【For INTERNATIONAL COLLABORATIVE RESEARCH 2016】

SUMMARY of

DATE: Day 1 Month 5 Year 20172016 RESEARCH RESULTS REPORTFor International Collaborative Research with IPR, Osaka University
Research Title

NMR analysis of α-synuclein and CNT

ApplicantName

Seung R. Paik

Affiliation

School of Chemical and Biological Engineering, Institute ofChemical Processes, College of Engineering, Seoul National
University, Seoul 151-744, Korea

Present Title

Professor

Research Collaborator (Host PI)

Prof. Fujiwara Toshimichi

SummaryCarbon nanotube (CNT), a cylindrical carbon allotrope with unique electrical, chemical,
mechanical, and optical properties, is a potential candidate for future cancer therapy due to its
exceptional cancer targeting property. However, it is necessary to reduce the hydrophobicity of CNT
for biological application because non-specific protein coagulation onto the hydrophobic surface
would cause toxic consequences. Previous studies have suggested that reducing the hydrophobicityof CNT with chemically modified PEG chains or hydrophobic amino acid residues ameliorates thecell cytotoxicity. Recently, we have found out that α-synuclein is capable of dispersing CNT stably
in aqueous solution, which cannot be done with other proteins. Since we expect that the complex ofα-synuclein and CNT could play a significant role on the photo-thermal cancer therapy, any detailed
information on the protein structure and the physical/chemical property of the complex would bevaluable as they have been assessed with instrumental analyses employing NMR, DSC, and CD. For
those analyses, single-walled carbon nanotube (SWNT) was used among various types of CNTs toincrease accuracy and resolution of the experiments. The complex of SWNT and α-synuclein(αS-SWNT) was prepared via a forced incubation between SWNT and α-synuclein using
tip-sonication in ice.
For the experiments, an optimal ratio between αS and SWNT had to be determined. When 1.0 mgof SWNT was treated with 1 ml of αS solution, at least 100 µM αS was required to disperse SWNT.
The far-UV CD spectra revealed a drastic conformational change of αS from random coil to α-helix.
The quantitative analysis of the spectra using BeStSel algorithm verified that the content of α-helix
increased from 5.7% to 26.5% while that of random coil decreased from 49.7% to 39.1%. The result
suggests that the main region of αS interacting with the hydrophobic surface of SWNT would be theN-terminus by considering the previous reports about the α-helix formation of αS upon its
interaction with lipid membranes. From the 1H-15N HSQC spectra of αS-SWNT, it was alsosuggested that the C-terminus of αS might be exposed outward with relatively high flexibility, whichcoincides with the result of far-UV CD spectra. However, the NMR spectra of αS-SWNT was notclear enough to identify the core structure of N-terminus due to high molecular weight of αS-SWNT.
In this respect, we are planning to conduct solid-state NMR to unveil more detailed information on
the structure of αS bound to SWNT as our future research. The DSC analysis result showed thethermal stability of αS-SWNT near the body temperature, which ensures bio-applicability of ourαS-SWNT system. In vivo evaluation of αS-SWNT for photo-thermal cancer therapy will be
conducted in the near future.