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COOH-terminal truncations and site-directed mutations enhance thermostability and chaperone-like activity of porcine alphaB-crystallin.

Liao JH, Lee JS, Wu SH, Chiou SH - Mol. Vis. (2009)

Bottom Line: The deletion of 12 residues from the COOH-terminal end greatly reduced the solubility, thermostability, and chaperone activity of alphaB-crystallin.On the contrary, the truncation of only 10 residues or less resulted in increased thermostability and enhanced anti-aggregation chaperone activity of alphaB-crystallin, with a maximal effect occurring on elimination of the last two residues.Our study clearly demonstrated that both the length and electrostatic charge of the COOH-terminal segment play crucial roles in governing the structural stability and chaperone activity of alphaB-crystallin.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.

ABSTRACT

Purpose: The COOH-terminal extension segment of alphaB-crystallin, a member of small heat shock protein (sHSP) family, appears to be a flexible polypeptide segment susceptible to proteolytic truncation and modifications under physiological conditions. To investigate its role on the structure and chaperone-like activity, we constructed various mutants of porcine alphaB-crystallin with either COOH-terminal serial truncations or site-specific mutagenesis on the last two residues.

Methods: The structures of these mutants were analyzed by circular dichroism (CD) spectroscopy, fluorescence spectra, mass spectrometry, Gel-permeation FPLC, and dynamic light-scattering spectrophotometry. Chaperone activity assays were performed under thermal and non-thermal stresses. The stability of proteins was examined by turbidity assays and CD spectra.

Results: All mutants showed similar secondary and tertiary structural features to the wild-type alphaB-crystallin as revealed by circular dichroism. However, truncations of the COOH-terminal segment generated crystallin aggregates with a molecular size slightly smaller than that of the wild-type alphaB-crystallin. The deletion of 12 residues from the COOH-terminal end greatly reduced the solubility, thermostability, and chaperone activity of alphaB-crystallin. On the contrary, the truncation of only 10 residues or less resulted in increased thermostability and enhanced anti-aggregation chaperone activity of alphaB-crystallin, with a maximal effect occurring on elimination of the last two residues. Moreover, displacing the last two lysines with glutamates or other neutral amino acids tended to show even higher chaperone activity than the deletion mutants.

Conclusions: Our study clearly demonstrated that both the length and electrostatic charge of the COOH-terminal segment play crucial roles in governing the structural stability and chaperone activity of alphaB-crystallin.

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Related in: MedlinePlus

Comparison of chaperone activities of wild-type αB-crystallin and its mutants under thermal denaturation. A: Porcine α-crystallin was used as a substrate for chaperone-activity assays of wild-type αB-crystallin and mutants at 65 °C. Mutant proteins and wild-type αB-crystallin showed different chaperone activities at a molar ratio of 2:3 (chaperone/γ-crystallin). The scattering curves at 360 nm in the presence of chaperoning crystallins are shown as follows: control solution without chaperone (closed square), wild-type αB-crystallin (closed circle), Δ1 (closed triangle), Δ2 (closed asterisk), Δ1/K174A (open square), K174A (open circle), K175A (open triangle), and K174/175A (open asterisk). It is noted that K174/175A and Δ1/K174A show the highest activity among all mutants. B: Comparison of chaperone activities of wild-type αB-crystallin and mutants with different electrostatic amino acids under identical conditions as in A. The scattering curves at 360 nm in the presence of chaperoning crystallins are shown as follows: control solution without chaperone (closed square), wild-type αB-crystallin (closed circle), Δ1 (closed triangle), Δ2 (closed rhombus), Δ1/K174A (open square), Δ1/K174S (open circle), Δ1/K174E (open triangle), K174/175A (open rhombus), and K174/175E (open asterisk). C: Comparison of chaperone activity (percentage protection) of wild-type αB-crystallin and mutants. Wild-type αB-crystallin was shown to possess poor protective activity and K174/175E shown to possess the best protective activity among all proteins. The final concentration of porcine α-crystallin is 5.5 μM. D: Chaperone activities of wild-type αB-crystallin and its mutants under thermal denaturation at 70 °C. Porcine α-crystallin was used as a substrate for chaperone-activity assays of wild-type αB-crystallin and its mutants with different electrostatic amino acids at 70 °C in a molar ratio of 2:3 (chaperone/γ-crystallin). The scattering curves at 360 nm in the presence of chaperoning crystallins are shown as follows: control solution without chaperone (closed square), wild-type αB-crystallin (closed circle), Δ1 (closed triangle), Δ2 (closed rhombus), Δ1/K174A (open square), Δ1/K174S (open circle), Δ1/K174E (open triangle), K174/175A (open rhombus), and K174/175E (open asterisk). Both Δ1/K174E and K174/175E show the best protective activity among all mutants under these conditions. The final concentration of porcine α-crystallin is 5.5 μM.
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f7: Comparison of chaperone activities of wild-type αB-crystallin and its mutants under thermal denaturation. A: Porcine α-crystallin was used as a substrate for chaperone-activity assays of wild-type αB-crystallin and mutants at 65 °C. Mutant proteins and wild-type αB-crystallin showed different chaperone activities at a molar ratio of 2:3 (chaperone/γ-crystallin). The scattering curves at 360 nm in the presence of chaperoning crystallins are shown as follows: control solution without chaperone (closed square), wild-type αB-crystallin (closed circle), Δ1 (closed triangle), Δ2 (closed asterisk), Δ1/K174A (open square), K174A (open circle), K175A (open triangle), and K174/175A (open asterisk). It is noted that K174/175A and Δ1/K174A show the highest activity among all mutants. B: Comparison of chaperone activities of wild-type αB-crystallin and mutants with different electrostatic amino acids under identical conditions as in A. The scattering curves at 360 nm in the presence of chaperoning crystallins are shown as follows: control solution without chaperone (closed square), wild-type αB-crystallin (closed circle), Δ1 (closed triangle), Δ2 (closed rhombus), Δ1/K174A (open square), Δ1/K174S (open circle), Δ1/K174E (open triangle), K174/175A (open rhombus), and K174/175E (open asterisk). C: Comparison of chaperone activity (percentage protection) of wild-type αB-crystallin and mutants. Wild-type αB-crystallin was shown to possess poor protective activity and K174/175E shown to possess the best protective activity among all proteins. The final concentration of porcine α-crystallin is 5.5 μM. D: Chaperone activities of wild-type αB-crystallin and its mutants under thermal denaturation at 70 °C. Porcine α-crystallin was used as a substrate for chaperone-activity assays of wild-type αB-crystallin and its mutants with different electrostatic amino acids at 70 °C in a molar ratio of 2:3 (chaperone/γ-crystallin). The scattering curves at 360 nm in the presence of chaperoning crystallins are shown as follows: control solution without chaperone (closed square), wild-type αB-crystallin (closed circle), Δ1 (closed triangle), Δ2 (closed rhombus), Δ1/K174A (open square), Δ1/K174S (open circle), Δ1/K174E (open triangle), K174/175A (open rhombus), and K174/175E (open asterisk). Both Δ1/K174E and K174/175E show the best protective activity among all mutants under these conditions. The final concentration of porcine α-crystallin is 5.5 μM.

Mentions: In order to investigate the roles of lysine174 and lysine175, we constructed various mutants and analyzed their chaperone activities at high temperature. At a molar ratio of 1:4 (chaperone/βL-crystallin) at 60 °C, the double mutant, K174/175A, showed an almost complete inhibition of βL-crystallin aggregation (Appendix 1; Figure S5). Mutant Δ1 showed slightly better chaperone activity than wild-type αB-crystallin. Both single-replacement mutants, K174A and K175A, showed better chaperone activities than Δ1 and wild-type αB-crystallin. Mutants Δ1/K174A and K174/175A showed better chaperone activities than Δ2. The results indicate that two COOH-terminal lysines do not appear to play positively enhancing roles regarding chaperone activity of αB-crystallin. We then compared the chaperone activities of various mutants by changing electrostatic-charge states at the COOH-terminal end (Appendix 1; Figure S5). Mutants Δ1/K174A, Δ1/K174S, Δ1/K174E, K174/175A, and K174/175E all show better chaperone activities than Δ2, indicating again that the positive lysine residues may not contribute much to the chaperone activity. When we increased the temperature to 65 °C and used γ-crystallin as a substrate at a molar ratio of 2:3 (chaperone/γ-crystallin), similar results to βL-crystallin were observed (Figure 7). These results are summarized in Figure 7C as percentages of protection, (Iγ-IB)/Iγx100, where Iγ is the intensity of scattered light for α-crystallin without chaperone protein in chaperone assays, and IB is the intensity of scattered light in the presence of αB-crystallin or various mutants. Wild-type αB-crystallin showed about 26% protection whereas Δ1/K174E and K174/175E showed about 96% protection under the same assay conditions. Mutant Δ1 showed only 34% protection and Δ2 about 75% protection. Most prominently Δ1/K174A, Δ1/K174S, and K174/175A each showed about 87% protection. It is of interest to find that K175A showed 71% and K174A 58% protection, emphasizing that replacing lysine at position 174 contributes much less in increasing chaperone activity than at position 175. We further increase the temperature to 70 oC and perform the same assay (Figure 7D). Wild-type αB-crystallin and Δ1 become extensively turbid after 20 min incubation in this assay. Mutants Δ1/K174A, Δ1/K174S, and K174/175A showed near 50% protection at 70 oC. Both Δ1/K174E and K174/175E still showed good protective activities under this condition.


COOH-terminal truncations and site-directed mutations enhance thermostability and chaperone-like activity of porcine alphaB-crystallin.

Liao JH, Lee JS, Wu SH, Chiou SH - Mol. Vis. (2009)

Comparison of chaperone activities of wild-type αB-crystallin and its mutants under thermal denaturation. A: Porcine α-crystallin was used as a substrate for chaperone-activity assays of wild-type αB-crystallin and mutants at 65 °C. Mutant proteins and wild-type αB-crystallin showed different chaperone activities at a molar ratio of 2:3 (chaperone/γ-crystallin). The scattering curves at 360 nm in the presence of chaperoning crystallins are shown as follows: control solution without chaperone (closed square), wild-type αB-crystallin (closed circle), Δ1 (closed triangle), Δ2 (closed asterisk), Δ1/K174A (open square), K174A (open circle), K175A (open triangle), and K174/175A (open asterisk). It is noted that K174/175A and Δ1/K174A show the highest activity among all mutants. B: Comparison of chaperone activities of wild-type αB-crystallin and mutants with different electrostatic amino acids under identical conditions as in A. The scattering curves at 360 nm in the presence of chaperoning crystallins are shown as follows: control solution without chaperone (closed square), wild-type αB-crystallin (closed circle), Δ1 (closed triangle), Δ2 (closed rhombus), Δ1/K174A (open square), Δ1/K174S (open circle), Δ1/K174E (open triangle), K174/175A (open rhombus), and K174/175E (open asterisk). C: Comparison of chaperone activity (percentage protection) of wild-type αB-crystallin and mutants. Wild-type αB-crystallin was shown to possess poor protective activity and K174/175E shown to possess the best protective activity among all proteins. The final concentration of porcine α-crystallin is 5.5 μM. D: Chaperone activities of wild-type αB-crystallin and its mutants under thermal denaturation at 70 °C. Porcine α-crystallin was used as a substrate for chaperone-activity assays of wild-type αB-crystallin and its mutants with different electrostatic amino acids at 70 °C in a molar ratio of 2:3 (chaperone/γ-crystallin). The scattering curves at 360 nm in the presence of chaperoning crystallins are shown as follows: control solution without chaperone (closed square), wild-type αB-crystallin (closed circle), Δ1 (closed triangle), Δ2 (closed rhombus), Δ1/K174A (open square), Δ1/K174S (open circle), Δ1/K174E (open triangle), K174/175A (open rhombus), and K174/175E (open asterisk). Both Δ1/K174E and K174/175E show the best protective activity among all mutants under these conditions. The final concentration of porcine α-crystallin is 5.5 μM.
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f7: Comparison of chaperone activities of wild-type αB-crystallin and its mutants under thermal denaturation. A: Porcine α-crystallin was used as a substrate for chaperone-activity assays of wild-type αB-crystallin and mutants at 65 °C. Mutant proteins and wild-type αB-crystallin showed different chaperone activities at a molar ratio of 2:3 (chaperone/γ-crystallin). The scattering curves at 360 nm in the presence of chaperoning crystallins are shown as follows: control solution without chaperone (closed square), wild-type αB-crystallin (closed circle), Δ1 (closed triangle), Δ2 (closed asterisk), Δ1/K174A (open square), K174A (open circle), K175A (open triangle), and K174/175A (open asterisk). It is noted that K174/175A and Δ1/K174A show the highest activity among all mutants. B: Comparison of chaperone activities of wild-type αB-crystallin and mutants with different electrostatic amino acids under identical conditions as in A. The scattering curves at 360 nm in the presence of chaperoning crystallins are shown as follows: control solution without chaperone (closed square), wild-type αB-crystallin (closed circle), Δ1 (closed triangle), Δ2 (closed rhombus), Δ1/K174A (open square), Δ1/K174S (open circle), Δ1/K174E (open triangle), K174/175A (open rhombus), and K174/175E (open asterisk). C: Comparison of chaperone activity (percentage protection) of wild-type αB-crystallin and mutants. Wild-type αB-crystallin was shown to possess poor protective activity and K174/175E shown to possess the best protective activity among all proteins. The final concentration of porcine α-crystallin is 5.5 μM. D: Chaperone activities of wild-type αB-crystallin and its mutants under thermal denaturation at 70 °C. Porcine α-crystallin was used as a substrate for chaperone-activity assays of wild-type αB-crystallin and its mutants with different electrostatic amino acids at 70 °C in a molar ratio of 2:3 (chaperone/γ-crystallin). The scattering curves at 360 nm in the presence of chaperoning crystallins are shown as follows: control solution without chaperone (closed square), wild-type αB-crystallin (closed circle), Δ1 (closed triangle), Δ2 (closed rhombus), Δ1/K174A (open square), Δ1/K174S (open circle), Δ1/K174E (open triangle), K174/175A (open rhombus), and K174/175E (open asterisk). Both Δ1/K174E and K174/175E show the best protective activity among all mutants under these conditions. The final concentration of porcine α-crystallin is 5.5 μM.
Mentions: In order to investigate the roles of lysine174 and lysine175, we constructed various mutants and analyzed their chaperone activities at high temperature. At a molar ratio of 1:4 (chaperone/βL-crystallin) at 60 °C, the double mutant, K174/175A, showed an almost complete inhibition of βL-crystallin aggregation (Appendix 1; Figure S5). Mutant Δ1 showed slightly better chaperone activity than wild-type αB-crystallin. Both single-replacement mutants, K174A and K175A, showed better chaperone activities than Δ1 and wild-type αB-crystallin. Mutants Δ1/K174A and K174/175A showed better chaperone activities than Δ2. The results indicate that two COOH-terminal lysines do not appear to play positively enhancing roles regarding chaperone activity of αB-crystallin. We then compared the chaperone activities of various mutants by changing electrostatic-charge states at the COOH-terminal end (Appendix 1; Figure S5). Mutants Δ1/K174A, Δ1/K174S, Δ1/K174E, K174/175A, and K174/175E all show better chaperone activities than Δ2, indicating again that the positive lysine residues may not contribute much to the chaperone activity. When we increased the temperature to 65 °C and used γ-crystallin as a substrate at a molar ratio of 2:3 (chaperone/γ-crystallin), similar results to βL-crystallin were observed (Figure 7). These results are summarized in Figure 7C as percentages of protection, (Iγ-IB)/Iγx100, where Iγ is the intensity of scattered light for α-crystallin without chaperone protein in chaperone assays, and IB is the intensity of scattered light in the presence of αB-crystallin or various mutants. Wild-type αB-crystallin showed about 26% protection whereas Δ1/K174E and K174/175E showed about 96% protection under the same assay conditions. Mutant Δ1 showed only 34% protection and Δ2 about 75% protection. Most prominently Δ1/K174A, Δ1/K174S, and K174/175A each showed about 87% protection. It is of interest to find that K175A showed 71% and K174A 58% protection, emphasizing that replacing lysine at position 174 contributes much less in increasing chaperone activity than at position 175. We further increase the temperature to 70 oC and perform the same assay (Figure 7D). Wild-type αB-crystallin and Δ1 become extensively turbid after 20 min incubation in this assay. Mutants Δ1/K174A, Δ1/K174S, and K174/175A showed near 50% protection at 70 oC. Both Δ1/K174E and K174/175E still showed good protective activities under this condition.

Bottom Line: The deletion of 12 residues from the COOH-terminal end greatly reduced the solubility, thermostability, and chaperone activity of alphaB-crystallin.On the contrary, the truncation of only 10 residues or less resulted in increased thermostability and enhanced anti-aggregation chaperone activity of alphaB-crystallin, with a maximal effect occurring on elimination of the last two residues.Our study clearly demonstrated that both the length and electrostatic charge of the COOH-terminal segment play crucial roles in governing the structural stability and chaperone activity of alphaB-crystallin.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.

ABSTRACT

Purpose: The COOH-terminal extension segment of alphaB-crystallin, a member of small heat shock protein (sHSP) family, appears to be a flexible polypeptide segment susceptible to proteolytic truncation and modifications under physiological conditions. To investigate its role on the structure and chaperone-like activity, we constructed various mutants of porcine alphaB-crystallin with either COOH-terminal serial truncations or site-specific mutagenesis on the last two residues.

Methods: The structures of these mutants were analyzed by circular dichroism (CD) spectroscopy, fluorescence spectra, mass spectrometry, Gel-permeation FPLC, and dynamic light-scattering spectrophotometry. Chaperone activity assays were performed under thermal and non-thermal stresses. The stability of proteins was examined by turbidity assays and CD spectra.

Results: All mutants showed similar secondary and tertiary structural features to the wild-type alphaB-crystallin as revealed by circular dichroism. However, truncations of the COOH-terminal segment generated crystallin aggregates with a molecular size slightly smaller than that of the wild-type alphaB-crystallin. The deletion of 12 residues from the COOH-terminal end greatly reduced the solubility, thermostability, and chaperone activity of alphaB-crystallin. On the contrary, the truncation of only 10 residues or less resulted in increased thermostability and enhanced anti-aggregation chaperone activity of alphaB-crystallin, with a maximal effect occurring on elimination of the last two residues. Moreover, displacing the last two lysines with glutamates or other neutral amino acids tended to show even higher chaperone activity than the deletion mutants.

Conclusions: Our study clearly demonstrated that both the length and electrostatic charge of the COOH-terminal segment play crucial roles in governing the structural stability and chaperone activity of alphaB-crystallin.

Show MeSH
Related in: MedlinePlus