Interaction of α-synuclein and a cell penetrating fusion peptide with higher eukaryotic cell membranes assessed by ¹⁹F NMR

Abstract

We show that fluorine NMR can be used to monitor the insertion and change in conformation of a ¹⁹F-labeled cell-penetrating peptide upon interacting with the cellular plasma membrane. α-Synuclein and a construct comprising a cell-penetrating peptide covalently attached to its N-terminus were studied. Important information about the interaction of the proteins with CHO-K1 cells was obtained by monitoring the diminution of ¹⁹F resonances of 3-fluoro-l-tyrosine labeled proteins. For α-synuclein, a decrease in the resonance from position 39 was observed indicating that only the N-terminal third region of the protein interacts with plasma membrane. However, when the fusion construct was incubated with the cells, a decrease in the resonance from the fusion peptide region was noted with no change in the resonances from α-synuclein region. Longer incubation, studied by using confocal fluorescence microscopy, revealed that the fusion construct translocates into the cells, but α-synuclein alone did not cross the membrane in significant amounts.

Figure 1

Schematic representation of the cytoplasmic transduction peptide (CTP), YGR₂AR₆, covalently attached to the N-terminus of α-synuclein. Red indicates the positions of tyrosines. Green shows the position of the fluorescent label.

Figure 2

NMR tubes containing an initial uniform suspension of ~1.5 × 10⁷ CHO-K1 cells/mL in F-12 media with 10% D₂O and various (w/v) concentrations of Ficoll at 37 °C after 3 h. The cells viability was greater than 90% for 20% Ficoll.

Figure 3

¹⁹F-labeled α-synuclein spectra in the presence (black) and absence (red) of CHO-K1 cells. (A) wild-type α-synuclein (B) Y125F α-synuclein. The protein concentration was 100 μM. Residue assignments are shown above the resonances.

Figure 4

¹⁹F-labeled Y39F α-synuclein spectra in the presence (black) and absence (red) of CHO-K1 cells. Residue assignments are shown above the resonances.

Figure 5

Interaction of ¹⁹F-labeled CTP-α-synuclein with CHO-K1 cells. (A) CTP-α-synuclein in cells media containing 20% (w/v) Ficoll and 10% D₂O (B) CTP-α-synuclein and CHO-K1 cells in the same media and (C) after 1 h incubation. The protein concentration was 100 μM. The arrow indicates the decrease in the resonance from Y-11.

Figure 6

CTP mediated delivery of fluorescently-labeled α-synuclein into CHO-K1 cells. From left to right: Alexa Fluor 488 fluorescence (green), Mito Tracker Red fluorescence (red), and merged images. No autofluorescence was noted.