@article {87967, title = {Mapping Distinct Sequences of Structure Formation Differentiating Multiple Folding Pathways of a Small Protein.}, journal = {J Am Chem Soc}, year = {2021}, month = {2021 Jan 12}, abstract = {

To determine experimentally how the multiple folding pathways of a protein differ, in the order in which the structural parts are assembled, has been a long-standing challenge. To resolve whether structure formation during folding can progress in multiple ways, the complex folding landscape of monellin has been characterized, structurally and temporally, using the multisite time-resolved FRET methodology. After an initial heterogeneous polypeptide chain collapse, structure formation proceeds on parallel pathways. Kinetic analysis of the population evolution data across various protein segments provides a clear structural distinction between the parallel pathways. The analysis leads to a phenomenological model that describes how and when discrete segments acquire structure independently of each other in different subensembles of protein molecules. When averaged over all molecules, structure formation is seen to progress as α-helix formation, followed by core consolidation, then β-sheet formation, and last end-to-end distance compaction. Parts of the protein that are closer in the primary sequence acquire structure before parts separated by longer sequence.

}, issn = {1520-5126}, doi = {10.1021/jacs.0c11097}, author = {Bhatia, Sandhya and Krishnamoorthy, Guruswamy and Udgaonkar, Jayant B} } @article {99931, title = {Microsecond dynamics during the binding-induced folding of an intrinsically disordered protein.}, journal = {J Mol Biol}, year = {2021}, month = {2021 Sep 16}, pages = {167254}, abstract = {

Tau is an intrinsically disordered protein implicated in many neurodegenerative diseases. The repeat domain fragment of tau, tau-K18, is known to undergo a disorder to order transition in the presence of lipid micelles and vesicles, in which helices form in each of the repeat domains. Here, the mechanism of helical structure formation, induced by a phospholipid mimetic, sodium dodecyl sulfate (SDS) at sub-micellar concentrations, has been studied using multiple biophysical probes. A study of the conformational dynamics of the disordered state, using photoinduced electron transfer coupled to fluorescence correlation spectroscopy (PET-FCS) has indicated the presence of an intermediate state, I, in equilibrium with the unfolded state, U. The cooperative binding of the ligand (L), SDS, to I has been shown to induce the formation of a compact, helical intermediate (IL) within the dead time (\~{}37 {\textmu}s) of a continuous flow mixer. Quantitative analysis of the PET-FCS data and the ensemble microsecond kinetic data, suggests that the mechanism of induction of helical structure can be described by a U <-> I <-> IL <-> FL mechanism, in which the final helical state, FL, forms from IL with a time constant of 50-200 {\textmu}s. Finally, it has been shown that the helical conformation is an aggregation-competent state that can directly form amyloid fibrils.

}, issn = {1089-8638}, doi = {10.1016/j.jmb.2021.167254}, author = {Sen, Sreemantee and Kumar, Harish and Udgaonkar, Jayant B} } @article {56128, title = {Mechanistic approaches to understand the prion-like propagation of aggregates of the human tau protein.}, journal = {Biochim Biophys Acta Proteins Proteom}, volume = {1867}, year = {2019}, month = {2019 Apr 12}, pages = {922-932}, abstract = {

The dynamic nature of the tau protein under physiological conditions is likely to be critical for it to perform its diverse functions inside a cell. Under some conditions, this intrinsically disordered protein assembles into pathogenic aggregates that are self-perpetuating, toxic and infectious in nature. The role of liquid-liquid phase separation in the initiation of the aggregation reaction remains to be delineated. Depending on the nature of the aggregate, its structure, and its localization, neurodegenerative disorders with diverse clinical features are manifested. The prion-like mechanism by which these aggregates propagate and spread across the brain is not well understood. Various factors (PTMs, mutations) have been strongly associated with the pathological aggregates of tau. However, little is known about how these factors modulate the pathological properties linked to aggregation. This review describes the current progress towards understanding the mechanism of propagation of tau aggregates.

}, issn = {1878-1454}, doi = {10.1016/j.bbapap.2019.04.004}, author = {Kumar, Harish and Udgaonkar, Jayant B} } @article {31050, title = {Mechanism of aggregation and membrane interactions of mammalian prion protein.}, journal = {Biochim Biophys Acta}, year = {2018}, month = {2018 Mar 04}, abstract = {

The cellular prion protein (PrP), which is present ubiquitously in all mammalian neurons, is normally found to be linked to the cell membrane through a glycosylphosphatidylinositol (GPI) anchor. The conformational conversion of PrPinto misfolded and aggregated forms is associated with transmissible neurodegenerative diseases known as prion diseases. The importance of different misfolded conformations in prion diseases, and the mechanism by which prion aggregates induce neurotoxicity remain poorly understood. Multiple studies have been shown that the toxicity of misfolded prion protein is directly correlated with its ability to interact with and perturb membranes. This review describes the current progress toward understanding prion protein misfolding and aggregation, as well as the interaction of prion protein aggregates with lipid membrane.

}, issn = {0006-3002}, doi = {10.1016/j.bbamem.2018.02.031}, author = {Sabareesan, Ambadi Thody and Mathew, M K and Udgaonkar, Jayant B} } @article {43838, title = {Mechanistic and Structural Origins of the Asymmetric Barrier to Prion-like Cross-Seeding between Tau-3R and Tau-4R.}, journal = {J Mol Biol}, year = {2018}, month = {2018 Sep 26}, abstract = {

The spread and deposition of infectious fibrillar protein aggregates in the brain via a prion-like mechanism, is a critical component in the patho-physiology of various neurodegenerative diseases, including the tauopathies. In tauopathies, two isoforms of tau, containing three and four microtubule binding repeats (MTBRs), are found to aggregate, and the type of isoform present in aggregates, determines the type of tauopathy. Cross-seeding between the two tau isoforms is limited by an asymmetric barrier, similar to the species barrier that restricts prion transmission across species, whose origin has remained unclear. In this study, the growth of the tau fibrils is shown to be describable by a two-step Michaelis-Menten-like model. Delineation of the mechanism as a Michaelis-Menten-like mechanism has enabled a quantitative understanding of the asymmetric seeding barrier that exists between two isoforms of tau, tau-K18 and tau-K19 (which differ in containing four and three MTBRs, respectively), wherein tau-K18 fibrils cannot seed tau-K19 monomer. Furthermore, high-resolution structural analysis of the two isoforms shows that the structural core is more ordered in tau-K19 than in tau-K18. Hence, the current work provides kinetic and structural rationales for asymmetric seeding barriers in general, and for the two tau isoforms in particular.

}, issn = {1089-8638}, doi = {10.1016/j.jmb.2018.09.010}, author = {Kumar, Harish and Udgaonkar, Jayant B} } @article {25254, title = {Modulation of the Extent of Cooperative Structural Change During Protein Folding by Chemical Denaturant.}, journal = {J Phys Chem B}, volume = {121}, year = {2017}, month = {2017 Sep 07}, pages = {8263-8275}, abstract = {

Protein folding and unfolding reactions invariably appear to be highly cooperative reactions, but the structural and sequence determinants of cooperativity are poorly understood. Importantly, it is not known whether cooperative structural change occurs throughout the protein, or whether some parts change cooperatively and other parts change noncooperatively. In the current study, hydrogen exchange mass spectrometry has been used to show that the mechanism of unfolding of the PI3K SH3 domain is similar in the absence and presence of 5 M urea. The data are well described by a four state N <-> IN <-> I2 <-> U model, in which structural changes occur noncooperatively during the N <-> IN and IN <-> I2 transitions, and occur cooperatively during the I2 <-> U transition. The nSrc-loop and RT-loop, as well as β strands 4 and 5 undergo noncooperative unfolding, while β strands 1, 2, and 3 unfold cooperatively in the absence of urea. However, in the presence of 5 M urea, the unfolding of β strand 4 switches to become cooperative, leading to an increase in the extent of cooperative structural change. The current study highlights the relationship between protein stability and cooperativity, by showing how the extent of cooperativity can be varied, using chemical denaturant to alter protein stability.

}, issn = {1520-5207}, doi = {10.1021/acs.jpcb.7b04473}, author = {Jethva, Prashant N and Udgaonkar, Jayant B} } @article {21473, title = {Modulation of the extent of structural heterogeneity in α-synuclein fibrils by the small molecule thioflavin T.}, journal = {J Biol Chem}, year = {2017}, month = {2017 Jul 31}, abstract = {

The transition of intrinsically disordered, monomeric α-synuclein into β-sheet-rich oligomers and fibrils is associated with multiple neurodegenerative diseases. Fibrillar aggregates possessing distinct structures that differ in their toxicity, have been observed in different pathological phenotypes. Understanding the mechanism of formation of various fibril polymorphs with differing cytotoxic effects, is essential for determining how the aggregation reaction could be modulated to favor non-toxic fibrils over toxic fibrils. In this study, two morphologically different α-synuclein fibrils, one helical and the other ribbon-like, are shown to form together. Surprisingly, a widely-used small molecule for probing aggregation reactions, thioflavin T (ThT), is found to tune the structural heterogeneity found in the fibrils. The ribbon-like fibrils formed in the presence of ThT are found to have a longer structural core than do the helical fibrils formed in the absence of ThT. The ribbon-like fibrils are also more toxic to cells. By facilitating the formation of ribbon-like fibrils over helical fibrils, ThT reduces the extent of fibril polymorphism. This study highlights the role of a small molecule such as ThT in selectively favoring the formation of a specific type of fibril, by binding to aggregates formed early on one of multiple pathways, thereby altering the structural core and external morphology of the fibrils formed.

}, issn = {1083-351X}, doi = {10.1074/jbc.M117.795617}, author = {Kumar, Harish and Singh, Jogender and Kumari, Pratibha and Udgaonkar, Jayant B} } @article {5240, title = {Microsecond rearrangements of hydrophobic clusters in an initially collapsed globule prime structure formation during the folding of a small protein.}, journal = {J Mol Biol}, year = {2016}, month = {2016 Jun 28}, abstract = {

To determine how polypeptide chain collapse initiates structure formation during protein folding is a long standing goal. It has been challenging to characterize experimentally the dynamics of the polypeptide chain which lead to the formation of a compact kinetic molten globule (MG) in about a millisecond. In this study, the sub-millisecond events that occur early during the folding of monellin (MNEI) from the guanidine hydrochloride-unfolded state have been characterized using multiple fluorescence and fluorescence resonance energy transfer probes. The kinetic MG is shown to form in a non-cooperative manner from the unfolded (U) state as a result of at least three different processes happening during the first millisecond of folding. Initial chain compaction completes within the first 37μs, and further compaction occurs only after structure formation commences at a few milliseconds of folding. Transient non-native and native-like hydrophobic clusters with side-chains of certain residues buried form during the initial chain collapse, and the non-native clusters quickly disassemble. Subsequently, partial chain-desolvation occurs, leading to the formation of a kinetic MG. The initial chain compaction and subsequent chain rearrangement appear to be barrier-less processes. The two structural rearrangements within the collapsed globule appear to prime the protein for the actual folding transition.

}, issn = {1089-8638}, doi = {10.1016/j.jmb.2016.06.015}, author = {Goluguri, Rama Reddy and Udgaonkar, Jayant B} } @article {5917, title = {Molecular crowding causes narrowing of population heterogeneity and restricts internal dynamics in a protein}, journal = {METHODS AND APPLICATIONS IN FLUORESCENCE}, volume = {4}, year = {2016}, month = {MAR 2016}, abstract = {

Macromolecular crowding is a distinguishing property of intracellular media. Knowledge on the structure and dynamics of a protein in a crowded environment is essential for a complete understanding of its function. Reduction in intermolecular space could cause structural and functional alterations. Here, we have studied a model protein barstar to see how polyethylene glycol (PEG)-induced crowding affects its various structural states (native, unfolded and molten-globule-like) with different extents of change in conformational heterogeneity. Intramolecular distances and distance distributions were determined by time-resolved Forster resonance energy transfer from Trp53 to several acceptor sites by analysis of fluorescence decay kinetics using the Maximum Entropy Method. We observed PEG-induced narrowing of population distributions along with shifting of populations towards more compact states. Structural compactness also resulted in the slowing down of internal dynamics of the protein as revealed by fluorescence anisotropy decay kinetics of the fluorophore IAEDANS attached at several sites.

}, author = {Mondal, Samsuzzoha and Kallianpur, Mamata V and Udgaonkar, Jayant B and Krishnamoorthy, G} } @article {999, title = {Molecular Mechanism of the Misfolding and Oligomerization of the Prion Protein: Current Understanding and Its Implications.}, journal = {Biochemistry}, volume = {54}, year = {2015}, month = {2015 Jul 28}, pages = {4431-42}, abstract = {

Prion diseases, also known as transmissible spongiform encephalopathies, make up a group of fatal neurodegenerative disorders linked with the misfolding and aggregation of the prion protein (PrP). Although it is not yet understood how the misfolding of PrP induces neurodegeneration, it is widely accepted that the formation of misfolded prion protein (termed PrP(Sc)) is both the triggering event in the disease and the main component of the infectious agent responsible for disease transmission. Despite the clear involvement of PrP(Sc) in prion diseases, the exact composition of PrP(Sc) is not yet well-known. Recent studies show that misfolded oligomers of PrP could, however, be responsible for neurotoxicity and/or infectivity in the prion diseases. Hence, understanding the molecular mechanism of formation of the misfolded oligomers of PrP is critical for developing an understanding about the prion diseases and for developing anti-prion therapeutics. This review discusses recent advances in understanding the molecular mechanism of misfolded oligomer formation by PrP and its implications for the development of anti-prion therapeutics.

}, keywords = {Animals, Humans, Models, Molecular, Prion Diseases, Protein Folding, Protein Multimerization, Protein Structure, Quaternary, Protein Structure, Secondary, PrPC Proteins, PrPSc Proteins}, issn = {1520-4995}, doi = {10.1021/acs.biochem.5b00605}, author = {Singh, Jogender and Udgaonkar, Jayant B} }