Biology subjects

Paul, S. S.

Publications and source records attributed to Paul, S. S..

4 recordsLinked to original sources

bioRxiv2025

Deciphering the mechanism of protein aggregation and effects of inhibitors using single-molecule mass photometry

Aggregation of misfolded proteins is a prominent feature of many diseases and hence an attractive drug target. However, the small oligomers that are critical early species in the aggregation cascade are difficult to monitor directly owing to their heterogeneity and transience, complicating efforts to define aggregation mechanisms and target oligomers therapeutically. Here, we observe changes in oligomer populations directly using single-molecule mass photometry (SMMP). Studying the pathogenic P301L mutant of tau protein linked to frontotemporal dementia, we globally fit the growth/decay kinetics for every oligomer population observed by SMMP to microscopic models of aggregation. A simple extension to the best-fit model also accounts for amyloid fibril kinetics, as monitored by Thioflavin T fluorescence, providing the first quantitative model of aggregation kinetics across all stages of the cascade based on direct observation. Crucially, we find that models fitting amyloid kinetics alone fail to capture oligomer behavior, implying that--contrary to standard practice--amyloid kinetics cannot be relied on to deduce aggregation mechanisms. Furthermore, there is no single rate-limiting nucleation step preceding rapid growth, as generally assumed, suggesting that standard models of aggregation are overly simplistic. Repeating the analysis in the presence of aggregation inhibitors allows identification of the discrete steps in the cascade affected by the inhibitors. This work presents a powerful new approach for defining protein aggregation mechanisms and the mechanism of action of inhibitors, with applications to understanding many diseases and developing novel therapeutics.

biophysics↗

bioRxiv2022

Quantifying oligomer populations in real time during protein aggregation using single-molecule mass photometry

Protein aggregation is a hallmark of many neurodegenerative diseases. The early stages of the aggregation cascade are crucial because small oligomers are thought to be key neurotoxic species, but they are difficult to study because they feature heterogeneous mixtures of transient states. We show how the populations of different oligomers can be tracked as they evolve over time during aggregation by using single-molecule mass photometry to measure individually the masses of the oligomers present in solution. Applying the approach to tau protein, whose aggregates are linked to diseases including Alzheimers and frontotemporal dementia, we found that tau existed in an equilibrium between monomers, dimers, and trimers before aggregation was triggered. Once aggregation commenced, the monomer population dropped continuously, paired first with a rise in the population of the smallest oligomers and then a steep drop as the protein was incorporated into larger oligomers and fibrils. Fitting these populations to kinetic models allowed different models of aggregation to be tested, identifying the most likely mechanism and quantifying the microscopic rates for each step in the mechanism. This work demonstrates a powerful new approach for characterizing previously inaccessible regimes in protein aggregation and building quantitative mechanistic models.

biophysics↗

bioRxiv2020

Structure and conformational cycle of a bacteriophage-encoded chaperonin

Chaperonins are ubiquitous molecular chaperones found in all domains of life. They form ring-shaped complexes that assist in the folding of substrate proteins in an ATP-dependent reaction cycle. Key to the folding cycle is the transient encapsulation of substrate proteins by the chaperonin. Here we present a structural and functional characterization of the chaperonin gp146 ({phi}EL) from the phage EL of Pseudomonas aeruginosa. {phi}EL, an evolutionary distant homolog of bacterial GroEL, is active in ATP hydrolysis and prevents the aggregation of denatured protein in a nucleotide-dependent manner. However, {phi}EL failed to refold the encapsulation-dependent model substrate rhodanese and did not interact with E. coli GroES, the lid-shaped co-chaperone of GroEL. {phi}EL forms tetradecameric double-ring complexes, which dissociate into single rings in the presence of ATP. Crystal structures of {phi}EL (at 3.54 and 4.03 [A]) in presence of ATP*BeFx revealed two distinct single-ring conformational states, both with open access to the ring cavity. One state showed uniform ATP-bound subunit conformations (symmetric state), whereas the second combined distinct ATP- and ADP-bound subunit conformations (asymmetric state). Cryo-electron microscopy of apo-{phi}EL revealed a double-ring structure composed of rings in the asymmetric state (3.45 [A] resolution). We propose that the phage chaperonin undergoes nucleotide-dependent conformational switching between double- and single rings and functions in aggregation prevention without substrate protein encapsulation. Thus, {phi}EL may represent an evolutionary more ancient chaperonin prior to acquisition of the encapsulation mechanism.

biochemistry↗

bioRxiv2019

Heme minimizes Parkinson’s disease-associated toxicity by inducing a conformational distortion in the oligomers of alpha-Synuclein

Aggregation of the intrinsically disordered protein alpha-Synuclein (-Syn) into insoluble fibrils with a cross-{beta} sheet amyloid structure plays a key role in the neuronal pathology of Parkinsons disease (PD). The fibrillation pathway of -Syn encompasses a multitude of transient oligomeric forms differing in size, secondary structure, hydrophobic exposure and toxicity. According to a recent solid state NMR study, the fibrillating unit of -Syn contains the core residues of the protein arranged into in-register parallel {beta} sheets with a unique Greek key topology. Here, we have shown that the physiologically available small molecule heme (hemin chloride) when added at sub-stoichiometric ratios to either monomeric or aggregated -Syn, arrests its aggregation in an oligomeric state, which is minimally toxic. Using cryo-EM, we observed that these heme-induced oligomers are mace-shaped and consist of approximately four monomers. However, the presence of a crucial twist or contortion in their Greek key structural architecture prevents further hierarchical appending into annular oligomers and protofilament formation. We confirm using a His50Gln mutant that the binding of heme onto His50 is crucial in inflicting the structural distortion and is responsible for the stabilization of the non-toxic and off-pathway -Syn oligomers. We believe that this study provides a novel strategy of developing a therapeutic solution of PD, which has been elusive so far.

biophysics↗