Investigation of the Polymeric Properties of α-Synuclein and Comparison with NMR Experiments: A Replica Exchange Molecular Dynamics Study

Abstract

Intrinsically disordered proteins (IDPs) have been shown to be involved in a number of cellular functions, in addition to their predominance in diseased states. α-synuclein may be described as one such IDP implicated in the pathology of Parkinson's disease. Understanding the conformational characteristics of the monomeric state of α-synuclein is necessary for understanding the role of the monomer conformation in aggregation. Polymer theories have been applied to investigate the statistical properties of homopolymeric IDPs. Here we use Replica Exchange Molecular Dynamics (REMD) simulations using temperature as a proxy for solvent quality to examine how well these theories developed for homopolymeric chains describe heteropolymeric α-synuclein. Our results indicate that α-synuclein behaves like a homopolymer at the extremes of solvent quality, while in the intermediate solvent regime, the uneven distribution of charged residues along the sequence strongly influences the conformations adopted by the chain. We refine the ensemble extracted from the REMD simulations of α-synuclein, which shows the best qualitative agreement with experiment, by fitting to the experimental NMR Residual Dipolar Couplings (RDCs) and Paramagnetic Relaxation Enhancements (PREs). Our results demonstrate that the detailed shape of the RDC patterns are sensitive to the angular correlations that are local in sequence while longer range anti-correlations which arise from packing constraints affect the RDC magnitudes.

Figure 1

(A) Primary sequence of human α-synuclein. The N, NAC and C-terminal regions are represented in blue, green and red respectively. (B–D) Representative conformations of α-synuclein selected for the low (B), intermediate (C), and high temperature (D) ensembles. The representative structures were chosen based on the top four clusters, determined using the hierarchical clustering method . The color scheme used here are the same as that in A.

Figure 2

Global shape and size descriptors for α-synuclein. Average radius of gyration, R_g (A) hydrodynamic radius, R_h (B) Asphericity, ™ (C) and shape parameter, S (D) plotted as a function of simulation temperature. Standard deviations within ensembles are represented as error bars.

Figure 3

Scaling of average internal distances (R_ij) plotted as a function of sequence separation |i–j| for the low (blue), intermediate (cyan), and high (red) temperature ensembles. The fits to the intermediate and high temperature ensembles, shown as solid lines, have scaling exponents of 0.46 and 0.57 respectively. Error bars represent standard deviations displayed here only for select residues along the sequence for clarity.

Figure 4

Ensemble averaged angular correlation function plotted as a function of sequence separation for the (A) low (blue), intermediate (cyan), and high (red) temperature ensembles, and the N (maroon), NAC (magenta) and C (green) terminal domains of the (B) low, (C) intermediate, and (D) high temperature ensembles.

Figure 5

Residual Dipolar Couplings calculated based on global alignment for the (A) low, (B) intermediate and (C) high temperature ensembles. The horizontal lines represent the calculated RDCs averaged over the chains, with values of 0.86, 1.65 and 2.5 Hz for the low, intermediate and high temperature ensembles respectively.

Figure 6

Correlation between the RDCs calculated from global (black) and local (red) alignments using LAWs length − 3(A), 5 (B), 9 (C), 15 (D) and 25 (E).

Figure 7

Conformational characteristics of the simulation ensembles correlating with experiment: PREs for the REMD ensemble (A, B, C), reconstructed ensemble (D, E, F) and NMR experimental data (G, H, I) for spin label at positions A19, A90 and G132 respectively. The dotted lines represent the theoretical PRE values calculated for α-synuclein with no long-range contacts, determined as described previously .

Figure 8

Residue density along the sequence for the intermediate temperature ensemble of α-synuclein. The residue density is calculated as the count of the average number of residues within 7Å of the side chains of any residue along the sequence.

Figure 9

Comparison of the experimental HN RDCs (black) with (A) the average RDC determined from global alignment of the intermediate temperature REMD ensemble (red), (B) RDCs calculated using local alignment for the weighted subset of the reconstructed ensemble (red).

Figure 10

Ensemble averaged helix (red) and beta strand (black) propensities for the original REMD (A) and reconstructed (B) α-synuclein ensembles. Helix propensities were calculated by combining the alpha pi and 3–10 conformations, while the strand propensities were obtained by combining the extended and bridge conformations .