DNA induces folding in α-synuclein: Understanding the mechanism using chaperone property of osmolytes

doi:10.1016/j.abb.2007.03.042

Archives of Biochemistry and Biophysics

Volume 464, Issue 1, 1 August 2007, Pages 57-69

https://doi.org/10.1016/j.abb.2007.03.042 Get rights and content

Abstract

α-Synuclein conformational modulation leading to fibrillation has been centrally implicated in Parkinson’s disease. Previously, we have shown that α-synuclein has DNA binding property. In the present study, we have characterized the effect of DNA binding on the conformation and fibrillation kinetics of α-synuclein. It was observed that single-stranded circular DNA induce α-helix conformation in α-synuclein while plasmid supercoiled DNA has dual effect inducing a partially folded conformation and α-helix under different experimental conditions. Interestingly, α-synuclein showed a specificity for GC* nucleotide sequence in its binding ability to DNA. The aggregation kinetics data showed that DNA which induced partially folded conformation in α-synuclein promoted the fibrillation while DNA which induced α-helix delayed the fibrillation, indicating that the partially folded intermediate conformation is critical in the aggregation process. Further, the mechanism of DNA-induced folding/aggregation of α-synuclein was studied using effect of osmolytes on α-synuclein as a model system. Among the five osmolytes used, Glycerol, trimethylamine-N-oxide, Betaine, and Taurine induced partially folded conformation and in turn enhanced the aggregation of α-synuclein. The ability of DNA and osmolytes in inducing conformational transition in α-synuclein, indicates that two factors are critical in modulating α-synuclein folding: (i) electrostatic interaction as in the case of DNA, and (ii) hydrophobic interactions as in the case of osmolytes. The property of DNA inducing α-helical conformation in α-synuclein and inhibiting the fibrillation may be of significance in engineering DNA-chip based therapeutic approaches to PD and other amyloid disorders.

Section snippets

Materials

α-Synuclein was purchased from rPeptides, USA, the purity of α-synuclein was confirmed by Gel filteration and mass-spectrometric analysis. Supercoiled DNA pUC 18 plasmid (Cesium chloride purified, 90% supercoiled structure), λDNA, Calf-thymus DNA, single-stranded circular (ssc)DNA (MP3), double-stranded circular (dsc)DNA (MP3), Tris and Hepes buffers were purchased from Bangalore Genei, India. Poly d(GC)·d(GC) and poly d(AT)·d(AT), Sarcosine, TMAO, Taurine, Betaine, Glycerol, and Copper grids

scDNA induces ordered conformation in α-synuclein

Circular dichroism spectroscopy (CD) was used to determine the effects of DNA binding on the secondary structure of α-synuclein. It was observed that scDNA caused a biphasic conformational transition in α-synuclein. Natively, α-synuclein is in random-coil conformation. On immediate mixing of the DNA and α-synuclein at room temperature a partial folding was induced in α-synuclein (Fig. 1A) while α-helix conformation was formed on long term incubation at 4 °C (Fig. 1B).

The CD spectrum of native

Discussion

α-Synuclein is a natively unfolded protein with little or no ordered structure under physiological conditions. At neutral pH, it is calculated to have 24 negative charges (15 of which are localized in the last third of the protein sequence), leading to a strong electrostatic repulsion, which hinders the folding of α-synuclein [39]. As previously reported [9], [10], α-synuclein at neutral pH has a far-UV-CD spectrum typical of an unfolded polypeptide chain, and reflecting the lack of ordered

Acknowledgments

The authors thank Director, CFTRI for his encouragement. The financial assistance by Department of Atomic Energy, India through BNRS project is gratefully acknowledged. M.L.H. is grateful to Council for Scientific and Industrial Research, India for Senior Research Fellowships. We thank Prof. R. Varadarajan, Molecular Biophysics Unit, Indian Institute of Science, Bangalore for reviewing the manuscript and providing valuable suggestions. We also thank Chairman, Molecular Biophysics Unit, Indian

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DNA induces folding in α-synuclein: Understanding the mechanism using chaperone property of osmolytes

Abstract

Section snippets

Materials

scDNA induces ordered conformation in α-synuclein

Discussion

Acknowledgments

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