Highly Disordered Amyloid-β Monomer Probed by Single-Molecule FRET and MD Simulation

Abstract

Monomers of amyloid-β (Aβ) protein are known to be disordered, but there is considerable controversy over the existence of residual or transient conformations that can potentially promote oligomerization and fibril formation. We employed single-molecule Förster resonance energy transfer (FRET) spectroscopy with site-specific dye labeling using an unnatural amino acid and molecular dynamics simulations to investigate conformations and dynamics of Aβ isoforms with 40 (Aβ40) and 42 residues (Aβ42). The FRET efficiency distributions of both proteins measured in phosphate-buffered saline at room temperature show a single peak with very similar FRET efficiencies, indicating there is apparently only one state. 2D FRET efficiency-donor lifetime analysis reveals, however, that there is a broad distribution of rapidly interconverting conformations. Using nanosecond fluorescence correlation spectroscopy, we measured the timescale of the fluctuations between these conformations to be ∼35 ns, similar to that of disordered proteins. These results suggest that both Aβ40 and Aβ42 populate an ensemble of rapidly reconfiguring unfolded states, with no long-lived conformational state distinguishable from that of the disordered ensemble. To gain molecular-level insights into these observations, we performed molecular dynamics simulations with a force field optimized to describe disordered proteins. We find, as in experiments, that both peptides populate configurations consistent with random polymer chains, with the vast majority of conformations lacking significant secondary structure, giving rise to very similar ensemble-averaged FRET efficiencies.

Electronic energy transfer in biomacromolecules

“…MD simulations have proven to be very useful in generating possible conformations of the system that help interpreting observed efficiencies in terms of well‐defined D / A separations and orientations . As it will be discussed in the following sections, MD simulations also allow investigating the interplay between EET and structural dynamics by properly postprocessing trajectories of electronic coupling values, thus allowing to generate the expected FRET observables in intermediate situations between the limiting dynamic and static averaging regimes.…”

“…This problem has been widely discussed in the application of FRET to measure distances in biosystems, given that the main source of uncertainty is related to the value adopted for the orientation factor κ. In this context, MD simulations have emerged as a powerful tool to examine the impact of electronic coupling fluctuations on FRET studies on a variety of biosystems and to aid in the characterization of the structural ensembles underlying static distributions of FRET rates . For example, simulations have shown some disagreement with the isotropically averaged 〈 κ 〉 = 2/3 value often assumed in FRET studies, or the occurrence of significant correlations between distances and orientations, neglected in the isotropic factor by assuming that 〈 κ 2 R −6 〉 = 〈 κ 2 〉〈 R −6 〉 .…”

Perspective: Chain dynamics of unfolded and intrinsically disordered proteins from nanosecond fluorescence correlation spectroscopy combined with single-molecule FRET

“…Interestingly, such IDPs typically exhibit global chain dynamics on similar time scales as chemically denatured proteins, in the range of ∼10-100 ns, 30,47,100,101 and they also show internal friction. 30,47,100 An interesting trend is illustrated by a plot of τ i versus the dimensions of the chains, which exhibits a decrease in τ i with increasing chain expansion (Fig.…”

“…85,[107][108][109] These developments have started to enable a more and more realistic and detailed view of unfolded proteins. 102 Since the reconfiguration of unfolded proteins in the absence of persistent interactions occurs on the sub-microsecond time scale, MD simulations can now access these dynamics even in explicit solvent, 11,12,101,110,111 providing a new opportunity for identifying the molecular contributions affecting them. As an example, Fig.…”

“…135 Finally, understanding unfolded state dynamics may also be important for the pathological aggregation of proteins involved in many neurodegenerative diseases. 101,137 In summary, a vast array of questions related to the dynamics of unfolded and intrinsically disordered proteins is waiting to be addressed, and a wide range of methods and concepts from polymer physics, chemical physics, and soft matter physics will be essential for tackling these challenges. Single-molecule FRET combined with nsFCS has helped to open a new window into previously inaccessible length scales and time scales.…”