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pH Induced Conformational Transitions in the Transforming Growth Factor β-Induced Protein (TGFβIp) Associated Corneal Dystrophy Mutants.

Murugan E, Venkatraman A, Lei Z, Mouvet V, Rui Yi Lim R, Muruganantham N, Goh E, Swee Lim Peh G, Beuerman RW, Chaurasia SS, Rajamani L, Mehta JS - Sci Rep (2016)

Bottom Line: R555W was more susceptible to acidic pH compared to H572R and displayed varying chemical stabilities with decreasing pH.Thermal denaturation studies at acidic pH showed that while WT did not undergo any conformational transition, the mutants exhibited a clear pH-dependent irreversible conversion from αβ conformation to β-sheet oligomers.Electron microscopy and dynamic light scattering studies showed that β-oligomers of H572R were larger compared to R555W.

View Article: PubMed Central - PubMed

Affiliation: Tissue Engineering and Stem Cell Group, Singapore Eye Research Institute, Singapore.

ABSTRACT
Most stromal corneal dystrophies are associated with aggregation and deposition of the mutated transforming growth factor-β induced protein (TGFβIp). The 4(th)_FAS1 domain of TGFβIp harbors ~80% of the mutations that forms amyloidogenic and non-amyloidogenic aggregates. To understand the mechanism of aggregation and the differences between the amyloidogenic and non-amyloidogenic phenotypes, we expressed the 4(th)_FAS1 domains of TGFβIp carrying the mutations R555W (non-amyloidogenic) and H572R (amyloidogenic) along with the wild-type (WT). R555W was more susceptible to acidic pH compared to H572R and displayed varying chemical stabilities with decreasing pH. Thermal denaturation studies at acidic pH showed that while WT did not undergo any conformational transition, the mutants exhibited a clear pH-dependent irreversible conversion from αβ conformation to β-sheet oligomers. The β-oligomers of both mutants were stable at physiological temperature and pH. Electron microscopy and dynamic light scattering studies showed that β-oligomers of H572R were larger compared to R555W. The β-oligomers of both mutants were cytotoxic to primary human corneal stromal fibroblast (pHCSF) cells. The β-oligomers of both mutants exhibit variations in their morphologies, sizes, thermal and chemical stabilities, aggregation patterns and cytotoxicities.

No MeSH data available.


Related in: MedlinePlus

pH sensitivity and stability of the non-amyloidogenic (R555W) phenotype.(a) Fluorescence emission spectra of R555W with decrease in pH. There was a clear decrease in emission maximum at 332 nm with decrease in pH (indicated by the black arrow). (b–e) Fluorescence emission spectra of R555W showing the reversibility to folded state after removal of urea. The R555W mutant was incubated with increasing concentrations of urea from 0.25 M to 8 M at various acidic conditions (pH 3, pH 4.5, pH 5.5) and pH 7 and the emission fluorescence before and after urea incubation was measured. The emission spectra before urea incubation 332 nm (black), after incubating with 8 M urea (red) and after removing urea by buffer exchange (blue). Unfolding of the protein is seen by the shifting of peaks (black arrow) from 332 nm to ~ 352 nm. The refolding of the protein after removal of urea is seen by the return of the emission maximum to ~332 nm (green arrow). (f–i) Investigation of the stability the non-amyloidogenic phenotype using urea denaturation studies. The R555W mutant was incubated with increasing concentrations of Urea from 0.25 M to 8 M at various acidic pH (pH 3, pH 4.5, pH 5.5) and pH 7, and the emission fluorescence was measured. The denaturation plots of ‘fraction unfolded vs urea concentration’ were plotted and fit into a two state model, with the parameters calculated as described in the methods section.
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f5: pH sensitivity and stability of the non-amyloidogenic (R555W) phenotype.(a) Fluorescence emission spectra of R555W with decrease in pH. There was a clear decrease in emission maximum at 332 nm with decrease in pH (indicated by the black arrow). (b–e) Fluorescence emission spectra of R555W showing the reversibility to folded state after removal of urea. The R555W mutant was incubated with increasing concentrations of urea from 0.25 M to 8 M at various acidic conditions (pH 3, pH 4.5, pH 5.5) and pH 7 and the emission fluorescence before and after urea incubation was measured. The emission spectra before urea incubation 332 nm (black), after incubating with 8 M urea (red) and after removing urea by buffer exchange (blue). Unfolding of the protein is seen by the shifting of peaks (black arrow) from 332 nm to ~ 352 nm. The refolding of the protein after removal of urea is seen by the return of the emission maximum to ~332 nm (green arrow). (f–i) Investigation of the stability the non-amyloidogenic phenotype using urea denaturation studies. The R555W mutant was incubated with increasing concentrations of Urea from 0.25 M to 8 M at various acidic pH (pH 3, pH 4.5, pH 5.5) and pH 7, and the emission fluorescence was measured. The denaturation plots of ‘fraction unfolded vs urea concentration’ were plotted and fit into a two state model, with the parameters calculated as described in the methods section.

Mentions: The tryptophan residue in R555W allowed us to measure the emission fluorescence. Examination of the emission fluorescence at ~332 nm of R555W in acidic pH showed a significant decrease in emission intensity with decreasing pH, however the emission maxima remained unchanged (Fig. 5a). To obtain a better insight into the effect of pH on R555W, we monitored the urea-induced unfolding of R555W. Increasing the urea concentration progressively shifts the emission maxima (~332 nm) to longer wavelengths (~352 nm), suggesting a clear transition from folded to unfolded conformations (5b–e). To confirm refolding of unfolded R555W, the unfolded protein at different pH conditions (pH 3.0, pH 4.5, pH 5.5 and pH 7.0) was diluted appropriately and emission spectra were recorded. The emission maxima (λmax) plotted with the unfolded and refolded domains were superimposable (Supplementary Fig. S3). (Fig. 5b–e). A clear reversal in fluorescence maxima from ~352 nm to ~332 nm was observed thereby allowing us to estimate the thermodynamic stability of the mutant protein in various pH. Figure 5f–i shows urea denaturation curves plotted as ‘fraction unfolded (yU) vs increasing urea concentrations’ as monitored by the changes in emission maxima (Δλmax) at various pH values (Supplementary Fig. S3) for the R555W mutant. The thermodynamic parameters derived from urea denaturation are shown in the table (Table 2). A significant decrease in free energy of unfolding from 13.8 ± 0.8 kJ/mole to 7.108 ± 1.5 kJ/mole was observed from pH 7.0 to pH 4.5 for the R555W mutant. However, for the H572R mutant, fluorescence studies could not be performed because of the absence of a tryptophan residue.


pH Induced Conformational Transitions in the Transforming Growth Factor β-Induced Protein (TGFβIp) Associated Corneal Dystrophy Mutants.

Murugan E, Venkatraman A, Lei Z, Mouvet V, Rui Yi Lim R, Muruganantham N, Goh E, Swee Lim Peh G, Beuerman RW, Chaurasia SS, Rajamani L, Mehta JS - Sci Rep (2016)

pH sensitivity and stability of the non-amyloidogenic (R555W) phenotype.(a) Fluorescence emission spectra of R555W with decrease in pH. There was a clear decrease in emission maximum at 332 nm with decrease in pH (indicated by the black arrow). (b–e) Fluorescence emission spectra of R555W showing the reversibility to folded state after removal of urea. The R555W mutant was incubated with increasing concentrations of urea from 0.25 M to 8 M at various acidic conditions (pH 3, pH 4.5, pH 5.5) and pH 7 and the emission fluorescence before and after urea incubation was measured. The emission spectra before urea incubation 332 nm (black), after incubating with 8 M urea (red) and after removing urea by buffer exchange (blue). Unfolding of the protein is seen by the shifting of peaks (black arrow) from 332 nm to ~ 352 nm. The refolding of the protein after removal of urea is seen by the return of the emission maximum to ~332 nm (green arrow). (f–i) Investigation of the stability the non-amyloidogenic phenotype using urea denaturation studies. The R555W mutant was incubated with increasing concentrations of Urea from 0.25 M to 8 M at various acidic pH (pH 3, pH 4.5, pH 5.5) and pH 7, and the emission fluorescence was measured. The denaturation plots of ‘fraction unfolded vs urea concentration’ were plotted and fit into a two state model, with the parameters calculated as described in the methods section.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4814907&req=5

f5: pH sensitivity and stability of the non-amyloidogenic (R555W) phenotype.(a) Fluorescence emission spectra of R555W with decrease in pH. There was a clear decrease in emission maximum at 332 nm with decrease in pH (indicated by the black arrow). (b–e) Fluorescence emission spectra of R555W showing the reversibility to folded state after removal of urea. The R555W mutant was incubated with increasing concentrations of urea from 0.25 M to 8 M at various acidic conditions (pH 3, pH 4.5, pH 5.5) and pH 7 and the emission fluorescence before and after urea incubation was measured. The emission spectra before urea incubation 332 nm (black), after incubating with 8 M urea (red) and after removing urea by buffer exchange (blue). Unfolding of the protein is seen by the shifting of peaks (black arrow) from 332 nm to ~ 352 nm. The refolding of the protein after removal of urea is seen by the return of the emission maximum to ~332 nm (green arrow). (f–i) Investigation of the stability the non-amyloidogenic phenotype using urea denaturation studies. The R555W mutant was incubated with increasing concentrations of Urea from 0.25 M to 8 M at various acidic pH (pH 3, pH 4.5, pH 5.5) and pH 7, and the emission fluorescence was measured. The denaturation plots of ‘fraction unfolded vs urea concentration’ were plotted and fit into a two state model, with the parameters calculated as described in the methods section.
Mentions: The tryptophan residue in R555W allowed us to measure the emission fluorescence. Examination of the emission fluorescence at ~332 nm of R555W in acidic pH showed a significant decrease in emission intensity with decreasing pH, however the emission maxima remained unchanged (Fig. 5a). To obtain a better insight into the effect of pH on R555W, we monitored the urea-induced unfolding of R555W. Increasing the urea concentration progressively shifts the emission maxima (~332 nm) to longer wavelengths (~352 nm), suggesting a clear transition from folded to unfolded conformations (5b–e). To confirm refolding of unfolded R555W, the unfolded protein at different pH conditions (pH 3.0, pH 4.5, pH 5.5 and pH 7.0) was diluted appropriately and emission spectra were recorded. The emission maxima (λmax) plotted with the unfolded and refolded domains were superimposable (Supplementary Fig. S3). (Fig. 5b–e). A clear reversal in fluorescence maxima from ~352 nm to ~332 nm was observed thereby allowing us to estimate the thermodynamic stability of the mutant protein in various pH. Figure 5f–i shows urea denaturation curves plotted as ‘fraction unfolded (yU) vs increasing urea concentrations’ as monitored by the changes in emission maxima (Δλmax) at various pH values (Supplementary Fig. S3) for the R555W mutant. The thermodynamic parameters derived from urea denaturation are shown in the table (Table 2). A significant decrease in free energy of unfolding from 13.8 ± 0.8 kJ/mole to 7.108 ± 1.5 kJ/mole was observed from pH 7.0 to pH 4.5 for the R555W mutant. However, for the H572R mutant, fluorescence studies could not be performed because of the absence of a tryptophan residue.

Bottom Line: R555W was more susceptible to acidic pH compared to H572R and displayed varying chemical stabilities with decreasing pH.Thermal denaturation studies at acidic pH showed that while WT did not undergo any conformational transition, the mutants exhibited a clear pH-dependent irreversible conversion from αβ conformation to β-sheet oligomers.Electron microscopy and dynamic light scattering studies showed that β-oligomers of H572R were larger compared to R555W.

View Article: PubMed Central - PubMed

Affiliation: Tissue Engineering and Stem Cell Group, Singapore Eye Research Institute, Singapore.

ABSTRACT
Most stromal corneal dystrophies are associated with aggregation and deposition of the mutated transforming growth factor-β induced protein (TGFβIp). The 4(th)_FAS1 domain of TGFβIp harbors ~80% of the mutations that forms amyloidogenic and non-amyloidogenic aggregates. To understand the mechanism of aggregation and the differences between the amyloidogenic and non-amyloidogenic phenotypes, we expressed the 4(th)_FAS1 domains of TGFβIp carrying the mutations R555W (non-amyloidogenic) and H572R (amyloidogenic) along with the wild-type (WT). R555W was more susceptible to acidic pH compared to H572R and displayed varying chemical stabilities with decreasing pH. Thermal denaturation studies at acidic pH showed that while WT did not undergo any conformational transition, the mutants exhibited a clear pH-dependent irreversible conversion from αβ conformation to β-sheet oligomers. The β-oligomers of both mutants were stable at physiological temperature and pH. Electron microscopy and dynamic light scattering studies showed that β-oligomers of H572R were larger compared to R555W. The β-oligomers of both mutants were cytotoxic to primary human corneal stromal fibroblast (pHCSF) cells. The β-oligomers of both mutants exhibit variations in their morphologies, sizes, thermal and chemical stabilities, aggregation patterns and cytotoxicities.

No MeSH data available.


Related in: MedlinePlus