@article {60327, title = {Monitoring site-specific conformational changes in real-time reveals a misfolding mechanism of the prion protein.}, journal = {Elife}, volume = {8}, year = {2019}, month = {2019 Jun 24}, abstract = {

During pathological aggregation, proteins undergo remarkable conformational re-arrangements to anomalously assemble into a heterogeneous collection of misfolded multimers, ranging from soluble oligomers to insoluble amyloid fibrils. Inspired by fluorescence resonance energy transfer (FRET) measurements of protein folding, an experimental strategy to study site-specific misfolding kinetics during aggregation, by effectively suppressing contributions from inter-molecular FRET, is described. Specifically, the kinetics of conformational changes across different secondary and tertiary structural segments of the mouse prion protein (moPrP) were monitored independently, after the monomeric units transformed into large oligomers O, which subsequently disaggregated reversibly into small oligomers O at pH 4. The sequence segments spanning helices α2 and α3 underwent a compaction during the formation of O and elongation into β-sheets during the formation of O. The β1-α1-β2 and α2-α3 subdomains were separated, and the helix α1 was unfolded to varying extents in both O and O.

}, issn = {2050-084X}, doi = {10.7554/eLife.44698}, author = {Sengupta, Ishita and Udgaonkar, Jayant} } @article {35145, title = {Structural mechanisms of oligomer and amyloid fibril formation by the prion protein.}, journal = {Chem Commun (Camb)}, year = {2018}, month = {2018 May 23}, abstract = {

Misfolding and aggregation of the prion protein is responsible for multiple neurodegenerative diseases. Works from several laboratories on folding of both the WT and multiple pathogenic mutant variants of the prion protein have identified several structurally dissimilar intermediates, which might be potential precursors to misfolding and aggregation. The misfolded aggregates themselves are morphologically distinct, critically dependent on the solution conditions under which they are prepared, but always β-sheet rich. Despite the lack of an atomic resolution structure of the infectious pathogenic agent in prion diseases, several low resolution models have identified the β-sheet rich core of the aggregates formed in vitro, to lie in the α2-α3 subdomain of the prion protein, albeit with local stabilities that vary with the type of aggregate. This feature article describes recent advances in the investigation of in vitro prion protein aggregation using multiple spectroscopic probes, with particular focus on (1) identifying aggregation-prone conformations of the monomeric protein, (2) conditions which trigger misfolding and oligomerization, (3) the mechanism of misfolding and aggregation, and (4) the structure of the misfolded intermediates and final aggregates.

}, issn = {1364-548X}, doi = {10.1039/c8cc03053g}, author = {Sengupta, Ishita and Udgaonkar, Jayant B} } @article {20954, title = {Expression and purification of single cysteine-containing mutant variants of the mouse prion protein by oxidative refolding.}, journal = {Protein Expr Purif}, year = {2017}, month = {2017 Jul 20}, abstract = {

The folding and aggregation of proteins has been studied extensively, using multiple probes. To facilitate such experiments, introduction of spectroscopically-active moieties in to the protein of interest is often necessary. This is commonly achieved by specifically labelling cysteine residues in the protein, which are either present naturally or introduced artificially by site-directed mutagenesis. In the case of the recombinant prion protein, which is normally expressed in inclusion bodies, the presence of the native disulfide bond complicates the correct refolding of single cysteine-containing mutant variants of the protein. To overcome this major bottleneck, a simple purification strategy for single tryptophan, single cysteine-containing mutant variants of the mouse prion protein is presented, with yields comparable to that of the wild type protein. The protein(s) obtained by this method are correctly folded, with a single reduced cysteine, and the native disulfide bond between residues C178 and C213 intact. The β-sheet rich oligomers formed from these mutant variant protein(s) are identical to the wild type protein oligomer. The current strategy facilitates sample preparation for a number of high resolution spectroscopic measurements for the prion protein, which specifically require thiol labelling.

}, issn = {1096-0279}, doi = {10.1016/j.pep.2017.07.014}, author = {Sengupta, Ishita and Udgaonkar, Jayant B} } @article {17517, title = {Salt-mediated oligomerization of the mouse prion protein monitored by real time NMR.}, journal = {J Mol Biol}, year = {2017}, month = {2017 May 11}, abstract = {

The prion protein forms {\ss}-rich soluble oligomers in vitro at pH4 in the presence of physiological concentrations of salt. In the absence of salt, oligomerization and misfolding does not take place in an experimentally tractable timescale. While it is well established that a lowering of pH facilitates misfolding and oligomerization of this protein, the role of salt remains poorly understood. Here, solution-state NMR was used to probe perturbations in the monomeric mouse prion protein moPrP) structure immediately upon salt-addition, prior to the commencement of the oligomerization reaction. The weak binding of salt at multiple sites dispersed all over the monomeric protein causes a weak non-specific perturbation of structure throughout the protein. The only significant perturbation occurs in the loop between helix 2 and 3 in and around the partially buried K193-E195 salt bridge. The disruption of this key electrostatic interaction is the earliest detectable change in the monomer before any major conformational change occurs and appears to constitute the trigger for the commencement of misfolding and oligomerization. Subsequently, the kinetics of monomer loss, due to oligomerization, was monitored at the individual residue level. The oligomerization reaction was found to be rate-limited by association and not conformational change, with an average reaction order of 2.6, across residues. Not surprisingly, salt accelerated the oligomerization kinetics, in a non-specific manner, by electrostatic screening of the highly charged monomers at acidic pH. Together, these results allowed a demarcation of the specific and non-specific effects of salt on prion protein misfolding and oligomerization.

}, issn = {1089-8638}, doi = {10.1016/j.jmb.2017.05.006}, author = {Sengupta, Ishita and Bhate, Suhas H and Das, Ranabir and Udgaonkar, Jayant B} }