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. 2014 Jan 28;9(1):e86983.
doi: 10.1371/journal.pone.0086983. eCollection 2014.

Investigation of intramolecular dynamics and conformations of α-, β- and γ-synuclein

Vanessa C Ducas  1 , Elizabeth Rhoades  2
Affiliations

Investigation of intramolecular dynamics and conformations of α-, β- and γ-synuclein

Vanessa C Ducas et al. PLoS One. .

Abstract

The synucleins are a family of natively unstructured proteins consisting of α-, β-, and γ-synuclein which are primarily expressed in neurons. They have been linked to a wide variety of pathologies, including neurological disorders, such as Parkinson's disease (α-synuclein) and dementia with Lewy bodies (α- and β-synuclein), as well as various types of cancers (γ-synuclein). Self-association is a key pathological feature of many of these disorders, with α-synuclein having the highest propensity to form aggregates, while β-synuclein is the least prone. Here, we used a combination of fluorescence correlation spectroscopy and single molecule Förster resonance energy transfer to compare the intrinsic dynamics of different regions of all three synuclein proteins to investigate any correlation with putative functional or dysfunctional interactions. Despite a relatively high degree of sequence homology, we find that individual regions sample a broad range of diffusion coefficients, differing by almost a factor of four. At low pH, a condition that accelerates aggregation of α-synuclein, on average smaller diffusion coefficients are measured, supporting a hypothesis that slower intrachain dynamics may be correlated with self-association. Moreover, there is a surprising inverse correlation between dynamics and bulkiness of the segments. Aside from this observation, we could not discern any clear relationship between the physico-chemical properties of the constructs and their intrinsic dynamics. This work suggests that while protein dynamics may play a role in modulating self-association or interactions with other binding partners, other factors, particularly the local cellular environment, may be more important.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Synuclein proteins sequences.
The various regions probed here are indicated by the colored boxes: green – AH construct (88% identity between αS and βS and 80% identity between αS and γS); purple – LF construct(74% identity between αS and βS and 59% identity between αS and γS); orange – NAC construct (48% identity between αS and βS and 41% identity between αS and γS); and blue – CT construct (27% identity between αS and βS and 25% identity between αS and γS). The sequence enclosed by the dashed line corresponds to an additional proline-rich construct (residues 102 to 126) probed in the C-terminal region of βS.
Figure 2
Figure 2. Representative autocorrelation curves of a single-labeled control construct (A) and of a double-labeled construct (B).
(A)The control autocorrelation curve shows the characteristic decay curve observed for the simple translational diffusion (τD) with no evidence for a second decay component. The TMR remains fluorescent (solid red circle), independent of the conformation of the protein. (B)The autocorrelation curve of the double-labeled construct has an additional fast decay component (τR), reflecting the intrachain diffusion that brings the fluorophores into close proximity, resulting in self-quenching (open red circles), which is relieved when the fluorophores diffuse apart (solid red circles). Red traces correspond to fits with Eq. 1 where A = 0 (no kinetic component; A) and with Eq. 1 (B), with accompanying residual plots.
Figure 3
Figure 3. Representative single-molecule FRET histogram of the CT construct of αS.
The major peak at ETeff∼0.84 arises from energy transfer between residues 92 and 115, while the peak at ETeff∼0 results from donor-only molecules.
Figure 4
Figure 4. τR and Mean ETeff as a function of the physico-chemical properties of the synucleins.
Net charge per residue (A and D), bulkiness per residue (B and E) and hydrophobicity per residue (C and F) at pH 7.4 (solid symbols) and/or at pH 3.5 (open symbols). Circles– AH constructs; squares – LF constructs; triangles – NAC constructs; inverted triangles – CT constructs.
Figure 5
Figure 5. Relative diffusion coefficient as a function of the physico-chemical properties of the synucleins.
Net charge per residue (A), bulkiness per residue (B), and hydrophobicity per residue (C) at pH 7.4 (solid symbols) and/or at pH 3.5 (open symbols). Circles– AH constructs; squares– LF constructs; triangles– NAC constructs; inverted triangles – CT constructs; star– βS CT construct 102–126 construct.
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