Conformational ensembles of fully disordered natural polypeptides represent the starting point of protein refolding initiated by transfer to folding conditions. Thus, understanding the transient properties and dimensions of such peptides under folding conditions is a necessary step in the understanding of their subsequent folding behavior. Such ensembles can also undergo alternative folding and form amyloid structures, which are involved in many neurological degenerative diseases. Here, we performed a structural study of this initial state using time-resolved fluorescence resonance energy transfer analysis of a series of eight partially overlapping double-labeled chain segments of the N-terminal and NAC domains of the α-synuclein molecule. The distributions of end-to-end distance and segmental intramolecular diffusion coefficients were simultaneously determined for eight labeled chain segments. We used the coefficient of variation, Cv, as a measure of the conformational heterogeneity (i.e., structural disorder). With the exception of two segments, the Cvs were characteristic of a fully disordered state of the chain. Subtle deviations from this behavior at the segment labeled in the NAC domain and the segment at the N termini reflected subtle conformational bias that might be related to the initiation of transition to amyloid aggregates. The chain length dependence of the mean segmental end-to-end distance followed a power law as predicted by Flory, but the dependence was steeper than previously predicted, probably due to the contribution of the excluded volume effect, which is more dominant for shorter-chain segments. The observed intramolecular diffusion coefficients (< 10 to ∼ 252/ns) are only an order of magnitude lower than the common diffusion coefficients of low molecular weight probes. This diffusion coefficient increased with chain length, probably due to the cumulative contributions of minor bond rotations along the chain. These results gave us a reference both for characteristics of a natural unfolded polypeptide at the moment of initiation of folding and for detection of possible initiation sites of the amyloid transition.
Bibliographical noteFunding Information:
Plasmid of the WT αS was kindly provided by Dr. Thomas Jovin of the Max Plank Institute for Biophysical Chemistry in Gottingen; his advice and encouragement are gratefully acknowledged. Valuable discussions with Dan Amir, Gershon Hazan, and Maik H. Jacob are also gratefully acknowledged. This study was supported by grants from the German Israel Binational Science Foundation (GIF), the Israel Science Foundation (ISF), the United States–Israel Binational Science Foundation (BSF), and the EU Marie Curie TOK.
- disordered proteins
- intramolecular diffusion
- random coil
- unfolded state
- α synuclein