14-3-3τ as a Modulator of Early α-Synuclein Multimerization and Amyloid Formation.

The aggregation of α-synuclein (αS) plays a key role in Parkinson's disease (PD) etiology. While the onset of PD is age-related, the cellular quality control system appears to regulate αS aggregation throughout most human life. Intriguingly, the protein 14-3-3τ has been demonstrated to delay αS aggregation and the onset of PD in various models. However, the molecular mechanisms behind this delay remain elusive. Our study confirms the delay in αS aggregation by 14-3-3τ, unveiling a concentration-dependent relation. Utilizing microscale thermophoresis (MST) and single-molecule burst analysis, we quantified the early αS multimers and concluded that these multimers exhibit properties that classify them as nanoscale condensates that form in a cooperative process, preceding the critical nucleus for fibril formation. Significantly, the αS multimer formation mechanism changes dramatically in the presence of scaffold protein 14-3-3τ. Our data modeling suggests that 14-3-3τ modulates the multimerization process, leading to the creation of mixed multimers or co-condensates, comprising both αS and 14-3-3τ. These mixed multimers form in a noncooperative process. They are smaller, more numerous, and distinctively not on the pathway to amyloid formation. Importantly, 14-3-3τ thus acts in the very early stage of αS multimerization, ensuring that αS does not aggregate but remains soluble and functional. This offers long-sought novel entries for the pharmacological modulation of PD.

Exploring Intra- and Inter-Regional Interactions in the IDP α-Synuclein Using smFRET and MD Simulations.

Theoretical concepts from polymer physics are often used to describe intrinsically disordered proteins (IDPs). However, amino acid interactions within and between regions of the protein can lead to deviations from typical polymer scaling behavior and even to short-lived secondary structures. To investigate the key interactions in the dynamic IDP α-synuclein (αS) at the amino acid level, we conducted single-molecule fluorescence resonance energy transfer (smFRET) experiments and coarse-grained molecular dynamics (CG-MD) simulations. We find excellent agreement between experiments and simulations. Our results show that a physiological salt solution is a good solvent for αS and that the protein is highly dynamic throughout its entire chain, with local intra- and inter-regional interactions leading to deviations from global scaling. Specifically, we observe expansion in the C-terminal region, compaction in the NAC region, and a slightly smaller distance between the C- and N-termini than expected. Our simulations indicate that the compaction in the NAC region results from hydrophobic aliphatic contacts, mostly between valine and alanine residues, and cation-π interactions between lysine and tyrosine. In addition, hydrogen bonds also seem to contribute to the compaction of the NAC region. The expansion of the C-terminal region is due to intraregional electrostatic repulsion and increased chain stiffness from several prolines. Overall, our study demonstrates the effectiveness of combining smFRET experiments with CG-MD simulations to investigate the key interactions in highly dynamic IDPs at the amino acid level.

Quantification of Dark Protein Populations in Fluorescent Proteins by Two-Color Coincidence Detection and Nanophotonic Manipulation.

Genetically encoded visible fluorescent proteins (VFPs) are a key tool used to visualize cellular processes. However, compared to synthetic fluorophores, VFPs are photophysically complex. This photophysical complexity includes the presence of non-emitting, dark proteins within the ensemble of VFPs. Quantitative fluorescence microcopy approaches that rely on VFPs to obtain molecular insights are hampered by the presence of these dark proteins. To account for the presence of dark proteins, it is necessary to know the fraction of dark proteins (fdark) in the ensemble. To date, fdark has rarely been quantified, and different methods to determine fdark have not been compared. Here, we use and compare two different methods to determine the fdark of four commonly used VFPs: EGFP, SYFP2, mStrawberry, and mRFP1. In the first, direct method, we make use of VFP tandems and single-molecule two-color coincidence detection (TCCD). The second method relies on comparing the bright state fluorescence quantum yield obtained by photonic manipulation to the ensemble-averaged fluorescence quantum yield of the VFP. Our results show that, although very different in nature, both methods are suitable to obtain fdark. Both methods show that all four VFPs contain a considerable fraction of dark proteins. We determine fdark values between 30 and 60% for the different VFPs. The high values for fdark of these commonly used VFPs highlight that fdark has to be accounted for in quantitative microscopy and spectroscopy.