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<p>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</p>1 aAcharya, Nirbhik1 aMishra, Prajna1 aJha, Santosh, Kumar  uhttps://library.ncl.res.in/content/evidence-dry-molten-globule-domains-ph-induced-equilibrium-folding-intermediate-001691nas a2200145   4500008004100000245011600041210006900157260000800226300001400234490000800248520112000256100001901376700002401395856012601419       2017                        eng d00aAlternatively packed dry molten globule-like intermediate in the native state ensemble of a multidomain protein0 aAlternatively packed dry molten globulelike intermediate in the   cOCT  a9336-93470 v1213 aIt has been difficult to quantify the degree of side-chain conformational heterogeneity in the native (N) state ensemble of proteins and the relative energetic contributions of the side-chain packing and the hydrophobic effect in protein stability. Here, we show using multiple site specific spectroscopic probes and tools of thermodynamics that the N state ensemble of a multidomain protein contains an equilibrium intermediate (I) whose interdomain region resembles a dry molten globule. In the I state, a tryptophan residue in the interdomain region is alternatively packed, but its secondary structure and intradomain packing are N-like. The I state also has a larger interdomain distance, but the domain-domain interface is dry and molten. Our results indicate that hydrophobic desolvation and side-chain packing are decoupled during protein folding and that interdomain packing interactions have an important energetic contribution in protein stability. Dynamic interconversion between alternatively packed N-like states could be important for multiple allosteric and ligand binding functions of this protein.1 aMishra, Prajna1 aJha, Santosh, Kumar  uhttps://library.ncl.res.in/content/alternatively-packed-dry-molten-globule-intermediate-native-state-ensemble-multidomain01700nas a2200157   4500008004100000245010000041210006900141260000800210300001600218490000700234520111600241100002101357700001901378700002401397856012101421       2017                        eng d00aDry molten globule-like intermediate during the base-induced unfolding of a multidomain protein0 aDry molten globulelike intermediate during the baseinduced unfol  cNOV  a30207-302160 v193 aThe nature of the initial structural events during the base-induced unfolding of the native (N) state of proteins is poorly understood. Combining site-specific fluorescence resonance energy transfer, size exclusion chromatography, dynamic fluorescence quenching, red-edge excitation shift and circular dichroism spectroscopy, we show here that an early intermediate during the base-induced unfolding of a multidomain protein, i.e., the B form, has features of a dry molten globule. We show that the N (sic) B transition involves protein expansion and loosening of packing of inter-domain helices near domains I and II without the disruption of intra-domain packing or any change in hydration of the inter-domain region which resembles a molten hydrocarbon. Surprisingly, the disruption of inter-domain packing accounts for 40-45% of the total change in free energy of complete unfolding. Our results show that the disruption of van der Waals packing can be decoupled in different regions of a protein and could occur prior to hydrophobic solvation during base-induced unfolding, challenging the existing notion.1 aAcharya, Nirbhik1 aMishra, Prajna1 aJha, Santosh, Kumar  uhttps://library.ncl.res.in/content/dry-molten-globule-intermediate-during-base-induced-unfolding-multidomain-protein00607nas a2200145   4500008004100000245012500041210006900166260000800235300001600243490000700259100002100266700001900287700002400306856013100330       2019                        eng d00aDry molten globule-like intermediate during the base-induced unfolding of a multidomain protein (vol 19, pg 20307, 2017)0 aDry molten globulelike intermediate during the baseinduced unfol  cSEP  a20499-204990 v211 aAcharya, Nirbhik1 aMishra, Prajna1 aJha, Santosh, Kumar  uhttps://library.ncl.res.in/content/dry-molten-globule-intermediate-during-base-induced-unfolding-multidomain-protein-vol-19-pg02444nas a2200157   4500008004100000022001400041245014400055210006900199260000900268300001200277490000700289520181700296100002202113700002402135856012702159       2019                        eng d  a0006-296000aFolding and aggregation energy landscapes of tethered RRM domains of human TDP-43 are coupled via a metastable molten globule-like oligomer0 aFolding and aggregation energy landscapes of tethered RRM domain  cFEB   a608-6200 v583 a<p>Stress-induced misfolding and intraneuronal aggregation of the highly conserved nucleic acid binding protein TDP-43 (transactive response DNA binding protein 43 kDa) and its fragments have been implicated in amyotrophic lateral sclerosis and several other neurodegenerative diseases. However, the physicochemical mechanism of its misfolding from the functional folded state is poorly understood. TDP-43 is a four-domain protein and performs the essential nucleic acid binding function with the help of its two tandem RNA recognition motif domains naturally tethered by a linker (called here the tethered RRM domain of TDP-43 or TDP-43(tRRM)). Here, we show that the monomeric native form of TDP-43(tRRM) remains in a pH-dependent and reversible thermodynamic equilibrium with a protonated, nanosized, 40-merit form (the A form). Under the stress-like low-pH condition, the A form becomes predominantly populated. In the A form, protein molecules have restricted dynamics of surface side-chain residues but native-like secondary structure. This self-assembled form possesses a loosely packed core in which the intrinsically disordered and aggregation-prone regions are in the proximity. The A form is metastable and swiftly aggregates into a highly stable amyloid-like protofibrillar form (beta form) mediated by the disorder-to-order transition of intrinsically disordered regions upon small environmental perturbations. Interestingly, the A form and the beta form are not formed when TDP-43(tRRM) is bound to DNA, indicating that the nucleic acid binding regions of the protein participate in their formation. Our results reveal how the energy landscapes of folding and aggregation of TDP-43(tRRM) are coupled by a metastable molten-globule like oligomeric form and modulated by stress-like conditions.</p>
1 aPillai, Meenakshi1 aJha, Santosh, Kumar  uhttps://library.ncl.res.in/content/folding-and-aggregation-energy-landscapes-tethered-rrm-domains-human-tdp-43-are-coupled01524nas a2200157   4500008004100000022001400041245009700055210006900152260000800221300001400229490000800243520094500251100001901196700002401215856012701239       2019                        eng d  a1520-610600aSlow motion protein dance visualized using red-edge excitation shift of a buried fluorophore0 aSlow motion protein dance visualized using rededge excitation sh  cFEB  a1256-12640 v1233 a<p>It has been extremely challenging to detect protein structures with a dynamic core, such as dry molten globules, that remain in equilibrium with the tightly packed native (N) state and that are important for a myriad of entropy-driven protein functions. Here, we detect the higher entropy conformations of a human serum protein, using red-edge excitation shift experiments. We covalently introduced a fluorophore inside the protein core and observed that in a subset of native population, the side chains of the polar and buried residues have different spatial arrangements than the mean population and that they solvate the fluorophore on a timescale much slower than the nanosecond timescale of fluorescence. Our results provide direct evidence for the dense fluidity of protein core and show that alternate side-chain packing arrangements exist in the core that might be important for multiple binding functions of this protein.</p>
1 aMishra, Prajna1 aJha, Santosh, Kumar  uhttps://library.ncl.res.in/content/slow-motion-protein-dance-visualized-using-red-edge-excitation-shift-buried-fluorophore02315nas 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<p>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.</p>
1 aPillai, Meenakshi1 aJha, Santosh, Kumar  uhttps://library.ncl.res.in/content/early-metastable-assembly-during-stress-induced-formation-worm-amyloid-fibrils-nucleic-acid02266nas a2200217   4500008004100000022001400041245010100055210006900156260000800225300001100233490000800244520150100252653003101753653003001784653002801814653002201842100001901864700001701883700002401900856012401924       2021                        eng d  a0301-462200apH-dependent protein stability switch coupled to the perturbed pKa of a single ionizable residue0 apHdependent protein stability switch coupled to the perturbed pK  cJUL  a1065910 v2743 a<p>The contribution of electrostatic interactions in protein stability has not been fully understood. Burial of an ionizable amino acid inside the hydrophobic protein core can affect its ionization equilibrium and shift its pKa differentially in the native (N) and unfolded (U) states of a protein and this coupling between the folding/ unfolding cycle and the ionization equilibria of the ionizable residue can substantially influence the protein stability. Here, we studied the coupling of the folding/unfolding cycle with the ionization of a buried ionizable residue in a multi-domain protein, Human Serum Albumin (HSA) using fluorescence spectroscopy. A pHdependent change in the stability of HSA was observed in the near native pH range (pH 6.0-9.0). The protonation-deprotonation equilibrium of a single thiol residue that is buried in the protein structure was identified to give rise to the pH-dependent protein stability. We quantified the pKa of the thiol residue in the N and the U states. The mean pKa of the thiol in the N state was upshifted by 0.5 units to 8.7 due to the burial of the thiol in the protein structure. Surprisingly, the mean pKa of the thiol in the U state was observed to be downshifted by 1.3 units to 6.9. These results indicate that some charged residues are spatially proximal to the thiol group in the U state. Our results suggest that, in addition to the N state, electrostatic interactions in the U state are important determinants of protein stability.</p>10aelectrostatic interactions10aFluorescence spectroscopy10aThermodynamic stability10aUnfolded proteins1 aMishra, Prajna1 aPatni, Divya1 aJha, Santosh, Kumar  uhttps://library.ncl.res.in/content/ph-dependent-protein-stability-switch-coupled-perturbed-pka-single-ionizable-residue02040nas a2200157   4500008004100000022001400041245011600055210006900171260000900240300001400249490000800263520144100271100001701712700002401729856012901753       2021                        eng d  a1520-610600aProtonation-deprotonation switch controls the amyloid-like misfolding of nucleic-acid-binding domains of TDP-430 aProtonationdeprotonation switch controls the amyloidlike misfold  cAUG   a8383-83940 v1253 aNutrient starvation stress acidifies the cytosol and leads to the formation of large protein assemblies and misfolded aggregates. However, how starvation stress is sensed at the molecular level and leads to protein misfolding is poorly understood. TDP-43 is a vital protein, which, under stress-like conditions, associates with stress granule proteins via its functional nucleic-acid-binding domains (TDP-43(t)(RRM)) and misfolds to form aberrant aggregates. Here, we show that the monomeric N form of TDP-43(t)(RRM) forms a misfolded amyloid-like protein assembly, beta form, in a pH-dependent manner and identified the critical protein side-chain residue whose protonation triggers its misfolding. We systematically mutated the three buried ionizable residues, D105, H166, and H256, to neutral amino acids to block the pH-dependent protonation-deprotonation titration of their side chain and studied their effect on the N-to-beta transition. We observed that D105A and H2S6Q resembled TDP-43(t)(RRM) in their pH-dependent misfolding behavior. However, H166Q retains the N-like secondary structure under low-pH conditions and does not show pH-dependent misfolding to the beta form. These results indicate that H166 is the critical side-chain residue whose protonation triggers the misfolding of TDP-43(t)(RRM) and shed light on how stress-induced misfolding of proteins during neurodegeneration could begin from site-specific triggers.1 aPatni, Divya1 aJha, Santosh, Kumar  uhttps://library.ncl.res.in/content/protonation-deprotonation-switch-controls-amyloid-misfolding-nucleic-acid-binding-domains01426nas a2200157   4500008004100000022001400041245008400055210006900139260000800208300001400216490000800230520087600238100002101114700002401135856010901159       2022                        eng d  a1520-610600aDry molten globule-like intermediates in protein folding, function, and disease0 aDry molten globulelike intermediates in protein folding function  cNOV  a8614-86220 v1263 a<p>
	The performance of a protein depends on its correct folding to the final functional native form. Hence, understanding the process of protein folding has remained an important field of research for the scientific community for the past five decades. Two important intermediate states, namely, wet molten globule (WMG) and dry molten globule (DMG), have emerged as critical milestones during protein folding-unfolding reactions. While much has been discussed about WMGs as a common unfolding intermediate, the evidence for DMGs has remained elusive owing to their near-native features, which makes them difficult to probe using global structural probes. This Review puts together the available literature and new evidence on DMGs to give a broader perspective on the universality of DMGs and discuss their significance in protein folding, function, and disease.</p>
1 aAcharya, Nirbhik1 aJha, Santosh, Kumar  uhttps://library.ncl.res.in/content/dry-molten-globule-intermediates-protein-folding-function-and-disease01837nas a2200217   4500008004100000022001400041245008900055210006900144260000800213300001100221490000800232520109800240653003301338653002401371653002701395653001701422653002301439100001901462700002401481856011401505       2022                        eng d  a0301-462200aNative state conformational heterogeneity in the energy landscape of protein folding0 aNative state conformational heterogeneity in the energy landscap  cAPR  a1067610 v2833 aThe native structure of proteins is central to various functions performed by cells. A vital part of the structure-function paradigm of proteins is their inherent flexibility and dynamics. The dynamic interconversion between the conformational substates in the heterogeneous native state basin of the energy landscape enables a single protein molecule to perform multiple functions. The dynamics among the substates are assisted by the motion of different structural elements of a protein out of which side-chains of amino acids hold a significant position due to their involvement in various functions such as molecular recognition and dynamic allostery. This review briefly describes the origin of conformational heterogeneity in the native state ensemble and the motions of different structural modules that assist the equilibrium dynamics of the conformational substates. The review then centers the discussion on conformational heterogeneity due to side-chain movements in proteins, the experimental methods to detect and characterize them and their role in performing multiple functions.10aConformational heterogeneity10aDry molten globules10aEnergy landscape model10aNative state10aSide-chain packing1 aMishra, Prajna1 aJha, Santosh, Kumar  uhttps://library.ncl.res.in/content/native-state-conformational-heterogeneity-energy-landscape-protein-folding02523nas 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<p>
	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.</p>
1 aPillai, Meenakshi1 aDas, Atanu1 aJha, Santosh, Kumar  uhttps://library.ncl.res.in/content/electrostatic-modulation-intramolecular-and-intermolecular-interactions-during-formation02483nas a2200217   4500008004100000022001400041245010300055210006900158260000800227300001200235490000700247520173400254653002401988653002602012653003002038653001302068653001402081100002202095700002402117856012402141       2023                        eng d  a0887-358500aMultistep molecular mechanism of amyloid-like aggregation of nucleic acid-binding domain of TDP-430 aMultistep molecular mechanism of amyloidlike aggregation of nucl  cMAY  a649-6640 v913 a<p>
	TDP-43 protein is associated with many neurodegenerative diseases and has been shown to adopt various oligomeric and fibrillar states. However, a detailed kinetic understanding of the structural transformation of the native form of the protein to the fibrillar state is missing. In this study, we delineate the temporal sequence of structural events during the amyloid-like assembly of the functional nucleic acid-binding domain of TDP-43. We kinetically mapped the aggregation process using multiple probes such as tryptophan and thioflavin T (ThT) fluorescence, circular dichroism (CD), and dynamic light scattering (DLS) targeting different structural events. Our data reveal that aggregation occurs in four distinct steps-very fast, fast, slow, and very slow. The ``very fast'' change results in partially unfolded forms that undergo conformational conversion, oligomerization and bind to ThT in the ``fast step'' to form higher order intermediates (HOI). The temporal sequence of the formation of ThT binding sites and conformational conversion depends upon the protein concentration. The HOI further undergoes structural rearrangement to form protofibrils in the ``slow'' step, which, consequently, assembles in the ``very slow'' step to form an amyloid-like assembly. The spectroscopic properties of the amyloid-like assembly across the protein concentration remain similar. Additionally, we observe no lag phase across protein concentration for all the probes studied, suggesting that the aggregation process follows a linear polymerization reaction. Overall, our study demonstrates that the amyloid-like assembly forms in multiple steps, which is also supported by the temperature dependence of the kinetics.</p>
10aaggregation pathway10aamyloid-like assembly10aconformational conversion10akinetics10aoligomers1 aPillai, Meenakshi1 aJha, Santosh, Kumar  uhttps://library.ncl.res.in/content/multistep-molecular-mechanism-amyloid-aggregation-nucleic-acid-binding-domain-tdp-4301942nas a2200229   4500008004100000022001400041245012400055210006900179260000800248300001100256490000800267520108800275653003101363653001701394653002901411653002301440653002901463653004001492100002401532700002401556856013201580       2023                        eng d  a0301-462200aShapeshifter TDP-43: molecular mechanism of structural polymorphism, aggregation, phase separation and their modulators0 aShapeshifter TDP43 molecular mechanism of structural polymorphis  cAPR  a1069720 v2953 a<p>
	TDP-43 is a nucleic acid-binding protein that performs physiologically essential functions and is known to un-dergo phase separation and aggregation during stress. Initial observations have shown that TDP-43 forms het-erogeneous assemblies, including monomer, dimer, oligomers, aggregates, phase-separated assemblies, etc. However, the significance of each assembly of TDP-43 concerning its function, phase separation, and aggregation is poorly known. Furthermore, how different assemblies of TDP-43 are related to each other is unclear. In this review, we focus on the various assemblies of TDP-43 and discuss the plausible origin of the structural het-erogeneity of TDP-43. TDP-43 is involved in multiple physiological processes like phase separation, aggregation, prion-like seeding, and performing physiological functions. However, the molecular mechanism behind the physiological process performed by TDP-43 is not well understood. The current review discusses the plausible molecular mechanism of phase separation, aggregation, and prion-like propagation of TDP-43.</p>
10aAmyloid versus non-amyloid10aCoacervation10aEnvironmental conditions10aPrion-like seeding10aStructural heterogeneity10aThermodynamic and kinetic mechanism1 aDoke, Abhilasha, A.1 aJha, Santosh, Kumar  uhttps://library.ncl.res.in/content/shapeshifter-tdp-43-molecular-mechanism-structural-polymorphism-aggregation-phase-separation02235nas a2200217   4500008004100000022001400041245012000055210006900175260000900244300001100253490000900264520144800273653002901721653002501750653003401775653001101809653002801820100001701848700002401865856012801889       2023                        eng d  a1570-963900aThermodynamic modulation of folding and aggregation energy landscape by DNA binding of functional domains of TDP-430 aThermodynamic modulation of folding and aggregation energy lands  cJUL   a1409160 v18713 a<p>
	TDP-43 is a vital nucleic acid binding protein which forms stress-induced aberrant aggregates in around 97% cases of ALS, a fatal neurodegenerative disease. The functional tandem RRM domain of the protein (TDP-43tRRM) has been shown to undergo amyloid-like aggregation under stress in a pH-dependent fashion. However, the underlying thermodynamic and molecular basis of aggregation and how the energy landscape of folding, stability, and aggregation are coupled and modulated by nucleic acid binding is poorly understood. Here, we show that the pH stress thermodynamically destabilizes the native protein and systematically populates the unfoldedlike aggregation-prone molecules which leads to amyloid-like aggregation. We observed that specific DNA binding inhibits aggregation and populates native-like compact monomeric state even under low-pH stress as measured by circular dichroism, ANS binding, size exclusion chromatography, and transmission electron microscopy. We show that DNA-binding thermodynamically stabilizes and populates the native state even under stress and reduces the population of unfolded-like aggregation-prone molecules which leads to systematic aggregation inhibition. Our results suggest that thermodynamic modulation of the folding and aggregation energy landscape by nucleic-acid-like molecules could be a promising approach for effective therapeutic intervention in TDP-43-associated proteinopathies.</p>
10aAmyloid-like aggregation10aNucleic acid binding10aRNA recognition motifs (RRMs)10aTDP-4310aThermodynamic stability1 aPatni, Divya1 aJha, Santosh, Kumar  uhttps://library.ncl.res.in/content/thermodynamic-modulation-folding-and-aggregation-energy-landscape-dna-binding-functional00617nas a2200169   4500008004100000022001400041245007800055210006900133260000800202300001600210490000600226653002400232653003800256100002200294700002400316856010700340       2024                        eng d  a2470-134300aConformational enigma of TDP-43 misfolding in neurodegenerative disorders0 aConformational enigma of TDP43 misfolding in neurodegenerative d  cSEP  a40286-402970 v910aDNA-BINDING PROTEIN10aFRONTOTEMPORAL LOBAR DEGENERATION1 aPillai, Meenakshi1 aJha, Santosh, Kumar  uhttps://library.ncl.res.in/content/conformational-enigma-tdp-43-misfolding-neurodegenerative-disorders02962nas a2200277   4500008004100000022001400041245013400055210006900189260000800258300001400266490000700280520195400287653002502241653002702266653002802293653003202321653002502353653003002378653003602408653002302444100002302467700002402490700002402514700001502538856013102553       2024                        eng d  a1948-719300aDeciphering the monomeric and dimeric conformational landscapes of the full-length TDP-43 and the impact of the C-terminal domain0 aDeciphering the monomeric and dimeric conformational landscapes   cNOV  a4305-43210 v153 a<p>
	The aberrant aggregation of TAR DNA-binding protein 43 kDa (TDP-43) in cells leads to the pathogenesis of multiple fatal neurodegenerative diseases. Decoding the proposed initial transition between its functional dimeric and aggregation-prone monomeric states can potentially design a viable therapeutic strategy, which is presently limited by the lack of structural detail of the full-length TDP-43. To achieve a complete understanding of such a delicate phase space, we employed a multiscale simulation approach that unearths numerous crucial features, broadly summarized in two categories: (1) state-independent features that involve inherent chain collapsibility, rugged polymorphic landscape dictated by the terminal domains, high beta-sheet propensity, structural integrity preserved by backbone-based intrachain hydrogen bonds and electrostatic forces, the prominence of the C-terminal domain in the intrachain cross-domain interfaces, and equal participation of hydrophobic and hydrophilic (charged and polar) residues in cross-domain interfaces; and (2) dimerization-modulated characteristics that encompass slower collapsing dynamics, restricted polymorphic landscape, the dominance of side chains in interchain hydrogen bonds, the appearance of the N-terminal domain in the dimer interface, and the prominence of hydrophilic (specifically polar) residues in interchain homo- and cross-domain interfaces. In our work, the ill-known C-terminal domain appears as the most crucial structure-dictating domain, which preferably populates a compact conformation with a high beta-sheet propensity in its isolated state stabilized by intrabackbone hydrogen bonds, and these signatures are comparatively faded in its integrated form. Validation of our simulated observables by a complementary spectroscopic approach on multiple counts ensures the robustness of the computationally predicted features of the TDP-43 aggregation landscape.</p>
10achain collapsibility10adomain-wisefluctuation10aelectrostatic dominance10ahydrogen bond switchability10along-range crosstalk10apersistent beta-character10aprotagonistic C-terminal domain10arugged phase space1 aTammara, Vaishnavi1 aDoke, Abhilasha, A.1 aJha, Santosh, Kumar1 aDas, Atanu  uhttps://library.ncl.res.in/content/deciphering-monomeric-and-dimeric-conformational-landscapes-full-length-tdp-43-and-impact-c02380nas a2200265   4500008004100000022001400041245016600055210006900221260000900290300001400299490000700313520144700320653002801767653002501795653002901820653001301849653001401862653001101876100001701887700002201904700002201926700001601948700002401964856012601988       2024                        eng d  a1948-719300aDNA-mediated formation of phase-separated coacervates of the nucleic acid-binding domain of TAR DNA-binding protein (TDP-43) prevents its amyloid-like misfolding0 aDNAmediated formation of phaseseparated coacervates of the nucle  cOCT   a4105-41220 v153 a<p>
	Sequestration of protein molecules and nucleic acids to stress granules is one of the most promising strategies that cells employ to protect themselves from stress. In vitro, studies suggest that the nucleic acid-binding domain of TDP-43 (TDP-43tRRM) undergoes amyloid-like aggregation to beta-sheet-rich structures in low pH stress. In contrast, we observed that the TDP-43tRRM undergoes complex coacervation in the presence of ssDNA to a dense and light phase, preventing its amyloid-like aggregation. The soluble light phase consists of monomeric native-like TDP-43tRRM. The microscopic data suggest that the dense phase consists of spherical coacervates with limited internal dynamics. We performed multiparametric analysis by employing various biophysical techniques and found that complex coacervation depends on the concentration and ratio of the participating biomolecules and is driven by multivalent interactions. The modulation of these forces due to environmental conditions or disease mutations regulates the extent of coacervation, and the weakening of interactions between TDP-43tRRM and ssDNA leads to amyloid-like aggregation of TDP-43tRRM. Our results highlight a competition among the native state, amyloid-like aggregates, and complex coacervates tuned by various environmental factors. Together, our results illuminate an alternate function of TDP-43tRRM in response to pH stress in the presence of the ssDNA.</p>
10aamyloid-likeaggregation10acomplex coacervation10aDNA-protein interactions10aDynamics10apH stress10aTDP-431 aPatni, Divya1 aPatil, Anjali, D.1 aKirmire, Mona, S.1 aJha, Anjali1 aJha, Santosh, Kumar  uhttps://library.ncl.res.in/content/dna-mediated-formation-phase-separated-coacervates-nucleic-acid-binding-domain-tar-dna02190nas 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<p>
	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.</p>
1 aDoke, Abhilasha, A.1 aJha, Santosh, Kumar  uhttps://library.ncl.res.in/content/electrostatics-choreographs-aggregation-dynamics-full-length-tdp-43-monomeric-amyloid02443nas a2200157   4500008004100000022001400041245014500055210006900200260000800269300001400277490000700291520180800298100002402106700002402130856013102154       2024                        eng d  a0006-296000aIdentification of a hidden, highly aggregation-prone intermediate of full-length TDP-43 that triggers its misfolding and amyloid aggregation0 aIdentification of a hidden highly aggregationprone intermediate   cNOV  a3100-31130 v633 a<p>
	In cells, TDP-43 is a crucial protein that can form harmful amyloid aggregates linked to fatal and incurable human neurodegenerative disorders. Normally, TDP-43 exists in a smaller soluble native state that prevents aggregation. However, aging and stress can destabilize this native state, leading to the formation of disease-causing amyloid aggregates via the formation of partially unfolded, high-energy intermediates with a greater tendency to aggregate. These intermediates are crucial in the early stages of amyloid formation and are challenging to study due to their low stability. Understanding the structure of these early aggregation-prone states of TDP-43 is essential for designing effective treatments for TDP-43 proteinopathies. Targeting these initial intermediates could be more effective than focusing on fully formed amyloid aggregates. By disrupting the aggregation process at this early stage, we may be able to prevent the progression of diseases related to TDP-43 aggregation. Hence, we decided to uncover the hidden, high-energy intermediates in equilibrium with the native states of TDP-43 by modulating the thermodynamic stability of the soluble native dimer (N form) and monomeric molten globular state (MG form) of full-length TDP-43. The thermodynamic modulation performed in the current study successfully revealed the highly aggregation-prone intermediate of full-length TDP-43, i.e., PUF. Moreover, we observed that along with high aggregation propensity, the aggregation kinetics and mechanisms of PUF differ from previously identified intermediates of full-length TDP-43 (the MG and I forms). The information regarding the initial aggregation-prone state of full-length TDP-43 could lead to therapies for amyloid diseases by halting early protein aggregation.</p>
1 aDoke, Abhilasha, A.1 aJha, Santosh, Kumar  uhttps://library.ncl.res.in/content/identification-hidden-highly-aggregation-prone-intermediate-full-length-tdp-43-triggers-its02274nas a2200133   4500008004100000022001400041245016200055210006900217260000800286520167500294100002001969700002401989856012702013       2024                        eng d  a1520-610600aMulti-site red-edge excitation shift reveals the residue-specific solvation dynamics during the native to amyloid-like transition of an amyloidogenic protein0 aMultisite rededge excitation shift reveals the residuespecific s  cDEC3 a<p>
	Changes in water-protein interactions are crucial for proteins to achieve functional and nonfunctional conformations during structural transitions by modulating local stability. Amyloid-like protein aggregates in deteriorating neurons are hallmarks of neurodegenerative disorders. These aggregates form through significant structural changes, transitioning from functional native conformations to supramolecular cross-beta-sheet structures via misfolded and oligomeric intermediates in a multistep process. However, the site-specific dynamics of water molecules from the native to misfolded conformations and further to oligomeric and compact amyloid structures remain poorly understood. In this study, we used the fluorescence method known as red-edge excitation shift (REES) to investigate the solvation dynamics at specific sites in various equilibrium conformations en route to the misfolding and aggregation of the functional domain of the TDP-43 protein (TDP-43tRRM). We generated three single tryptophan-single cysteine mutants of TDP-43tRRM, with the cysteines at different positions and tryptophan at a fixed position. Each sole cysteine was fluorescently labeled and used as a site-specific fluorophore along with the single tryptophan, creating four monitorable sites for REES studies. By investigating the site-specific extent of REES, we developed a residue-specific solvation dynamics map of TDP-43tRRM during its misfolding and aggregation. Our observations revealed that solvation dynamics progressively became more rigid and heterogeneous to varying extents at different sites during the transition from native to amyloid-like conformations.</p>
1 aMore, Sonal, R.1 aJha, Santosh, Kumar  uhttps://library.ncl.res.in/content/multi-site-red-edge-excitation-shift-reveals-residue-specific-solvation-dynamics-during02704nas a2200253   4500008004100000022001400041245012700055210006900182260000800251300001400259490000700273520183800280653002002118653002702138653002202165653001902187653002402206653001402230100002202244700002202266700001502288700002402303856012302327       2024                        eng d  a1948-719300aPathological mutations D169G and P112H electrostatically aggravate the amyloidogenicity of the functional domain of TDP-430 aPathological mutations D169G and P112H electrostatically aggrava  cNOV  a4267-42830 v153 a<p>
	Aggregation of TDP-43 is linked to the pathogenesis of many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Notably, electrostatic point mutations such as D169G and P112H, located within the highly conserved functional tandem RNA recognition motif (RRM) domains of the TDP-43 protein (TDP-43tRRM), have been identified in diseased patients as well. In this study, we address how the electrostatic mutations alter both the native state stability and aggregation propensity of TDP-43tRRM. The mutants D169G and P112H show increased chemical stability compared to the TDP-43tRRM at physiological pH. However, at low pH, both the mutants undergo a conformational change to form amyloid-like fibrils, though with variable rates-the P112H mutant being substantially faster than the other two sequences (TDP-43tRRM and D169G mutant) showing comparable rates. Moreover, among the three sequences, only the P112H mutant undergoes a strong ionic strength-dependent aggregability trend. These observations signify the substantial contribution of the excess charge of the P112H mutant to its unique aggregation process. Complementary simulated observables with atomistic resolution assign the experimentally observed sequence-, pH-, and ionic strength-dependent aggregability pattern to the degree of thermal lability of the mutation site-containing RRM1 domain and its extent of dynamical anticorrelation with the RRM2 domain whose combination eventually dictate the extent of generation of aggregation-prone partially unfolded conformational ensembles. Our choice of a specific charge-modulated pathogenic mutation-based experiment-simulation-combination approach unravels the otherwise hidden residue-wise contribution to the individual steps of this extremely complicated multistep aggregation process.</p>
10aamyloid fibrils10aconformational changes10adisease mutations10aelectrostatics10aProtein aggregation10astability1 aPillai, Meenakshi1 aPatil, Anjali, D.1 aDas, Atanu1 aJha, Santosh, Kumar  uhttps://library.ncl.res.in/content/pathological-mutations-d169g-and-p112h-electrostatically-aggravate-amyloidogenicity02594nas a2200229   4500008004100000022001400041245014700055210006900202260000800271300001400279490000700293520172200300653002902022653004402051653003302095653001802128100002402146700002202170700001602192700002402208856013202232       2026                        eng d  a1948-719300aStructural and mechanistic heterogeneity of the phase separation and aggregation of full-length TDP-43 is governed by environmental conditions0 aStructural and mechanistic heterogeneity of the phase separation  cAPR  a1362-13740 v173 a<p>
	TAR DNA-binding protein 43 (TDP-43) is an essential physiological protein implicated in several fatal neurodegenerative disorders. Interestingly, the nature of TDP-43 aggregates varies across patients and disease conditions, suggesting an underlying heterogeneity in its self-assembly behavior. In this study, we investigated two native-like states of full-length TDP-43: the native dimer (N form) and the native-like oligomer (O form). These are compact, folded states with similar secondary structures but differ in size. We found that the N and O forms respond differently to external perturbations and form distinct self-assemblies under stress conditions. Under electrostatic stress, both N and O forms undergo phase separation but produce condensates with markedly different morphologies and dynamics. The underlying mechanisms driving their phase separation are different. Under thermal stress, both forms convert into amyloid aggregates, but again with clearly different morphologies, biochemical properties, and aggregation pathways. These results demonstrate that multiple conformations of TDP-43 respond to distinct perturbations by assembling into structurally and mechanistically different higher-order assemblies. Our findings highlight how the interplay among the structural state, solvation environment, and self-assembly mechanism governs the heterogeneity of TDP-43 assemblies, offering new insights into their physiological roles and pathological relevance. This study suggests that the heterogeneity observed in patients associated with TDP-43 aggregation may arise from differences in the cellular stresses experienced by the protein and the corresponding assembly mechanisms engaged.</p>
10aAmyloid-like aggregation10amolecular mechanism of phase separation10anative-like phase separation10aSelf-assembly1 aDoke, Abhilasha, A.1 aKirmire, Mona, S.1 aJha, Anjali1 aJha, Santosh, Kumar  uhttps://library.ncl.res.in/content/structural-and-mechanistic-heterogeneity-phase-separation-and-aggregation-full-length-tdp-43