Welcome to The Jha Lab
1.Characterization of the nature and role of the dry molten globule-like intermediate states during protein folding and misfolding reactions
In this project, Dr. Nirbhik Acharya investigated dry molten globule-like intermediates in protein folding, unfolding, and misfolding. Traditional views suggest side-chain disruptions and hydrophobic core solvation during unfolding, but an alternative model proposes that side-chain packing first loosens to form a dry molten globule-like state (DMG), followed by water penetration to create a wet molten globule-like state (WMG).
In the first part, he showed DMG-like intermediates during pH-induced unfolding of human serum albumin (HSA), highlighting base-induced expansion in the inter-domain region and non-cooperativity in the native-DMG transition.
In subsequent work, he reported amyloid-like aggregation initiation from a DMG-like state in the neurodegenerative disease-related protein TDP-43tRRM. His research characterises the amyloidogenic DMG-like state and maps amyloid fibril cores using high-resolution techniques.
2.Thermodynamic and spectroscopic characterization of near-native states in protein folding
In this study, Dr. Prajna Mishra explored a protein's native state ensemble using spectroscopic probes and thermodynamics. Nature employs flexibility and dynamics for efficient protein function. Her research supported the conformational selection model, highlighting the significance of side-chain arrangements in protein stability.
In the first part, she found an equilibrium intermediate (I) in HSA with dry molten globule-like features. This I state displayed alternative packing of a tryptophan residue, N-like secondary structure, and played a role in protein stability.
In the next part, she used Red-Edge Excitation Shift to observe slow-motion protein dynamics in a buried fluorophore. Her findings revealed higher entropy conformations and diverse side-chain packing in the core, vital for various protein functions.
In collaboration with Dr. Divya Patni, they discovered a pH-dependent protein stability switch linked to a single buried ionizable residue's altered pKa. They found a pH-dependent protein stability switch linked to a buried ionizable residue's altered pKa in Human Serum Albumin (HSA). The thiol's pKa increased by 0.5 units in the native state and decreased by 1.3 units in the unfolded state, indicating the importance of electrostatic interactions in both states for protein stability.
3.Mapping the energy landscape of amyloid-like aggregation of the nucleic acid binding domain of TDP-43 (tRRM)
In this project Meenakshi Pillai, PhD navigated through the folding and aggregation energy landscape of TDP-43tRRM and provided the molecular mechanism of aggregation of TDP-43-tRRM.
Dr. Meenakshi Pillai studied structural changes in TDP-43-tRRM in response to pH and ionic strength variations. Two intermediates, A and L forms, were identified with disrupted side-chain packing but intact secondary structure. They exhibit wet molten globule characteristics and can transition back to the native state under reduced stress or convert to amyloid-like aggregates (β form) under persistent stress.
In her next project, she examined the transition from the native state to the β form over time, revealing significant structural events, including side-chain disruption, conformational change, and alterations in hydrodynamic radii. The aggregation process exhibited a multi-step sequential model dependent on protein concentration.
She further analyzed the impact of disease-associated mutations D169G and P112H, finding that while they didn't alter the global structure, they influenced stability and aggregation kinetics.
Ionic strength and pH influenced TDP-43-tRRM structure, leading to various species: monomers, dry molten globules, native-like oligomers, amyloid aggregates, and amorphous aggregates. Salt concentration affected the aggregation process, lowering the activation barrier and modulating the transition state
In her last part of project, she mapped the internal core of the aggregates using hydrogen-deuterium exchange and mass spectrometry, identifying protected regions as the core of the amyloid-like aggregates.
4.Thermodynamic regulation of the amyloid-like aggregation of the functional domains of TDP-43
In her project, Dr. Divya Patni investigated the factors driving protein aggregation in TDP-43 proteinopathies, neurodegenerative diseases. She discovered that TDP-43-tRRM transitions from its native state to an amyloid-like form (β form) in response to low pH stress. The pH-induced transition is linked to the protonation of a buried histidine residue at a pKa value of ~4.0, especially histidine 166. Mutating this histidine to glutamine (H166Q) inhibited aggregation, forming a molten globule with native-like secondary structure but disrupted tertiary structure under low pH stress.
In the subsequent part of her work, Dr. Divya Patni examined pH-dependent changes in TDP-43tRRM stability through thermodynamic measurements. It is most stable at pH 6.5-8.0 but becomes less stable below pH 5.0, leading to disruption in tertiary structure and increased misfolding into the β form. She found that binding to ss(TG)6 stabilizes TDP-43tRRM under all pH conditions and prevents it from accessing the aggregation energy landscape under low pH stress, maintaining its secondary and tertiary structure and regulating aggregation.
5.Understanding the molecular mechanism of misfolding and aggregation of full-length TDP-43
A current PhD student, Abhilasha Doke, is working on this project where she aims to understand the influence of environmental conditions on the folding and aggregation energy landscape of full-length TDP-43.
In her initial work, she demonstrated that TDP-43, a protein linked to neurodegenerative diseases, can sense environmental stress and undergo amyloid-like aggregation. She achieved the unique purification of full-length TDP-43 without solubilization tags or mutations. Using thermodynamics, kinetics, and spectroscopic probes, she revealed that TDP-43 has a responsive proteostasis network, maintaining equilibrium between structural states sensitive to environmental changes. Under extreme stress, it can form amyloid-like aggregates through an amyloidogenic intermediate. Notably, native ligands like single-stranded DNA inhibit aggregation and drive equilibrium toward a stable protein-DNA complex.
Currently she is working on understanding the molecular mechanism of aggregation of full-length TDP-43 using different biophysical tools and kinetics as a probe.