In Vitro Phosphorylation Does not Influence the Aggregation Kinetics of WT α-Synuclein in Contrast to Its Phosphorylation Mutants
Abstract
:1. Introduction
2. Results and Discussion
2.1. Construction and Production of the α-SYN Phosphorylation Mutants S129A/D and Y125F/E
2.2. In Vitro Aggregation Properties of the Phosphorylation Mutants
2.3. In Vitro Phosphorylation of α-SYN on Different Residues
2.4. Structural Properties of in Vitro Phosphorylated α-SYN
2.5. The Influence of pS129- and pY125-α-SYN on Its in Vitro Fibril Formation
2.6. Does FKBP12 Still Accelerate the Fibrillization of Phosphorylated α-SYN?
2.7. Discussion
2.7.1. The Y125 Phosphorylation Mutants Display Different Aggregation Properties Compared to in Vitro Phosphorylated α-SYN
2.7.2 . In Vitro Phosphorylation of α-SYN on S129 or Y125 Does not Affect the Fibrillization Process
2.7.3. FKBP12 Mediated Acceleration of Fibrillization Is Unaltered for Phosphorylated α-SYN
3. Experimental Section
3.1. Purification of α-Synuclein (α-SYN)
3.2. Construction of Phosphorylation Mutants of α-Synuclein (α-SYN)
3.3. SDS PAGE
3.4. Fluorescence Correlation Spectroscopy (FCS)
3.5. Circular Dichroism
3.6. Turbidity and Thioflavin T Measurements
3.7. Data Fitting and Statistical Analysis of ThioT and Turbidity Data
3.8. Phosphorylation Assay
3.9. Mass Spectrometry
3.10. Western Blot Analysis
3.11. Transmission Electron Microscopy (TEM)
3.12. Peptidyl Prolyl Cis-Trans Isomerase Activity of FKBP12
4. Conclusions
Supporting Information
Fluorescent Labeling
Fluorescence Correlation Spectroscopy (FCS)
Mass Spectrometry
Peptidyl prolyl Cis-Trans Isomerase Activity of FKBP12
Acknowledgments
Conflicts of Interest
References
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Schreurs, S.; Gerard, M.; Derua, R.; Waelkens, E.; Taymans, J.-M.; Baekelandt, V.; Engelborghs, Y. In Vitro Phosphorylation Does not Influence the Aggregation Kinetics of WT α-Synuclein in Contrast to Its Phosphorylation Mutants. Int. J. Mol. Sci. 2014, 15, 1040-1067. https://doi.org/10.3390/ijms15011040
Schreurs S, Gerard M, Derua R, Waelkens E, Taymans J-M, Baekelandt V, Engelborghs Y. In Vitro Phosphorylation Does not Influence the Aggregation Kinetics of WT α-Synuclein in Contrast to Its Phosphorylation Mutants. International Journal of Molecular Sciences. 2014; 15(1):1040-1067. https://doi.org/10.3390/ijms15011040
Chicago/Turabian StyleSchreurs, Sarah, Melanie Gerard, Rita Derua, Etienne Waelkens, Jean-Marc Taymans, Veerle Baekelandt, and Yves Engelborghs. 2014. "In Vitro Phosphorylation Does not Influence the Aggregation Kinetics of WT α-Synuclein in Contrast to Its Phosphorylation Mutants" International Journal of Molecular Sciences 15, no. 1: 1040-1067. https://doi.org/10.3390/ijms15011040