Segmental Conformational Disorder and Dynamics in the Intrinsically Disordered Protein α-Synuclein and Its Chain Length Dependence

We hypothesize that the first step of aggregation of disordered proteins, such as α-synuclein, is controlled by the rate of backbone reconfiguration. When reconfiguration is fast, bimolecular association is not stable, but as reconfiguration slows, association is more stable and subsequent aggregation is faster. To investigate this hypothesis, we have measured the rate of intramolecular diffusion in α-synuclein, a protein involved in Parkinson's disease, under solvent conditions that accelerate or decelerate aggregation. Using the method of tryptophan-cysteine (Trp-Cys) quenching, the rate of intramolecular contact is measured in four different loops along the chain length. This intrinsically disordered protein is highly diffusive at low temperature at neutral pH, when aggregation is slow, and compacts and diffuses more slowly at high temperature or low pH, when aggregation is rapid. Diffusion also slows with the disease mutation A30P. This work provides unique insights into the earliest steps of α-synuclein aggregation pathway and should provide the basis for the development of drugs that can prevent aggregation at the initial stage.unfolded state | Alzheimer's disease | amyloid T he protein α-synuclein is the primary component of intracellular aggregants known as Lewy bodies, a hallmark of Parkinson's disease, and likely plays a central role in neuronal cell death (1). When recombinantly expressed and purified it is intrinsically disordered in solution and highly prone to aggregation (2). Recent work has shown that the native structure in human cells is likely a helical tetramer that is resistant to aggregation, but when this tetramer is denatured the monomers cannot autonomously refold and aggregate very quickly (3, 4). Thus it is likely that the aggregation pathway passes through the monomer. In vitro, the aggregation propensity of α-synuclein is very sensitive to solution conditions such as temperature, pH, salt concentration, and solution cofactors. There has been significant work to discover the structures that form in association with lipids, structures of final aggregates, and structures that may be the aggregation precursor (5-15). However, the underlying reason for why monomeric α-synuclein is so prone to aggregation is still unknown. We propose that a physical influence on aggregation is the internal dynamics of the disordered chain, that is, the rate of intramolecular diffusion.Over the past decade, a number of groups have measured intramolecular diffusion in a variety of sequences using measurements of intramolecular contact between two probes placed in the chain. All unstructured peptides, including fragments of α-synuclein, diffuse relatively rapidly, approximately 1-3 × 10 −6 cm 2 s −1 (16-18). These rates are less than 10 times slower than free diffusion of individual amino acids and about the same as translational diffusion of the entire chain, suggesting that the chain does not exert significant drag on the movement of individual segments (19), although there are measurable tail effects when ...

Section: Discussionsupporting

confidence: 45%

mentioning

confidence: 99%

Aggregation of α-synuclein is kinetically controlled by intramolecular diffusion

Ahmad

Chen

Lapidus

2012

“…The observed fastest diffusion coefficients, in high denaturant, are similar to those measured for unstructured peptides and proteins (34)(35)(36)(37)54). The slowest diffusion coefficient, observed for pH 7, 1.5 M GdnHCl, the lowest possible denaturant concentration in which the measurement of unfolded state dynamics could be achieved, is an order of magnitude slower and similar to other foldable proteins near their denaturation midpoint (33).…”

Section: Resultssupporting

confidence: 75%

“…) (34)(35)(36)(37)(38). In contrast, the unfolded polypeptide chains of Protein L and AcBP have been observed to diffuse very slowly, D ∼10 −10 and 10 −9 cm 2 s −1 (39,40), respectively.…”

mentioning

confidence: 98%

Prion protein dynamics before aggregation

Srivastava

Lapidus

2017

Prion diseases, like Alzheimer's disease and Parkinson disease, are rapidly progressive neurodegenerative disorders caused by misfolding followed by aggregation and accumulation of protein deposits in neuronal cells. Here we measure intramolecular polypeptide backbone reconfiguration as a way to understand the molecular basis of prion aggregation. Our hypothesis is that when reconfiguration is either much faster or much slower than bimolecular diffusion, biomolecular association is not stable, but as the reconfiguration rate becomes similar to the rate of biomolecular diffusion, the association is more stable and subsequent aggregation is faster. Using the technique of Trp-Cys contact quenching, we investigate the effects of various conditions on reconfiguration dynamics of the Syrian hamster and rabbit prion proteins. This protein exhibits behavior in all three reconfiguration regimes. We conclude that the hamster prion is prone to aggregation at pH 4.4 because its reconfiguration rate is slow enough to expose hydrophobic residues on the same time scale that bimolecular association occurs, whereas the rabbit sequence avoids aggregation by reconfiguring 10 times faster than the hamster sequence.protein folding | protein aggregation | intramolecular diffusion | prion disease | astemizole P rion disease, also known as transmissible spongiform encephalopathy (TSE) is attributed to misfolding followed by ordered aggregation and accumulation of protein deposits in neuronal cells (1-3). According to the "protein-only" hypothesis, the central event in prion (PrP) pathogenesis is the conformational conversion of the cellular α-helical rich isoform, PrP C , into misfolded β-sheet rich isoform, PrP Sc (3-6). The infectious form PrP Sc is believed to catalyze the conversion of PrP C , thereby permitting transmission between individuals and species (3, 7-10). However, there is very little known about PrP C before ordered aggregation. No putative aggregation precursor structure has been definitively identified; the protein may even be completely unstructured. It is believed that PrP C repeatedly cycles between the cell surface (∼pH 7.0) and endocytic compartment that has a pH as low as 4. 4 (11, 12). Aggregated prion has been isolated from this compartment so it has been suggested that aggregation initiates in late endosomes (13,14). Also, the reduction of the disulfide bridge has been reported to augment misfolding in vitro and may play a significant role in prion pathogenesis (15)(16)(17)(18)(19)(20). However, the physical basis for aggregation of prion, particularly under certain solvent conditions, is still poorly understood.Protein folding is a diffusive search of the unfolded polypeptide chain over the energy landscape in conformational space for a minimum energy state (21). Intramolecular diffusion (diffusion of one part of the chain relative to another) plays a critical role by determining the rate at which an unfolded polypeptide chain finds its native state (22-25). It has been measured for various peptides and pro...

“…This feature suggested that VirR contains an intrinsically disordered region. Such regions may acquire structure in the presence of a binding partner (28,29). Thus, we turned to NMR spectroscopy, using a truncated protein in which the highly hydrophobic stretch spanning amino acid residues 20-42 was deleted, along with the preceding 19 residues in the N terminus.…”

Section: Virr Regulates Release Of Immunomodulatory Factorsmentioning

confidence: 99%

Genetic regulation of vesiculogenesis and immunomodulation in Mycobacterium tuberculosis

Rath

Huang

Wang

et al. 2013

119

Mycobacterium tuberculosis (Mtb) restrains immune responses well enough to escape eradication but elicits enough immunopathology to ensure its transmission. Here we provide evidence that this host-pathogen relationship is regulated in part by a cytosolic, membrane-associated protein with a unique structural fold, encoded by the Mtb gene rv0431. The protein acts by regulating the quantity of Mtb-derived membrane vesicles bearing Toll-like receptor 2 ligands, including the lipoproteins LpqH and SodC. We propose that rv0431 be named "vesiculogenesis and immune response regulator." TLR2 | macrophages