Subunit Exchange of Small Heat Shock Proteins
2000; Elsevier BV; Volume: 275; Issue: 2 Linguagem: Inglês
10.1074/jbc.275.2.1035
ISSN1083-351X
AutoresMichael P. Bova, Hassane S. Mchaourab, Yun Han, B K Fung,
Tópico(s)Bee Products Chemical Analysis
ResumoαA-Crystallin, a member of the small heat shock protein (sHsp) family, is a large multimeric protein composed of 30–40 identical subunits. Its quaternary structure is highly dynamic, with subunits capable of freely and rapidly exchanging between oligomers. We report here the development of a fluorescence resonance energy transfer method for measuring structural compatibility between αA-crystallin and other proteins. We found that Hsp27 and αB-crystallin readily exchanged with fluorescence-labeled αA-crystallin, but not with other proteins structurally unrelated to sHsps. Truncation of 19 residues from the N terminus or 10 residues from the C terminus of αA-crystallin did not significantly change its subunit organization or exchange rate constant. In contrast, removal of the first 56 or more residues converts αA-crystallin into a predominantly small multimeric form consisting of three or four subunits, with a concomitant loss of exchange activity. These findings suggest residues 20–56 are essential for the formation of large oligomers and the exchange of subunits. Similar results were obtained with truncated Hsp27 lacking the first 87 residues. We further showed that the exchange rate is independent of αA-crystallin concentration, suggesting subunit dissociation may be the rate-limiting step in the exchange reaction. Our findings reveal a quarternary structure of αA-crystallin, consisting of small multimers of αA-crystallin subunits in a dynamic equilibrium with the oligomeric complex. αA-Crystallin, a member of the small heat shock protein (sHsp) family, is a large multimeric protein composed of 30–40 identical subunits. Its quaternary structure is highly dynamic, with subunits capable of freely and rapidly exchanging between oligomers. We report here the development of a fluorescence resonance energy transfer method for measuring structural compatibility between αA-crystallin and other proteins. We found that Hsp27 and αB-crystallin readily exchanged with fluorescence-labeled αA-crystallin, but not with other proteins structurally unrelated to sHsps. Truncation of 19 residues from the N terminus or 10 residues from the C terminus of αA-crystallin did not significantly change its subunit organization or exchange rate constant. In contrast, removal of the first 56 or more residues converts αA-crystallin into a predominantly small multimeric form consisting of three or four subunits, with a concomitant loss of exchange activity. These findings suggest residues 20–56 are essential for the formation of large oligomers and the exchange of subunits. Similar results were obtained with truncated Hsp27 lacking the first 87 residues. We further showed that the exchange rate is independent of αA-crystallin concentration, suggesting subunit dissociation may be the rate-limiting step in the exchange reaction. Our findings reveal a quarternary structure of αA-crystallin, consisting of small multimers of αA-crystallin subunits in a dynamic equilibrium with the oligomeric complex. heat shock protein 4-acetamido-4′-((iodoacetyl)amino)stilbene 2,2′-disulfonic acid Lucifer Yellow iodoacetamide polymerase chain reaction polyacrylamide gel electrophoresis small heat shock protein fluorescence resonance energy transfer 3-(N-morpholino)propanesulfonic acid α-Crystallin and Hsp271 are members of the small heat shock protein (sHsp) family (1.Ingolia T.D. Craig E.A. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 2360-2364Crossref PubMed Scopus (708) Google Scholar, 2.Nene V. Dunne D.W. Johnson K.S. Taylor D.W. Cordinley J.S. Mol. Biochem. Parasitol. 1986; 21: 179-188Crossref PubMed Scopus (90) Google Scholar, 3.Lindquist S. Craig E.A. Annu. Rev. Genet. 1988; 22: 631-677Crossref PubMed Scopus (4556) Google Scholar, 4.Klemenz R. Frohli E. Steiger R.H. Schafer R. Aoyama A. Proc. Natl. Acad. Sci. U. S. 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In this study we further demonstrate by this method that αA-crystallin can form a reversible hetero-oligomeric complex with Hsp27 and αB-crystallin, but not with other proteins unrelated to the sHsp family. In addition, we have identified the N-terminal region of αA-crystallin and Hsp27 to be essential for subunit exchange and oligomerization. Based on the results of our studies, we further propose an exchange mechanism that may explain the oligomeric organization of αA-crystallin subunits. Lucifer Yellow iodoacetamide (LYI) and 4-acetamido-4′-((iodoacetyl)amino) stilbene-2,2′-disulfonic acid (AIAS) were purchased from Molecular Probes, Eugene, OR. Rhodanese was obtained from Sigma and used in the experiments without further purification. Restriction enzymes and Taq polymerase were purchased from New England Biolabs and Promega, respectively.Escherichia coli strain BL21DE3 and the pET 20b+ expression vector were obtained from Novagen. Purified β-crystallin, γ-crystallin, and human recombinant αB-crystallin were generous gifts from Dr. Joseph Horwitz, University of California, Los Angeles, CA. cDNA constructs of αA-crystallin and Hsp27 mutants were prepared by PCR amplification using the appropriate primers. The PCR reactions were carried out in a 25-μl volume containing 10 mm Tris-HCl, pH 8.3, 50 mm KCl, 1.5 mm MgCl2, 50 μm dNTP, 1.0 μm primers, 2.5 units ofTaq polymerase, and 100 ng of αA-crystallin or Hsp27 cDNA as a template. Amplification was performed for 25 cycles with conditions for denaturation at 94 °C for 1 min, annealing at 54 °C for 2 min, and extension at 72 °C for 3 min. The PCR products were gel-purified and ligated into the pET 20b+expression vector. The correct constructs were confirmed by DNA sequencing. BL21DE3 cells harboring the pET 20b+ αA-crystallin or Hsp27 constructs were grown in 500 ml of LB broth to a cell density between 0.6–1.0 optical density at 600 nm and induced with 0.5 mmisopropyl-β-d-thiogalactopyranoside for 3 h. Cells were harvested by centrifugation at 4,000 × g for 10 min; resuspended in 20 ml of ice-cold buffer containing 0.1m NaCl, 2 mm EDTA, 50 mm Tris, pH 7.9; and lysed by sonication. The cell lysates were clarified by centrifugation at 15,000 × g for 30 min to remove the particulates, followed by filtration through a 0.2-μm filter. Polyethyleneimine was then added to the filtrate with rapid stirring to form a 0.12% solution. After incubation on ice for 2 min, the mixture was centrifuged at 15,000 × g for 10 min to remove the precipitated DNA. The clear supernatant containing the soluble recombinant protein was applied onto a Mono-Q column (Amersham Pharmacia Biotech) pre-equilibrated with 0.1 m NaCl, 20 mm Tris, pH 8.5, and eluted using a linear gradient of 0.1–1 m NaCl in the same buffer. Fractions containing the recombinant protein were pooled, concentrated by ultrafiltration, and further purified by Superose 6 gel filtration chromatography (Amersham Pharmacia Biotech) equilibrated with 100 mm NaCl, 50 mm sodium phosphate, pH 7.5. All the wild-type and truncated proteins of αA-crystallin and Hsp27 were purified by the procedure described above except αA-crystallin-(56–173), αA-crystallin-(65–173), and αA-crystallin-(85–173), which required an additional step following the Mono-Q ion exchange chromatography. In this case, the fractions containing mutant protein were pooled, concentrated, and adjusted to 1m ammonium sulfate, pH 7.0. The protein solution was then applied to a phenyl-Sepharose HP column containing a 1-ml bed volume pre-equilibrated with 1 m ammonium sulfate, 50 mm sodium phosphate, pH 7.0, and eluted using a linear gradient of 1.0–0.1 m ammonium sulfate in the same buffer. Fractions containing these mutant proteins were pooled, concentrated, and further purified by gel filtration chromatography as described earlier. αA-crystallin was labeled with AIAS or LYI under the conditions as described previously (67.Bova M.P. Ding L.L. Horwitz J. Fung B.K.-K. J. Biol. Chem. 1997; 272: 29511-29517Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar). The specific labeling of the single cysteine residue was confirmed by titration with 5,5′-dithiobis(2-nitrobenzoic acid). AIAS-labeled αA-crystallin-(56–173) was prepared using the same conditions as the wild type, with the exception that the purified truncated protein was labeled with AIAS for 3 h at room temperature. LYI-labeled αA-crystallin-(56–173) was prepared similarly. However, the reaction time was extended for an additional 1 h at 37 °C. Hsp27 was labeled with AIAS in a reaction mixture containing 1 mg/ml recombinant protein, 3.2 mm AIAS, 100 mm NaCl, 20 mm MOPS, pH 7.9 for 3 h at 37 °C. Hsp27 was labeled with 8.4 mm LYI for 9 h at 37 °C with the same buffer conditions. Unreacted AIAS or LYI were separated from the labeled proteins on a G-25 Sephadex desalting column equilibrated with Buffer A (100 mm NaCl, 50 mm sodium phosphate, pH 7.5). The final protein preparations were separated on SDS-PAGE, and the absence of free fluorescent label was confirmed by fluorescence imaging of the unstained gels. In some preparations, gel filtration of the fluorescent αA-crystallin under denaturing condition was also employed to assure the covalent attachment of the label to the protein. Proteins with a quaternary structure compatible with αA-crystallin were determined by a FRET method based on reversibility of the subunit exchange reaction. αA-crystallin mixtures containing an equal amount of donor and acceptor was prepared by incubating LYI-labeled αA-crystallin (20 μm) and AIAS-labeled αA-crystallin (20 μm) in Buffer A at 37 °C for 4 h. The complete mixing of the two subunit populations was confirmed by monitoring the quenching of the emission intensity of AIAS fluorescence at 415 nm. A 100-μl aliquot of the test protein at concentration specified in the figure legend was then added to an equal volume of fluorescent αA-crystallin in Buffer A. At various time periods, 40 μl of the protein mixture was removed, diluted 50-fold with the same buffer, and the AIAS emission intensity at 415 nm were determined. The rate was calculated from the equationF(t)/F(0) = A +Be -kt, where k is defined as the transfer rate constant, and F(t) andF(0) correspond to the emission intensity at time =t and 0, respectively. The constants, A andB, were determined using the initial condition where A + B = 1 at t = 0 and the final value ofA = F(∞)/F(0) att = ∞. It is important to note that sinceF(∞)/F(0) > 1, the constant Bis a negative number, in agreement with the decrease in energy transfer between donors and acceptors as the fluorescent αA-crystallin is diluted by the test protein. The rate constant was determined by non-linear regression analysis of the data using the Biomedical Statistical Package Program. Protein concentrations were determined by Coomassie Blue binding (68.Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (222440) Google Scholar) using γ-globulin as a standard. SDS-PAGE of proteins was performed by the method of Laemmli (69.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (212859) Google Scholar). The concentrations of LYI and AIAS were determined from their absorption spectra using molar extinction coefficients of 13,000 cm−1m−1 at 435 nm and 35,000 cm−1m−1 at 335 nm, respectively. The oligomeric structure of wild-type αA-crystallin and its mutant proteins were determined by gel filtration on a Superose 6 column using thyroglobulin, bovine γ-globlulin, chicken ovalbumin, equine carbonic anhydrase, equine myoglobin fragment, and bovine pancreatic trypsin inhibitor as standards. The far UV circular dichroism spectra of wild-type αA-crystallin and αA-crystallin-(56–173) were recorded in a cuvette with a 0.2-mm path length at room temperature using a Jasco J-600 spectropolarimeter. The secondary structure was estimated using the self-consistent method of Sreerama and Woody (70.Sreerama N. Woody R.W. Anal. Biochem. 1993; 209: 32Crossref PubMed Scopus (952) Google Scholar). We have previously reported the application of FRET to monitor the exchange of αA-crystallin subunits (67.Bova M.P. Ding L.L. Horwitz J. Fung B.K.-K. J. Biol. Chem. 1997; 272: 29511-29517Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar). In this earlier study, we labeled recombinant αA-crystallin with AIAS, which served as an energy donor, and LYI, which serves as an energy acceptor. Upon mixing of the two populations of labeled αA-crystallin, we observed a time-dependent decrease in AIAS fluorescence at 415 nm, indicating an exchange reaction that brings the two labeled αA-crystallin subunits closer to each other. We further showed that subunit exchange is reversible, as revealed by the rapid recovery of the AIAS emission intensity as the fluorescent αA-crystallin was diluted with unlabeled αA-crystallin. We surmised that the recovery of donor fluorescence can be utilized to monitor the exchange of αA-crystallin with other proteins having similar structure and biophysical properties. Fig. 1 shows a more extensive study of diluting the mixture of AIAS-labeled and LYI-labeled αA-crystallin with unlabeled recombinant αA-crystallin. To prepare the fluorescent αA-crystallin mixture, we incubated an equal amount of AIAS-labeled and LYI-labeled αA-crystallin together at 37 °C for at least 4 h. Measurement of the donor quenching indicated that the subunits from these two populations of αA-crystallin were completely scrambled. When unlabeled αA-crystallin were added to the fluorescent αA-crystallin mixture, we observed a time-dependent increase in AIAS emission intensity at 415 nm (Fig. 1, upper panel). This result indicated that the fluorescent αA-crystallin subunits were rapidly exchanging with the unlabeled αA-crystallin, resulting in a greater separation between the AIAS-labeled and LYI-labeled αA-crystallin subunits. Moreover, the final level of AIAS emission intensity is proportional to the amount of unlabeled αA-crystallin added (Fig. 1, lower panel). The intensity reaches a maximum at a molar ratio of approximately 10 unlabeled αA-crystallin to 1 AIAS-labeled αA-crystallin. Under this condition, the distance between AIAS-labeled and LYI-labeled αA-crystallin was so far apart that there was no observable energy transfer between donors and acceptors. This quantifiable relationship between the donor emission intensity and amount of added αA-crystallin demonstrated the feasibility of using this simple dilution method to identify proteins structurally compatible with αA-crystallin. We found that the transfer rate constant, as determined by fitting the data shown in Fig.1 to the exponential functionF(t)/F(0) = A +Be −kt, was independent of the αA-crystallin concentration (data not shown). The average value in the range of 2.5–100 μm αA-crystallin was 6.36 ± 0.47 × 10−4 s−1. This interesting observation was independently confirmed by measuring the subunit exchange between two populations of fluorescent αA-crystallin. Fig.2 shows that the exchange rate, as determined by measuring the decrease in AIAS emission at 415 nm 15 min after mixing different concentrations of AIAS-labeled αA-crystallin and LYI-labeled αA-crystallin, is relatively constant over a 90-fold difference in αA-crystallin concentration. We have examined the interaction of αA-crystallin with other proteins in the lens using the dilution method. We found that the addition of either α-crystallin purified from bovine lens or recombinant αB-crystallin to premixed AIAS-labeled and LYI-labeled αA-crystallin resulted in a time-dependent increase of donor fluorescence (Fig. 3). The transfer rate constant between fluorescent αA-crystallin and unlabeled αB-crystallin was 6.3 × 10−4 s−1, which is identical to the rate constant between fluorescent αA-crystallin with unlabeled αA-crystallin at the same concentration. In contrast, β-crystallin and γ-crystallin did not exchange with αA-crystallin, suggesting that there was no measurable interaction between these proteins in their native state (Fig. 3). This result suggests the existence of a common structural interface that allows an unrestricted exchange between the subunits of αA- and αB-crystallin. We have previously reported the expression of full-length αA-crystallin cDNA in E. coli BL21DE3 cells and shown that the recombinant protein retains its chaperone activity, multimeric organization and subunit exchange properties (67.Bova M.P. Ding L.L. Horwitz J. Fung B.K.-K. J. Biol. Chem. 1997; 272: 29511-29517Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar). In this study we used the same expression system to produce a number of truncation mutants in order to determine which region of αA-crystallin is essential for subunit exchange. We found that most mutants were expressed in the soluble fraction at levels as high as 40% of the total cell proteins. The high level of expression allowed the purification of the truncated αA-crystallin to near homogeneity by successive Mono-Q ion exchange chromatography and gel filtration chromatography (Fig. 4, upper panel). We further characterized the size distribution of the truncation mutants by gel filtration (Fig. 4, lower panel). Our results indicated that the removal of up to 10 amino acids from the C terminus did not significantly change the subunit organization of αA-crystallin. These C-terminal truncation mutants form oligomers with an average molecular mass of 700 kDa. Similarly, αA-crystallin-(20–173) retained its high molecular mass oligomeric structure, as indicated by an average molecular mass of about 560 kDa. In contrast, αA-crystallin-(56–173), αA-crystallin-(65–173), and αA-crystallin-(85–173) were predominantly smaller in size, with average molecular masses of 43, 60, and 48 kDa, respectively (Fig. 4, lower panel). In addition, the elution profiles of αA-crystallin-(56–173) reveals a narrow size distribution (Fig. 5, upper panel), which is markedly different from the broad size distribution of wild-type αA-crystallin (8.Jaffe N.S. Horwitz J. Lens and Cataract. Gower Medical Publishing, 1991Google Scholar, 67.Bova M.P. Ding L.L. Horwitz J. Fung B.K.-K. J. Biol. Chem. 1997; 272: 29511-29517Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar). A similar narrow size distribution was also observed for αA-crystallin-(65–173) and αA-crystallin-(85–173) (data not shown). The discrete size distribution and apparent molecular mass of 43 kDa indicates that αA-crystallin-(56–173) exists as small multimers consisting of three or four subunits.Figure 5Characterization of αA-crystallin-(56–173) by gel filtration and far UV circular dichroism measurement. Upper panel, gel filtration chromatography of αA-crystallin-(56–173) performed on a Superose 6 HR 10/30 column. The apparent molecular mass of 43 kDa was determined from a standard curve constructed as described under "Experimental Procedures." Lower panel, far UV circular dichroism spectra of wild-type αA-crystallin (dotted line) and αA-crystallin-(56–173) (solid line). The data represent the average of 16 scans from a sample containing 1 mg/ml purified protein.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The organization of αA-crystallin-(56–173) into small multimers is not due to denaturation or marked changes of the protein secondary structure. The far UV circular dichr
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