@article {78265, title = {α-Synuclein aggregation nucleates through liquid-liquid phase separation.}, journal = {Nat Chem}, year = {2020}, month = {2020 Jun 08}, abstract = {

α-Synuclein (α-Syn) aggregation and amyloid formation is directly linked with Parkinson{\textquoteright}s disease pathogenesis. However, the early events involved in this process remain unclear. Here, using the in vitro reconstitution and cellular model, we show that liquid-liquid phase separation of α-Syn precedes its aggregation. In particular, in vitro generated α-Syn liquid-like droplets eventually undergo a liquid-to-solid transition and form an amyloid hydrogel that contains oligomers and fibrillar species. Factors known to aggravate α-Syn aggregation, such as low pH, phosphomimetic substitution and familial Parkinson{\textquoteright}s disease mutations, also promote α-Syn liquid-liquid phase separation and its subsequent maturation. We further demonstrate α-Syn liquid-droplet formation in cells. These cellular α-Syn droplets eventually transform into perinuclear aggresomes, the process regulated by microtubules. This work provides detailed insights into the phase-separation behaviour of natively unstructured α-Syn and its conversion to a disease-associated aggregated state, which is highly relevant in Parkinson{\textquoteright}s disease pathogenesis.

}, issn = {1755-4349}, doi = {10.1038/s41557-020-0465-9}, author = {Ray, Soumik and Singh, Nitu and Kumar, Rakesh and Patel, Komal and Pandey, Satyaprakash and Datta, Debalina and Mahato, Jaladhar and Panigrahi, Rajlaxmi and Navalkar, Ambuja and Mehra, Surabhi and Gadhe, Laxmikant and Chatterjee, Debdeep and Sawner, Ajay Singh and Maiti, Siddhartha and Bhatia, Sandhya and Gerez, Juan Atilio and Chowdhury, Arindam and Kumar, Ashutosh and Padinhateeri, Ranjith and Riek, Roland and Krishnamoorthy, G and Maji, Samir K} } @article {62759, title = {Observation of Continuous Contraction and a Metastable Misfolded State during the Collapse and Folding of a Small Protein.}, journal = {J Mol Biol}, year = {2019}, month = {2019 Jul 19}, abstract = {

To obtain proper insight into how structure develops during a protein folding reaction, it is necessary to understand the nature and mechanism of the polypeptide chain collapse reaction, which marks the initiation of folding. Here, the time-resolved fluorescence resonance energy transfer technique, in which the decay of the fluorescence light intensity with time is used to determine the time evolution of the distribution of intra-molecular distances, has been utilized to study the folding of the small protein, monellin. It is seen that when folding begins, about one-third of the protein molecules collapse into a molten globule state (I), from which they relax by continuous further contraction to transit to the native state. The larger fraction gets trapped into a metastable misfolded state. Exit from this metastable state occurs via collapse to the lower free energy I state. This exit is slow, on a time scale of seconds, because of activation energy barriers. The trapped misfolded molecules as well as the I molecules contract continuously and slowly as structure develops. A phenomenological model of Markovian evolution of the polymer chain undergoing folding, incorporating these features, has been developed, which fits well the experimentally observed time evolution of distance distributions. The observation that the "wrong turn" to the misfolded state has not been eliminated by evolution belies the common belief that the folding of functional protein sequences is very different from that of a random heteropolymer, and the former have been selected by evolution to fold quickly.

}, issn = {1089-8638}, doi = {10.1016/j.jmb.2019.07.024}, author = {Bhatia, Sandhya and Krishnamoorthy, G and Dhar, Deepak and Udgaonkar, Jayant B} } @article {29630, title = {Site-specific time-resolved FRET reveals local variations in the unfolding mechanism in an apparently two-state protein unfolding transition.}, journal = {Phys Chem Chem Phys}, volume = {20}, year = {2018}, month = {2018 Jan 31}, pages = {3216-3232}, abstract = {

Protein folding/unfolding transitions between the native (N) and unfolded (U) states are usually describable as two-state, only because of the dominant presence of the N and/or U states, because of which high energy intermediates remain undetected. Delineation of the cooperativity underlying these transitions, and characterization of high energy intermediates that are populated sparsely, have been difficult challenges, especially under equilibrium conditions, and require the use of a sensitive probe that reports on both the structures and population distributions of the partially unfolded intermediates. In this study, the use of multisite time-resolved FRET to monitor structural change in five specific segments of the small protein monellin, has brought out local deviations from two-state behavior during unfolding. It is shown that in some segments of the protein structure, denaturant-induced unfolding proceeds first by gradual expansion of the N state, then by an all-or-none transition from the N state ensemble to the U state ensemble, followed finally by expansion of the U state. Segments encompassing the sole helix appear, however, to unfold completely through a gradual transition from the N to U states. Finally, it is shown that equilibrium unfolding of monellin is not only heterogeneous, but that the degree of non-cooperativity differs between the sole α-helix and different parts of the β-sheet.

}, issn = {1463-9084}, doi = {10.1039/c7cp06214a}, author = {Bhatia, Sandhya and Krishnamoorthy, G and Udgaonkar, Jayant B} } @article {5917, title = {Molecular crowding causes narrowing of population heterogeneity and restricts internal dynamics in a protein}, journal = {METHODS AND APPLICATIONS IN FLUORESCENCE}, volume = {4}, year = {2016}, month = {MAR 2016}, abstract = {

Macromolecular crowding is a distinguishing property of intracellular media. Knowledge on the structure and dynamics of a protein in a crowded environment is essential for a complete understanding of its function. Reduction in intermolecular space could cause structural and functional alterations. Here, we have studied a model protein barstar to see how polyethylene glycol (PEG)-induced crowding affects its various structural states (native, unfolded and molten-globule-like) with different extents of change in conformational heterogeneity. Intramolecular distances and distance distributions were determined by time-resolved Forster resonance energy transfer from Trp53 to several acceptor sites by analysis of fluorescence decay kinetics using the Maximum Entropy Method. We observed PEG-induced narrowing of population distributions along with shifting of populations towards more compact states. Structural compactness also resulted in the slowing down of internal dynamics of the protein as revealed by fluorescence anisotropy decay kinetics of the fluorophore IAEDANS attached at several sites.

}, author = {Mondal, Samsuzzoha and Kallianpur, Mamata V and Udgaonkar, Jayant B and Krishnamoorthy, G} }