@article { ISI:000367968700030, title = {Evidence for dry molten globule-like domains in the pH-induced equilibrium folding intermediate of a multidomain protein}, journal = {Journal of Physical Chemistry Letters}, volume = {7}, number = {1}, year = {2016}, month = {JAN}, pages = {173-179}, publisher = {AMER CHEMICAL SOC}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, abstract = {

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

}, issn = {1948-7185}, doi = {10.1021/acs.jpclett.5b02545}, author = {Acharya, Nirbhik and Mishra, Prajna and Jha, Santosh Kumar} } @article {44156, title = {Alternatively packed dry molten globule-like intermediate in the native state ensemble of a multidomain protein}, journal = {Journal of Physical Chemistry B}, volume = {121}, year = {2017}, month = {OCT}, pages = {9336-9347}, type = {Article}, abstract = {It 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.}, doi = {10.1021/acs.jpcb.7b07032}, author = {Mishra, Prajna and Jha, Santosh Kumar} } @article {44095, title = {Dry molten globule-like intermediate during the base-induced unfolding of a multidomain protein}, journal = {Physical Chemistry Chemical Physics}, volume = {19}, year = {2017}, month = {NOV}, pages = {30207-30216}, type = {Article}, abstract = {The 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.}, doi = {10.1039/c7cp06614g}, author = {Acharya, Nirbhik and Mishra, Prajna and Jha, Santosh Kumar} } @article {46274, title = {Dry molten globule-like intermediate during the base-induced unfolding of a multidomain protein (vol 19, pg 20307, 2017)}, journal = {Physical Chemistry Chemical Physics}, volume = {21}, year = {2019}, month = {SEP}, pages = {20499-20499}, type = {Correction}, doi = {10.1039/c9cp90219h}, author = {Acharya, Nirbhik and Mishra, Prajna and Jha, Santosh Kumar} } @article {46199, title = {Folding and aggregation energy landscapes of tethered RRM domains of human TDP-43 are coupled via a metastable molten globule-like oligomer}, journal = {Biochemistry}, volume = {58}, year = {2019}, month = {FEB }, pages = {608-620}, type = {Article}, abstract = {

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.

}, issn = {0006-2960}, doi = {10.1021/acs.biochem.8b01013}, author = {Pillai, Meenakshi and Jha, Santosh Kumar} } @article {46196, title = {Slow motion protein dance visualized using red-edge excitation shift of a buried fluorophore}, journal = {Journal of Physical Chemistry B}, volume = {123}, year = {2019}, month = {FEB}, pages = {1256-1264}, type = {Article}, abstract = {

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.

}, issn = {1520-6106}, doi = {10.1021/acs.jpcb.8b11151}, author = {Mishra, Prajna and Jha, Santosh Kumar} } @article {46988, title = {Early metastable assembly during the stress-induced formation of worm-like amyloid fibrils of nucleic acid binding domains of TDP-43}, journal = {Biochemistry}, volume = {59}, year = {2020}, month = {JAN}, pages = {315-328}, type = {Article}, abstract = {

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 {\textquoteleft}{\textquoteleft}low-pH form{\textquoteright}{\textquoteright} or the {\textquoteleft}{\textquoteleft}L form{\textquoteright}{\textquoteright}). 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 {\textquoteleft}{\textquoteleft}beta form{\textquoteright}{\textquoteright} 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.

}, issn = {0006-2960}, doi = {10.1021/acs.biochem.9b00780}, author = {Pillai, Meenakshi and Jha, Santosh Kumar} } @article {47344, title = {pH-dependent protein stability switch coupled to the perturbed pKa of a single ionizable residue}, journal = {Biophysical Chemistry}, volume = {274}, year = {2021}, month = {JUL}, pages = {106591}, type = {Article}, abstract = {

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.

}, keywords = {electrostatic interactions, Fluorescence spectroscopy, Thermodynamic stability, Unfolded proteins}, issn = {0301-4622}, doi = {10.1016/j.bpc.2021.106591}, author = {Mishra, Prajna and Patni, Divya and Jha, Santosh Kumar} } @article {47663, title = {Protonation-deprotonation switch controls the amyloid-like misfolding of nucleic-acid-binding domains of TDP-43}, journal = {Journal of Physical Chemistry B}, volume = {125}, year = {2021}, month = {AUG }, pages = {8383-8394}, type = {Article}, abstract = {Nutrient 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.}, issn = {1520-6106}, doi = {10.1021/acs.jpcb.1c03262}, author = {Patni, Divya and Jha, Santosh Kumar} } @article {48016, title = {Dry molten globule-like intermediates in protein folding, function, and disease}, journal = {Journal of Physical Chemistry B}, volume = {126}, year = {2022}, month = {NOV}, pages = {8614-8622}, type = {Review}, abstract = {

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.

}, issn = {1520-6106}, doi = {10.1021/acs.jpcb.2c04991}, author = {Acharya, Nirbhik and Jha, Santosh Kumar} } @article {47825, title = {Native state conformational heterogeneity in the energy landscape of protein folding}, journal = {Biophysical Chemistry}, volume = {283}, year = {2022}, month = {APR}, pages = {106761}, type = {Article}, abstract = {The 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.}, keywords = {Conformational heterogeneity, Dry molten globules, Energy landscape model, Native state, Side-chain packing}, issn = {0301-4622}, doi = {10.1016/j.bpc.2022.106761}, author = {Mishra, Prajna and Jha, Santosh Kumar} } @article {48494, title = {Electrostatic modulation of intramolecular and intermolecular interactions during the formation of an amyloid-like assembly}, journal = {Biochemistry}, volume = {62}, year = {2023}, month = {MAY}, pages = {1890-1905}, type = {Article}, abstract = {

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.

}, issn = {0006-2960}, doi = {10.1021/acs.biochem.3c00014}, author = {Pillai, Meenakshi and Das, Atanu and Jha, Santosh Kumar} } @article {48492, title = {Shapeshifter TDP-43: molecular mechanism of structural polymorphism, aggregation, phase separation and their modulators}, journal = {Biophysical Chemistry}, volume = {295}, year = {2023}, month = {APR}, pages = {106972}, type = {Review}, abstract = {

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.

}, keywords = {Amyloid versus non-amyloid, Coacervation, Environmental conditions, Prion-like seeding, Structural heterogeneity, Thermodynamic and kinetic mechanism}, issn = {0301-4622}, doi = {10.1016/j.bpc.2023.106972}, author = {Doke, Abhilasha A. and Jha, Santosh Kumar} } @article {48725, title = {Thermodynamic modulation of folding and aggregation energy landscape by DNA binding of functional domains of TDP-43}, journal = {Biochimica Et Biophysica Acta-Proteins and Proteomics}, volume = {1871}, year = {2023}, month = {JUL }, pages = {140916}, type = {Article}, abstract = {

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.

}, keywords = {Amyloid-like aggregation, Nucleic acid binding, RNA recognition motifs (RRMs), TDP-43, Thermodynamic stability}, issn = {1570-9639}, doi = {10.1016/j.bbapap.2023.140916}, author = {Patni, Divya and Jha, Santosh Kumar} }