Intracellular aggregation of the human amyloid protein α-synuclein is causally linked to Parkinson's disease. While the isolated protein is intrinsically disordered, its native structure in mammalian cells is not known. Here we use nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy to derive atomic-resolution insights into the structure and dynamics of α-synuclein in different mammalian cell types. We show that the disordered nature of monomeric α-synuclein is stably preserved in non-neuronal and neuronal cells. Under physiological cell conditions, α-synuclein is amino-terminally acetylated and adopts conformations that are more compact than when in buffer, with residues of the aggregation-prone non-amyloid-β component (NAC) region shielded from exposure to the cytoplasm, which presumably counteracts spontaneous aggregation. These results establish that different types of crowded intracellular environments do not inherently promote α-synuclein oligomerization and, more generally, that intrinsic structural disorder is sustainable in mammalian cells.

Protein structure investigations are usually carried out in vitro under conditions far from their native environment in the cell. Differences between in-cell and in vitro structures of proteins can be generated by crowding effects, local pH changes, specific and nonspecific protein and ligand binding events, and chemical modifications. Double electron-electron resonance (DEER), in conjunction with site-directed spin-labeling, has emerged in the past decade as a powerful technique for exploring protein conformations in frozen solutions. The major challenges facing the application of this methodology to in-cell measurements are the instabilities of the standard nitroxide spin labels in the cell environment and the limited sensitivity at conventional X-band frequencies. We present a new approach for in-cell DEER distance measurement in human cells, based on the use of: (i) reduction resistant Gd(3+) chelates as spin labels, (ii) high frequency (94.9 GHz) for sensitivity enhancement, and (iii) hypo-osmotic shock for efficient delivery of the labeled protein into the cell. The proof of concept is demonstrated on doubly labeled ubiquitin in HeLa cells.

Mn(2+) chelating tags for Mn(2+)-Mn(2+) distance measurements by pulse EPR spectroscopy were developed. They feature a stable C-S conjugation to the protein, high reactivity towards cysteine thiols and short and rigid linkers that can be used in distance measurements with high resolution under reductive conditions. Double electron-electron resonance measurements at 95 GHz on ubiquitin labeled with these tags showed the expected narrow distance distribution.

It is an open question whether the conformations of proteins sampled in dilute solutions are the same as in the cellular environment. Here we address this question by double electron‐electron resonance (DEER) distance measurements with Gd(III) spin labels to probe the conformations of calmodulin (CaM) in vitro, in cell extract, and in human HeLa cells. Using the CaM mutants N53C/T110C and T34C/T117C labeled with maleimide‐DOTA‐Gd(III) in the N‐ and C‐terminal domains, we observed broad and varied interdomain distance distributions. The in vitro distance distributions of apo‐CaM and holo‐CaM in the presence and absence of the IQ target peptide can be described by combinations of closed, open, and collapsed conformations. In cell extract, apo‐ and holo‐CaM bind to target proteins in a similar way as apo‐ and holo‐CaM bind to IQ peptide in vitro. In HeLa cells, however, in the presence or absence of elevated in‐cell Ca2+ levels CaM unexpectedly produced more open conformations and very broad distance distributions indicative of many different interactions with in‐cell components. These results show‐case the importance of in‐cell analyses of protein structures.

No abstract