Limits...
Structure based aggregation studies reveal the presence of helix-rich intermediate during α-Synuclein aggregation.

Ghosh D, Singh PK, Sahay S, Jha NN, Jacob RS, Sen S, Kumar A, Riek R, Maji SK - Sci Rep (2015)

Bottom Line: Using a variety of complementary biophysical techniques monitoring entire pathway of nine different synucleins, we found that transition of unstructured conformation into β-sheet rich fibril formation involves helix-rich intermediates.A multidimensional NMR study characterizing the intermediate accompanied with site-specific fluorescence study suggests that the N-terminal and central portions mainly participate in the helix-rich intermediate formation while the C-terminus remained in an extended conformation.However, significant conformational transitions occur at the middle and at the C-terminus during helix to β-sheet transition as evident from Trp fluorescence study.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosciences and Bioengineering, IIT Bombay, Powai, Mumbai, India 400076.

ABSTRACT
Mechanistic understanding of nucleation dependent polymerization by α-synuclein (α-Syn) into toxic oligomers and amyloids is important for the drug development against Parkinson's disease. However the structural and morphological characterization during nucleation and subsequent fibrillation process of α-Syn is not clearly understood. Using a variety of complementary biophysical techniques monitoring entire pathway of nine different synucleins, we found that transition of unstructured conformation into β-sheet rich fibril formation involves helix-rich intermediates. These intermediates are common for all aggregating synucleins, contain high solvent-exposed hydrophobic surfaces, are cytotoxic to SHSY-5Y cells and accelerate α-Syn aggregation efficiently. A multidimensional NMR study characterizing the intermediate accompanied with site-specific fluorescence study suggests that the N-terminal and central portions mainly participate in the helix-rich intermediate formation while the C-terminus remained in an extended conformation. However, significant conformational transitions occur at the middle and at the C-terminus during helix to β-sheet transition as evident from Trp fluorescence study. Since partial helix-rich intermediates were also observed for other amyloidogenic proteins such as Aβ and IAPP, we hypothesize that this class of intermediates may be one of the important intermediates for amyloid formation pathway by many natively unstructured protein/peptides and represent a potential target for drug development against amyloid diseases.

Show MeSH

Related in: MedlinePlus

Secondary structural changes monitored by FTIR spectroscopy.Curve fitted FTIR spectra in the region corresponding to amide-I band showing the different secondary structural content of α-Syn species at different stages of aggregation (Upper panel). FTIR spectral data suggest predominate random coil structure at the beginning that eventually converted to β-sheet rich structure via α-helical intermediate during α-Syn aggregation. Corresponding CD spectra of the identical samples were shown in lower panel.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4363886&req=5

f4: Secondary structural changes monitored by FTIR spectroscopy.Curve fitted FTIR spectra in the region corresponding to amide-I band showing the different secondary structural content of α-Syn species at different stages of aggregation (Upper panel). FTIR spectral data suggest predominate random coil structure at the beginning that eventually converted to β-sheet rich structure via α-helical intermediate during α-Syn aggregation. Corresponding CD spectra of the identical samples were shown in lower panel.

Mentions: To further confirm that the aggregation of α-Syn involves a helix-rich intermediate formation, WT α-Syn aggregation was monitored by FTIR and CD spectroscopy. The LMW α-Syn at a concentration of 300 μM was incubated at 37°C with slight agitation (~50 r.p.m). Simultaneously, FTIR and CD spectroscopy was performed at regular intervals. The FTIR spectra in the range of 1600 cm−1 to 1700 cm−1 (amide-I band) were analyzed to determine the protein secondary structure59. After immediate dissolution and preparation (time point 0), FTIR spectra of soluble α-Syn showed a major absorbance peak at 1649 cm−1, indicating the presence of mostly random coil-like structure. The deconvolution of FTIR spectra at this point showed ~87% of random coil structure, consistent with CD spectra (88% random coil by CD deconvolution using CDPro software57 (Figure 4). With the progression of time when the incubated solution showed helical conformation in CD, FTIR spectra were measured. At 60 h, the intensity of the absorbance peak at 1649 cm−1 decreased and showed several other FTIR absorption peaks with a major absorption peak appeared at 1656 cm−1 indicating the formation of a helix-rich intermediate. This absorption peak at 1656 cm−1 was most prominent at 70 h. The deconvolution of FTIR spectra at 60 h and 70 h showed ~28% and 50% helicity, respectively. The deconvolution of CD spectra by CDpro57 showed 18% and 32% helix at 60 h and 70 h, respectively. On further incubation, another major peak appeared at 1630 cm−1 (as shown for 80 h) and intensity of the peak at 1656 and 1630 cm−1 became almost equal suggesting a significant population of both helix-rich intermediate and β-sheet-rich structure in the mixture (~39% helix and ~34% β-sheet content). The deconvolution of corresponding CD spectra at 80 h also revealed almost equal amount of helix (~28.6%) and β-sheet (~25.3%) content. The subsequent incubation at time point 95 h, for which the CD spectra of the solution predominately showed β-sheet secondary structure (Fig 4)), the FTIR study of the solution showed major absorption peaks at 1635 cm−1 corresponding to β-sheet-rich amyloid fibrils (~62% β-sheet content). Therefore, the FTIR data confirmed the findings of the CD data thereby strengthening the notion that a helix-rich intermediate is formed during α-Syn fibrillation.


Structure based aggregation studies reveal the presence of helix-rich intermediate during α-Synuclein aggregation.

Ghosh D, Singh PK, Sahay S, Jha NN, Jacob RS, Sen S, Kumar A, Riek R, Maji SK - Sci Rep (2015)

Secondary structural changes monitored by FTIR spectroscopy.Curve fitted FTIR spectra in the region corresponding to amide-I band showing the different secondary structural content of α-Syn species at different stages of aggregation (Upper panel). FTIR spectral data suggest predominate random coil structure at the beginning that eventually converted to β-sheet rich structure via α-helical intermediate during α-Syn aggregation. Corresponding CD spectra of the identical samples were shown in lower panel.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4363886&req=5

f4: Secondary structural changes monitored by FTIR spectroscopy.Curve fitted FTIR spectra in the region corresponding to amide-I band showing the different secondary structural content of α-Syn species at different stages of aggregation (Upper panel). FTIR spectral data suggest predominate random coil structure at the beginning that eventually converted to β-sheet rich structure via α-helical intermediate during α-Syn aggregation. Corresponding CD spectra of the identical samples were shown in lower panel.
Mentions: To further confirm that the aggregation of α-Syn involves a helix-rich intermediate formation, WT α-Syn aggregation was monitored by FTIR and CD spectroscopy. The LMW α-Syn at a concentration of 300 μM was incubated at 37°C with slight agitation (~50 r.p.m). Simultaneously, FTIR and CD spectroscopy was performed at regular intervals. The FTIR spectra in the range of 1600 cm−1 to 1700 cm−1 (amide-I band) were analyzed to determine the protein secondary structure59. After immediate dissolution and preparation (time point 0), FTIR spectra of soluble α-Syn showed a major absorbance peak at 1649 cm−1, indicating the presence of mostly random coil-like structure. The deconvolution of FTIR spectra at this point showed ~87% of random coil structure, consistent with CD spectra (88% random coil by CD deconvolution using CDPro software57 (Figure 4). With the progression of time when the incubated solution showed helical conformation in CD, FTIR spectra were measured. At 60 h, the intensity of the absorbance peak at 1649 cm−1 decreased and showed several other FTIR absorption peaks with a major absorption peak appeared at 1656 cm−1 indicating the formation of a helix-rich intermediate. This absorption peak at 1656 cm−1 was most prominent at 70 h. The deconvolution of FTIR spectra at 60 h and 70 h showed ~28% and 50% helicity, respectively. The deconvolution of CD spectra by CDpro57 showed 18% and 32% helix at 60 h and 70 h, respectively. On further incubation, another major peak appeared at 1630 cm−1 (as shown for 80 h) and intensity of the peak at 1656 and 1630 cm−1 became almost equal suggesting a significant population of both helix-rich intermediate and β-sheet-rich structure in the mixture (~39% helix and ~34% β-sheet content). The deconvolution of corresponding CD spectra at 80 h also revealed almost equal amount of helix (~28.6%) and β-sheet (~25.3%) content. The subsequent incubation at time point 95 h, for which the CD spectra of the solution predominately showed β-sheet secondary structure (Fig 4)), the FTIR study of the solution showed major absorption peaks at 1635 cm−1 corresponding to β-sheet-rich amyloid fibrils (~62% β-sheet content). Therefore, the FTIR data confirmed the findings of the CD data thereby strengthening the notion that a helix-rich intermediate is formed during α-Syn fibrillation.

Bottom Line: Using a variety of complementary biophysical techniques monitoring entire pathway of nine different synucleins, we found that transition of unstructured conformation into β-sheet rich fibril formation involves helix-rich intermediates.A multidimensional NMR study characterizing the intermediate accompanied with site-specific fluorescence study suggests that the N-terminal and central portions mainly participate in the helix-rich intermediate formation while the C-terminus remained in an extended conformation.However, significant conformational transitions occur at the middle and at the C-terminus during helix to β-sheet transition as evident from Trp fluorescence study.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosciences and Bioengineering, IIT Bombay, Powai, Mumbai, India 400076.

ABSTRACT
Mechanistic understanding of nucleation dependent polymerization by α-synuclein (α-Syn) into toxic oligomers and amyloids is important for the drug development against Parkinson's disease. However the structural and morphological characterization during nucleation and subsequent fibrillation process of α-Syn is not clearly understood. Using a variety of complementary biophysical techniques monitoring entire pathway of nine different synucleins, we found that transition of unstructured conformation into β-sheet rich fibril formation involves helix-rich intermediates. These intermediates are common for all aggregating synucleins, contain high solvent-exposed hydrophobic surfaces, are cytotoxic to SHSY-5Y cells and accelerate α-Syn aggregation efficiently. A multidimensional NMR study characterizing the intermediate accompanied with site-specific fluorescence study suggests that the N-terminal and central portions mainly participate in the helix-rich intermediate formation while the C-terminus remained in an extended conformation. However, significant conformational transitions occur at the middle and at the C-terminus during helix to β-sheet transition as evident from Trp fluorescence study. Since partial helix-rich intermediates were also observed for other amyloidogenic proteins such as Aβ and IAPP, we hypothesize that this class of intermediates may be one of the important intermediates for amyloid formation pathway by many natively unstructured protein/peptides and represent a potential target for drug development against amyloid diseases.

Show MeSH
Related in: MedlinePlus