Intrinsically
disordered proteins (IDPs) do not autonomously fold
into well-defined three-dimensional structures and are best described
as a heterogeneous ensemble of rapidly interconverting conformers.
It is challenging to elucidate their complex dynamic signatures using
a single technique. In this study, we employed sensitive fluorescence
depolarization kinetics by following picosecond time-resolved fluorescence
anisotropy decays to directly capture the essential dynamical features
of intrinsically disordered α-synuclein (α-syn) site-specifically
labeled with thiol-active fluorophores. By utilizing a long-lifetime
(≥10 ns) anisotropic label, we were able to discern three distinct
rotational components of α-syn. The subnanosecond component
represents the local wobbling-in-cone motion of the fluorophore, whereas
the slower (∼1.4 ns) component corresponds to the short-range
backbone dynamics governed by collective torsional fluctuations in
the Ramachandran Φ–Ψ dihedral space. This backbone
dihedral rotational time scale is sensitive to the local chain stiffness
and slows down in the presence of an adjacent proline residue. We
also observed a small-amplitude (≤10%) slower rotational correlation
time (6–10 ns) that represents the long-range correlated dynamics
involving a much longer segment of the polypeptide chain. These intrinsic
dynamic signatures of IDPs will provide critical mechanistic underpinnings
in a mosaic of biophysical phenomena involving internal friction,
allosteric interactions, and phase separation.
Protein folding and dynamics are governed by an intricate interplay of thermal and viscosity-mediated effects. The solvent viscosity contributes to the frictional drag in protein dynamics. In addition to this viscosity-dependent effect, there is also an intriguing viscosity-independent component that represents the intrinsic resistance of the polypeptide chain to changing its conformation. This solvent-independent component is termed internal friction. A longstanding question is what is the fundamental molecular origin of internal friction in highly solvated and rapidly fluctuating intrinsically disordered proteins (IDPs) devoid of any persistent intrachain interactions? Here, we present a unique case to directly demonstrate that sequence-specific backbone dihedral barriers control local internal friction in an archetypal IDP, namely, α-synuclein. We performed site-directed fluorescence depolarization kinetics using picosecond time-resolved fluorescence anisotropy measurements to directly observe the directional decorrelation arising due to short-range backbone torsional fluctuations in the dihedral space. A linear viscosity-dependent model of the dihedral relaxation time yielded a finite zero-viscosity intercept that corresponds to internal friction. Our site-specific dynamic readouts were able to detect localized sequence-specific frictional components that are otherwise skewed in viscosity-dependent long-range chain fluctuations. Our results revealed the presence of low internal friction in nonproline sequence segments. In contrast, a proline introduces torsional stiffness in the segment exhibiting high internal friction that can be compensated by a conformationally flexible glycine. Such an intriguing interplay of local dihedral dynamics can modulate sequence-dependent internal friction in a wide range of IDPs involved in a myriad of important events including folding, binding, assembly, and phase transitions.
Amyloid fibrils are highly ordered nanoscopic protein aggregates comprising a cross-b amyloid core and are associated with deadly human diseases. Structural studies have revealed the supramolecular architecture of a variety of diseaseassociated amyloids. However, the critical role of transient intermolecular interactions between the disordered polypeptide segments of protofilaments in directing the supramolecular structure and nanoscale morphology remains elusive. Here, we present a unique case to demonstrate that interchain excitation energy migration via intermolecular homo-Fö rster resonance energy transfer can decipher the architecture of amyloid fibrils of human a-synuclein. Site-specific homo-Fö rster resonance energy transfer efficiencies measured by fluorescence depolarization allowed us to construct a two-dimensional proximity correlation map that defines the supramolecular packing of a-synuclein within the fibrils. These studies captured unique heteroterminal cross talks between the fuzzy interprotofilament interfaces of the parallel-in-register amyloid spines. Our results will find applications in discerning the broader role of protein disorder and fuzziness in steering the distinct polymorphic amyloids that exhibit strain-specific disease phenotypes.
The accumulation of toxic soluble oligomers of the amyloid-β peptide (Aβ) is a key step in the pathogenesis of Alzheimer’s disease. There are mainly two conformationally distinct oligomers, namely, prefibrillar...
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