed. The scale bar is 500 nm. B, a zoomed-in region of the image reveals disc-shaped particles. The lines indicate particles whose heights were measured (red, blue, and green). The scale bar is 100 nm. C, size distribution of LHCP aggregates, measured from several regions on the surface. The red line is a Gaussian fit to the data, which gave a mean area of the particle of 214 nm2. D, height distribution of" @default.
• W2145875360 created "2016-06-24" @default.
• W2145875360 creator A5000584662 @default.
• W2145875360 creator A5004914237 @default.
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• W2145875360 date "2013-05-01" @default.
• W2145875360 modified "2023-10-16" @default.
• W2145875360 title "Mechanism of an ATP-independent Protein Disaggregase" @default.
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