Limits...
Role of acidic residues in helices TH8-TH9 in membrane interactions of the diphtheria toxin T domain.

Ghatak C, Rodnin MV, Vargas-Uribe M, McCluskey AJ, Flores-Canales JC, Kurnikova M, Ladokhin AS - Toxins (Basel) (2015)

Bottom Line: Thermal unfolding and fluorescence measurements, complemented with molecular dynamics simulations, suggest that the mutant E362Q is more susceptible to acid destabilization because of disruption of native intramolecular contacts.Both mutants adopt a final functional state upon further acidification.We conclude that these acidic residues are involved in the pH-dependent action of the T domain, and their replacements can be used for fine tuning the pH range of membrane interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS 66160, USA. c.ghatak79@gmail.com.

ABSTRACT
The pH-triggered membrane insertion of the diphtheria toxin translocation domain (T domain) results in transferring the catalytic domain into the cytosol, which is relevant to potential biomedical applications as a cargo-delivery system. Protonation of residues is suggested to play a key role in the process, and residues E349, D352 and E362 are of particular interest because of their location within the membrane insertion unit TH8-TH9. We have used various spectroscopic, computational and functional assays to characterize the properties of the T domain carrying the double mutation E349Q/D352N or the single mutation E362Q. Vesicle leakage measurements indicate that both mutants interact with the membrane under less acidic conditions than the wild-type. Thermal unfolding and fluorescence measurements, complemented with molecular dynamics simulations, suggest that the mutant E362Q is more susceptible to acid destabilization because of disruption of native intramolecular contacts. Fluorescence experiments show that removal of the charge in E362Q, and not in E349Q/D352N, is important for insertion of TH8-TH9. Both mutants adopt a final functional state upon further acidification. We conclude that these acidic residues are involved in the pH-dependent action of the T domain, and their replacements can be used for fine tuning the pH range of membrane interactions.

Show MeSH

Related in: MedlinePlus

Thermal unfolding measurements of the WT T domain (black) and the mutants E362Q (red) and E349Q/D352N (blue) show that the single mutation causes loss of thermal stability in solution at pH 8.0 (A) and 6.5 (B). The double mutation affects the stability of the T domain only at the more acidic pH. Measurements were reproduced at least three times for each mutant at each pH.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4417968&req=5

toxins-07-01303-f003: Thermal unfolding measurements of the WT T domain (black) and the mutants E362Q (red) and E349Q/D352N (blue) show that the single mutation causes loss of thermal stability in solution at pH 8.0 (A) and 6.5 (B). The double mutation affects the stability of the T domain only at the more acidic pH. Measurements were reproduced at least three times for each mutant at each pH.

Mentions: Next, we characterized the membrane insertion pathway of the mutants using methods developed in previous studies to investigate the T domain WT and mutants [18,22,23,24,25,30]. The first step in the pathway of the WT protein involves a conformational change in solution detectable through changes in the thermodynamic stability [21]. We examined the folding and thermodynamic stability of the mutants in solution using CD spectroscopy. At pH 8 and 6.5, the WT protein and both mutants show CD spectra typical of α-helical proteins (Supplemental Data, Figure S1). However, the molar ellipticity at 222 nm is reduced by 2.0 and 2.5 mdeg·cm2/dmol for the mutant E362Q at pH 8 and 6.5, respectively (about 14% and 19% less ellipticity than the WT protein). We studied the thermal stability by monitoring the molar ellipticity at 222 nm as a function of the temperature. The mutant E362Q shows a reduced stability at pH 8 and 6.5 (Figure 3), as evidenced by the shift in the thermal unfolding curves and the quantitative analysis of thermal unfolding (Table 1). The double mutation affects the thermal stability only at pH 6.5, but the changes are not as prominent as those induced by the single mutation. Reliable thermal unfolding measurements are experimentally challenging below pH 6.5, because the T domain tends to aggregate [19,20,31,32]. Nonetheless, our results clearly indicate that the mutant E362Q is less resistant to acid destabilization than the double mutant E349Q/D352N or the WT T domain.


Role of acidic residues in helices TH8-TH9 in membrane interactions of the diphtheria toxin T domain.

Ghatak C, Rodnin MV, Vargas-Uribe M, McCluskey AJ, Flores-Canales JC, Kurnikova M, Ladokhin AS - Toxins (Basel) (2015)

Thermal unfolding measurements of the WT T domain (black) and the mutants E362Q (red) and E349Q/D352N (blue) show that the single mutation causes loss of thermal stability in solution at pH 8.0 (A) and 6.5 (B). The double mutation affects the stability of the T domain only at the more acidic pH. Measurements were reproduced at least three times for each mutant at each pH.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4417968&req=5

toxins-07-01303-f003: Thermal unfolding measurements of the WT T domain (black) and the mutants E362Q (red) and E349Q/D352N (blue) show that the single mutation causes loss of thermal stability in solution at pH 8.0 (A) and 6.5 (B). The double mutation affects the stability of the T domain only at the more acidic pH. Measurements were reproduced at least three times for each mutant at each pH.
Mentions: Next, we characterized the membrane insertion pathway of the mutants using methods developed in previous studies to investigate the T domain WT and mutants [18,22,23,24,25,30]. The first step in the pathway of the WT protein involves a conformational change in solution detectable through changes in the thermodynamic stability [21]. We examined the folding and thermodynamic stability of the mutants in solution using CD spectroscopy. At pH 8 and 6.5, the WT protein and both mutants show CD spectra typical of α-helical proteins (Supplemental Data, Figure S1). However, the molar ellipticity at 222 nm is reduced by 2.0 and 2.5 mdeg·cm2/dmol for the mutant E362Q at pH 8 and 6.5, respectively (about 14% and 19% less ellipticity than the WT protein). We studied the thermal stability by monitoring the molar ellipticity at 222 nm as a function of the temperature. The mutant E362Q shows a reduced stability at pH 8 and 6.5 (Figure 3), as evidenced by the shift in the thermal unfolding curves and the quantitative analysis of thermal unfolding (Table 1). The double mutation affects the thermal stability only at pH 6.5, but the changes are not as prominent as those induced by the single mutation. Reliable thermal unfolding measurements are experimentally challenging below pH 6.5, because the T domain tends to aggregate [19,20,31,32]. Nonetheless, our results clearly indicate that the mutant E362Q is less resistant to acid destabilization than the double mutant E349Q/D352N or the WT T domain.

Bottom Line: Thermal unfolding and fluorescence measurements, complemented with molecular dynamics simulations, suggest that the mutant E362Q is more susceptible to acid destabilization because of disruption of native intramolecular contacts.Both mutants adopt a final functional state upon further acidification.We conclude that these acidic residues are involved in the pH-dependent action of the T domain, and their replacements can be used for fine tuning the pH range of membrane interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS 66160, USA. c.ghatak79@gmail.com.

ABSTRACT
The pH-triggered membrane insertion of the diphtheria toxin translocation domain (T domain) results in transferring the catalytic domain into the cytosol, which is relevant to potential biomedical applications as a cargo-delivery system. Protonation of residues is suggested to play a key role in the process, and residues E349, D352 and E362 are of particular interest because of their location within the membrane insertion unit TH8-TH9. We have used various spectroscopic, computational and functional assays to characterize the properties of the T domain carrying the double mutation E349Q/D352N or the single mutation E362Q. Vesicle leakage measurements indicate that both mutants interact with the membrane under less acidic conditions than the wild-type. Thermal unfolding and fluorescence measurements, complemented with molecular dynamics simulations, suggest that the mutant E362Q is more susceptible to acid destabilization because of disruption of native intramolecular contacts. Fluorescence experiments show that removal of the charge in E362Q, and not in E349Q/D352N, is important for insertion of TH8-TH9. Both mutants adopt a final functional state upon further acidification. We conclude that these acidic residues are involved in the pH-dependent action of the T domain, and their replacements can be used for fine tuning the pH range of membrane interactions.

Show MeSH
Related in: MedlinePlus