01762nas a2200169 4500008004100000022001400041245012500055210006900180260007100249300001200320490000600332520106900338100002101407700001901428700002401447856012101471 2016 eng d a1948-718500aEvidence for dry molten globule-like domains in the pH-induced equilibrium folding intermediate of a multidomain protein0 aEvidence for dry molten globulelike domains in the pHinduced equ a1155 16TH ST, NW, WASHINGTON, DC 20036 USAbAMER CHEMICAL SOCcJAN a173-1790 v73 a

The role of van der Waals (vdW) packing interactions compared to the hydrophobic effect in stabilizing the functional structure of proteins is poorly understood. Here we show, using fluorescence resonance energy transfer, dynamic fluorescence quenching, red-edge excitation shift, and near- and far-UV circular dichroism, that the pH-induced structural perturbation of a multidomain protein leads to the formation of a state in which two out of the three domains have characteristics of dry molten globules, that is, the domains are expanded compared to the native protein with disrupted packing interactions but have dry cores. We quantitatively estimate the energetic contribution of vdW interactions and show that they play an important role in the stability of the native state and cooperativity of its structural transition, in addition to the hydrophobic effect. Our results also indicate that during the pH-induced unfolding, side-chain unlocking and hydrophobic solvation occur in two distinct steps and not in concerted manner, as commonly believed

1 aAcharya, Nirbhik1 aMishra, Prajna1 aJha, Santosh, Kumar uhttps://library.ncl.res.in/content/evidence-dry-molten-globule-domains-ph-induced-equilibrium-folding-intermediate-002315nas a2200157 4500008004100000022001400041245013700055210006900192260000800261300001200269490000700281520169200288100002201980700002402002856013102026 2020 eng d a0006-296000aEarly metastable assembly during the stress-induced formation of worm-like amyloid fibrils of nucleic acid binding domains of TDP-430 aEarly metastable assembly during the stressinduced formation of  cJAN a315-3280 v593 a

TDP-43 protein travels between the cytosol and the nucleus to perform its nucleic acid binding functions through its two tandem RNA recognition motif domains (TDP-43(tRRM)). When exposed to various environmental stresses, it forms abnormal aggregates in the cytosol of neurons, which are the hallmarks of amyotrophic lateral sclerosis and other TDP-43 proteinopathies. However, the nature of early structural changes upon stress sensing and the consequent steps during the course of aggregation are not well understood. In this study, we show that under low-pH conditions, mimicking starvation stress, TDP-43(tRRm) undergoes a conformational opening reaction linked to the protonation of buried ionizable residues and grows into a metastable oligomeric assembly (called the ``low-pH form'' or the ``L form''). In the L form, the protein molecules have disrupted tertiary structure, solvent-exposed hydrophobic patches, and mobile side chains but the native-like secondary structure remains intact. The L form structure is held by weak interactions and has a steep dependence on ionic strength. In the presence of as little as 15 mM KCl, it fully misfolds and further oligomerizes to form a beta-sheet rich ``beta form'' in at least two distinct steps. The beta form has an ordered, stable structure that resembles worm-like amyloid fibrils. The unstructured regions of the protein gain structure during L (sic) beta conversion. Our results suggest that TDP-43(tRRm) could function as a stress sensor and support a recent model in which stress sensing during neurodegeneration occurs by assembly of proteins into metastable assemblies that are precursors to the solid aggregates.

1 aPillai, Meenakshi1 aJha, Santosh, Kumar uhttps://library.ncl.res.in/content/early-metastable-assembly-during-stress-induced-formation-worm-amyloid-fibrils-nucleic-acid02523nas a2200169 4500008004100000022001400041245012800055210006900183260000800252300001400260490000700274520188300281100002202164700001502186700002402201856012802225 2023 eng d a0006-296000aElectrostatic modulation of intramolecular and intermolecular interactions during the formation of an amyloid-like assembly0 aElectrostatic modulation of intramolecular and intermolecular in cMAY a1890-19050 v623 a

The mechanism of protein aggregation can be broadly viewedas ashift from the native-state stabilizing intramolecular to the aggregated-phasesustaining intermolecular interactions. Understanding the role ofelectrostatic forces on the extent of modulation of this switch hasrecently evolved as a topic of monumental significance as proteinaggregation has lately been connected to charge modifications of anaging proteome. To decipher the distinctive role of electrostaticforces on the extremely complicated phase separation landscape, weopted for a combined in vitro-in silico approach to ascertainthe structure-dynamics-stability-aggregabilityrelationship of the functional tandem RRM domains of the ALS-relatedprotein TDP-43 (TDP-43(tRRM)), under a bivariate solutioncondition in terms of pH and salt concentration. Under acidic pH conditions,the native TDP-43(tRRM) protein creates an aggregation-proneentropically favorable partially unfolded conformational landscapedue to enthalpic destabilization caused by the protonation of theburied ionizable residues and consequent overwhelming fluctuationsof selective segments of the sequence leading to anti-correlated movementsof the two domains of the protein. The evolved fluffy ensemble witha comparatively exposed backbone then easily interacts with incomingprotein molecules in the presence of salt via typical amyloid-aggregate-likeintermolecular backbone hydrogen bonds with a considerable contributionoriginating from the dispersion forces. Subsequent exposure to excesssalt at low pH conditions expedites the aggregation process via anelectrostatic screening mechanism where salt shows preferential bindingto the positively charged side chain. The applied target observable-specificapproach complementarity unveils the hidden information landscapeof an otherwise complex process with unquestionable conviction.

1 aPillai, Meenakshi1 aDas, Atanu1 aJha, Santosh, Kumar uhttps://library.ncl.res.in/content/electrostatic-modulation-intramolecular-and-intermolecular-interactions-during-formation02190nas a2200157 4500008004100000022001400041245011300055210006900168260000800237300001400245490000700259520159300266100002401859700002401883856012501907 2024 eng d a0006-296000aElectrostatics choreographs the aggregation dynamics of full-length TDP-43 via a monomeric amyloid precursor0 aElectrostatics choreographs the aggregation dynamics of fullleng cMAY a1553-15680 v633 a

TDP-43 is a ubiquitously expressed, multidomain functional protein that is distinctively known to form aggregates in many fatal neurodegenerative disorders. However, the information for arresting TDP-43 aggregation is missing due to a lack of understanding of the molecular mechanism of the aggregation and structural properties of TDP-43. TDP-43 is inherently prone to aggregation and has minimal protein solubility. Multiple studies have been performed on the smaller parts of TDP-43 or the full-length protein attached to a large solubilization tag. However, the presence of co-solutes or solubilization tags is observed to interfere with the molecular properties and aggregation mechanism of full-length TDP-43. Notably, this study populated and characterized the native, dimeric state of TDP-43 without the interference of co-solutes or protein modifications. We observed that the electrostatics of the local environment is capable of the partial unfolding and monomerization of the native dimeric state of TDP-43 into an amyloidogenic molten globule. By employing the tools of thermodynamics and kinetics, we reveal the structural characteristics and temporal order of the early intermediates and transition states during the transition of the molten globule to beta-rich, amyloid-like aggregates of TDP-43, which is governed by the electrostatics of the environment. The current advanced understanding of the nature of native and early aggregation-prone intermediates, early steps, and the influence of electrostatics in TDP-43 aggregation is essential for drug design.

1 aDoke, Abhilasha, A.1 aJha, Santosh, Kumar uhttps://library.ncl.res.in/content/electrostatics-choreographs-aggregation-dynamics-full-length-tdp-43-monomeric-amyloid