Research Summary

The overarching goal of the Mukhopadhyay lab is to investigate a wide range of fascinating aspects of intrinsically disordered proteins/regions (IDPs/IDRs) that undergo biomolecular condensation via liquid-liquid phase separation (LLPS) and amyloid formation. Traditionally, protein function was thought to be dependent on a unique well-defined 3D structure that is encoded by the amino acid sequence. However, intense investigations over the past two decades have revealed that a large fraction of the proteome of an organism consists of polypeptide segments that lack a well-defined structure under physiological conditions. These proteins can be fully disordered (IDPs) and/or can contain regions that are disordered (IDRs). IDPs challenge the tenets of the traditional structure-function paradigm and exist as a rapidly interconverting conformational ensemble rather than a single well-defined structure. The conformational plasticity allows them to adopt different structures depending on their binding partners. Therefore, a single polypeptide sequence is capable of accomplishing a range of functions. It proposed that retaining disorder is an evolutionary strategy that allows complex functions within a compact genome of higher organisms. Additionally, the dysfunction of many IDPs is associated with a range of deadly diseases such as Alzheimer's and Parkinson's diseases and cancers. In our research, we utilize a diverse array of tools and concepts from physical chemistry to chemical biology to biophysics and molecular biology to study the intriguing behavior of IDPs. We are interested in delineating the fundamental mechanism by which liquid-like condensates are formed and dissipated. We are also studying the liquid-to-solid phase transition into amyloid-like aggregates that are associated with deadly neurodegenerative diseases such as Alzheimer's, amyotrophic lateral sclerosis (ALS), and so forth. We are interested in the following specific aspects of IDPs:

• Conformational characteristics of IDPs and IDRs

• Biomolecular condensates

• Mechanism of amyloid formation

• Functional prions and amyloids

• Interactions of IDPs with lipid membrane

• Hydration water in IDPs

• Development and adaptation of new methodologies

See below for details on these sections.

Conformational characteristics of IDPs and IDRs

As stated above, IDPs exist as a structural ensemble comprising shapeshifting polypeptide chains constantly undergoing conformational fluctuations on a wide range of timescales. In our lab, we utilize various existing and emerging biophysical tools such as circular dichroism (CD), dynamic light scattering (DLS), steady-state and time-resolved fluorescence, and Raman spectroscopy to characterize the conformational plasticity, heterogeneity, distributions, dimension, and dynamics. Using site-specific fluorescence depolarization kinetics by picosecond time-resolved fluorescence anisotropy measurements, we are studying the inherent backbone dihedral dynamics associated with IDPs. These unique measurements also permit us to detect local structural propensity, long-range correlated motions, chain expansion and compaction, oligomerization, and amyloid formation.  

Biomolecular condensation via liquid-liquid phase separation (LLPS)

Components in cells are compartmentalized by membrane-bound organelles. A growing body of intense current research is beginning to reveal that in addition to these conventional organelles, cells also contain a variety of membrane-less organelles that are formed via biomolecular condensation or liquid-liquid phase separation (LLPS) of IDPs/IDRs/RNAs. These on-demand liquid organelles spatiotemporally control cellular biochemistry. Such types of condensates or coacervates of much simpler molecules may have been involved in prebiotic chemistry and the chemical origin of life. We are interested in delineating the fundamental mechanism by which these liquid-like condensates are formed and dissipated. Our research aims at decoding the inner workings of these liquid droplets derived from a wide range of IDPs/IDRs such as FUS, tau, yeast prion, Pmel repeat domain, N-terminal IDR of the prion protein (PrP), and a few bacterial and viral proteins. We are also studying the liquid-to-solid phase transition into amyloid-like aggregates that are associated with deadly neurodegenerative diseases such as Alzheimer's, amyotrophic lateral sclerosis (ALS), and so forth. 

Prion protein biology

Prion diseases are a family of fatal neurodegenerative disorders that correspond to transmissible spongiform encephalopathies and are manifested as infectious, genetic, and sporadic diseases in both humans and animals. The causative agent of prion diseases is proteinaceous infectious particles comprising amyloid-like aggregates of a misfolded isoform of a membrane-anchored protein called the prion protein (PrP). The key step in this pathogenesis is the misfolding of PrP from a largely α-helical normal cellular form (PrPC) to a predominantly b-sheet-rich, aggregated, protease-resistant, scrapie form (PrPSc), which self-replicates via the autocatalytic self-templating mechanism. Our previous results captured the key molecular events that govern the course of amyloid formation from PrP in the presence of a lipid membrane mimetic. We have been able to detect and characterize two distinct forms of oligomeric intermediates. Our results show that highly structured oligomers are benign off-pathway intermediates, whereas, structurally-labile on-pathway oligomers serve as amyloidogenic precursors. These amyloid-competent intermediates conformationally mature into polymorphic amyloid nanostructures that exhibit profound toxicity to mammalian cells. Additionally, by using excitation energy migration via homo-FRET, we were able to monitor lipid membrane-mediated oligomerization of PrP. We have also studied interactions of PrP with conformationally-distinct oligomers of amyloid-b peptides that have been linked to Alzheimer’s disease. Our latest results demonstrate that aggregation of PrP can also occur via LLPS-mediated non-canonical pathway. We are currently investigating the role of phase transitions in promoting self-replicating amyloid formation.

Interactions of IDPs with lipid membrane

Interactions of IDPs with lipid membranes have been associated with both function and disease. Many IDPs undergo binding-induced folding in the presence of membranes. For instance, α-synuclein is known to undergo folding upon binding to negatively charged lipids. In order to decipher the structural organization of the lipid-protein assembly as well as to unequivocally define the localization of the protein on the membrane surface, we have recorded along the polypeptide chain length a variety of sensitive site-specific fluorescence readouts including the red-edge excitation shift which originates from a highly ordered micro-environment such as the membrane-water interface comprising a thin (~15 Å) layer of motionally restricted water molecules. The results from our experiments allowed us to construct a dynamic hydration map that depicts the intriguing structural arrangement of α-synuclein bound to the membrane surface. Additionally, using time-resolved fluorescence anisotropy measurements, we were able to record the translational diffusion of α-synuclein on the membrane surface. Such structural and dynamical modulations of α-synuclein on the membrane could potentially be related to its physiological function as well as to the onset of Parkinson’s disease.

Functional prions and amyloids

Nature utilizes prions and amyloids for beneficial purposes from bacteria to humans. Unlike pathological amyloids, functional amyloids have evolved to tightly regulate toxicity. Curli, a functional amyloid protein, found on the extracellular matrix of E.coli can bypass the toxic consequences and allow curli biogenesis to occur on the bacterial lipopolysaccharide (LPS) membrane. We elucidated the key role of the membrane in modulating the aggregation of curli subunits. Our results showed that the electrostatic interaction between the protein and LPS promotes protein-protein interaction that results in efficiently sequestering the amyloid fold on the membrane surface without significant accumulation of toxic oligomeric intermediates. Our study hinted at a plausible strategy by which bacteria can overcome the toxicity during curli biogenesis. Additionally, we are also studying the self-perpetuating conformational conversion of a yeast prion protein, Sup35. Our latest results demonstrate an intriguing interplay of interactions between yeast prion and heat-shock proteins resulting in modulation in amyloid formation. We also elucidated the molecular origin of an unusual biphasic assembly process of a pH-responsive, intrinsically disordered repeat domain of a melanosomal protein (Pmel) that regulates the melanin synthesis in melanosomes. Our findings revealed that this unusual assembly kinetics is greatly influenced by an intriguing dual Hofmeister effect. At mildly acidic pH, typical of melanosomes, highly amyloidogenic oligomeric units assemble into metastable, dendritic, fractal networks, and this self-assembly process follows the forward Hofmeister series. However, the subsequent condensation and maturation of fractal networks into highly-ordered amyloid fibrils follow an inverse Hofmeister series due to fragmentation coupled with secondary nucleation processes. Our results showed that Hofmeister ions exert a strong influence on the aggregation kinetics and nanoscale morphology during amyloid assembly. Additionally, our intriguing observation reveals the unique role of Hofmeister ions in modulating the autocatalytic amplification process. These findings have broad implications in complex amyloidogenesis pathways of a multitude of intrinsically disordered proteins involved in disease and physiology.

Hydration water in IDPs

Water forms the natural matrix of biomolecules and drives a wide variety of crucial interactions within the cell. The hydration water layer, termed as ‘biological water’, present in the vicinity of proteins shares an intimate relationship with protein functions and plays critical roles in folding, binding, assembly, regulation, and catalysis. However, little is known about the behavior of water in IDPs. Our results showed that collapsed IDP globules prefer chain-chain interactions as opposed to chain-solvent interactions and possess a highly ordered water network akin to water clusters found under nanoconfinement. Subsequently, using site-specific ultrafast experiments, we were able to construct the time-resolved snapshots of water molecules in the hydration layer of a-synuclein. The hydration map revealed site-specific solvation dynamics and the presence of quasi-bound water molecules around the amyloidogenic segment of the protein. Our results on solvation dynamics demonstrated that amyloid formation is associated with extensive rearrangement and rigidification of the water structure around the polypeptide chain and the entropic release plays a pivotal role in directing the course of amyloid assembly.

Development and adaptation of new methodologies

A diverse array of existing and emerging spectroscopic and microscopic tools is being developed, adapted, and utilized in our lab to study the behavior of IDPs and the mechanism of phase separation and amyloid formation. The fluorescence depolarization by following the picosecond time-resolved fluorescence anisotropy has been one of the most utilized methodologies in our lab. Using this approach, we have been able to discern various essential modes of chain dynamics (local, segmental/backbone dihedral, long-range, global dynamics) of IDPs and their assemblies on the picosecond-nanosecond timescale. Recently, using anisotropy decay measurements, we have been able to probe energy migration via homo-FRET that illuminates the inner workings of a-synuclein amyloids. These unique and sensitive measurements yielded a 2D-proximity correlation map of a large number of intermolecular distances and detected unique, previously unobserved cross-talks between the hetero-terminal, disordered, fuzzy, inter-protofilament interfaces of the parallel-in-register amyloid spines. A relay of transient interactions via dense, dynamic, fuzzy, and mesh-like network enclosing the ordered amyloid-core can modulate the higher-order packing and nanoscale morphology which governs the amyloid polymorphism responsible for distinct strain-specific disease phenotypes in pathological amyloids. We are also involved in developing and adapting nano-biophysical tools, super-resolution near-field scanning optical microscopy, and ultrasensitive Raman spectroscopic techniques to elucidate the inner workings of amyloids and phase-separated liquid droplets.