Spatially resolved DNP-assisted NMR illuminates the conformational ensemble of α-synuclein in intact viable cells

Abstract

The protein α-syn adopts a wide variety of conformations including an intrinsically disordered monomeric form and an α-helical rich membrane-associated form that is thought to play an important role in cellular membrane processes. However, despite the high affinity of α-syn for membranes, evidence that the α-helical form is adopted inside cells has been indirect. DNP-assisted solid state NMR on frozen cellular samples can report on protein conformations inside cells. Moreover, by controlling the distribution of the DNP polarization agent throughout the cellular biomass, such experiments can provide quantitative information upon the entire structural ensemble or provide information about spatially resolved sub-populations. Using DNP-assisted magic angle spinning (MAS) NMR we establish that purified α-syn in the membrane-associated and intrinsically disordered forms have distinguishable spectra. We then introduced isotopically labeled monomeric α-syn into cells. When the DNP polarization agent is dispersed homogenously throughout the cell, we found that a minority of the α-syn inside cells adopted a highly α-helical rich conformation. When the DNP polarization agent is peripherally localized, we found that the α-helical rich conformation predominates. Thus, we provide direct evidence that α-helix rich conformations of α-syn are adopted near the cellular periphery inside cells under physiological conditions. Moreover, we demonstrate how selectively altering the spatial distribution of the DNP polarization agent can be a powerful tool to observe spatially distinct structural ensembles. This approach paves the way for more nuanced investigations into the conformations that proteins adopt in different areas of the cell.

Keywords: Biophysics and Structural Biology; DNP methods; DNP solid-state NMR; a-synuclein; in-cell NMR.

Figure 1:

Purified samples of uniformly ¹³C labeled α-syn in the nanodisc-associated form (red) and the frozen intrinsically disordered form (blue) were distinguished by ¹³C-¹³C correlation spectroscopy (DARR, 20 ms mixing) under DNP conditions (left column). The spectra of 75 μM ¹³C labeled α-syn inside intact HEK293 cells where the polarization agent AMUPol was introduced by electroporation (center column) resembles the spectra of the purified intrinsically disordered monomer while the spectra of 75 μM a-syn inside cells where AMUPol was introduced by incubation in 10 mM AMUPol (right column) shares features with the nanodisc associated form. Peak centers for the nanodisc-associated form are annotated with a red circle and peak centers for the frozen monomer are annotated with a blue circle. Spinning side bands are marked with an *. All spectra were recorded at 600 MHz with 12 kHz MAS at 104 K.

Figure 2:

A) α-syn in the nanodisc-associated α-helix rich (red) and frozen monomeric (blue) forms are differentiated by ¹⁵N-filtered ¹³C spectra collected under DNP conditions on specifically threonine labeled samples. Forward labeling with threonine resulted in ~10% isotope scrambling to glycine. Open circles indicate database chemical shifts for α-helical (red) and random coil (blue) conformations for both threonine and glycine C^α. B) Cartoon representation of the primary sequence of α-syn with the predicted regions of intrinsic disorder (lines) and α-helices (squiggles) are annotated with the location of glycines, alanines and threonines. Boxes indicate the chaperone binding region for the intrinsically disordered monomeric form (left) and the disordered tail of the membrane associated form of α-syn (right).

Figure 3:

Altering the spatial distribution of AMUPol in cellular samples highlights conformations present in different regions of the cells. A) Delivery of AMUPol via electroporation of cells in the presence of 20 mM AMUPol followed by removal of the extracellular AMUPol prior to data collection reports quantitively on the entire structural ensemble. In contrast, delivery of AMUPol by other methods results in a spatial bias in the resulting spectra. B) Delivery of AMUPol via incubation of cells in 10 mM AMUPol. C) Delivery of AMUPol to cells that had been electroporated in buffer and allowed to recover for 15 minutes before delivery via incubation in 30 mM AMUPol. D) Delivery of AMUPol to cells via incubation in 30 mM AMUPol. The ¹⁵N-filtered ¹³C spectra of threonine labeled α-syn inside cells (green) is plotted with the spectra of the nanodisc-associated α-helical rich α-syn (red) and the frozen intrinsically disordered monomeric forms (blue) that are scaled by the weighting that resulted in the best fit linear combination (orange). Insets in each panel are cartoon representation of the AMUPol distribution (brown) in the cell and the interstitial space for each AMUPol delivery method. Darker shades represent higher AMUPol concentrations. DNP enhancements (top right corner) and estimated extracellular (top right) and intracellular (bottom right) AMUPol concentrations are annotated. Spectra were recorded at 600 MHz with 12 kHz MAS at 104 K.

Figure 4:

Representative fluorescent microscopy images depicting the distribution of α-syn inside HEK293 cells when AMUPol was delivered by electroporation (A&C) and incubation (B&D). Cells were prepared identically to those used for NMR spectroscopy except cells were fixed and immunostained for α-syn (green) and stained with DAPI (blue). The a-syn was homogenously dispersed throughout most of the cells regardless of AMUPol delivery method, but the a-syn was more concentrated near the cellular periphery (arrowheads) for cells when AMUPol was delivered by incubation.