@article {133180, title = {Replacement of the native cis prolines by alanine leads to simplification of the complex folding mechanism of a small globular protein.}, journal = {Biophys J}, year = {2023}, month = {2023 Aug 18}, abstract = {

The folding mechanism of MNEI, a single-chain variant of naturally occurring double-chain monellin, is complex, with multiple parallel refolding channels. To determine whether its folding energy landscape could be simplified, the two native cis-prolines, Pro41 and Pro93, were mutated, singly and together, to Ala. The stability of P93A was the same as that of the wild-type protein, pWT; however, P41A and P41AP93A were destabilized by \~{}0.9\ kcal mol. The effects of the mutations on the very fast, fast, slow, and very slow phases of folding were studied. They showed that heterogeneity in the unfolded state arises due to cis to trans isomerization of the Gly92-Pro93 peptide bond. The Pro41 to Ala mutation abolished the very slow phase of folding, whereas surprisingly, the Pro93 to Ala mutation abolished the very fast phase of folding. Double-jump, interrupted folding experiments indicated that two sequential trans to cis proline isomerization steps, of the Gly92-Pro93 peptide bond followed by the Arg40-Pro41 peptide bond, lead to the formation of the native state. They also revealed the accumulation of a late native-like intermediate, N, which differs from the native state in the isomeric status of the Arg40-Pro41 bond, as well as in a few tertiary contacts as monitored by near-UV CD measurements. The Pro to Ala mutations not only eliminated the cis to trans Pro isomerization reaction in the unfolded state, but also the two trans to cis Pro isomerization reactions during folding. By doing so, and by differentially affecting the relative stabilities of folding intermediates, the mutations resulted in a simplification of the folding mechanism. The two Pro to Ala mutations together accelerate folding to such an extent that the native state forms more than 1000-fold faster than in the case of pWT.

}, issn = {1542-0086}, doi = {10.1016/j.bpj.2023.08.012}, author = {Kaushik, Anushka and Udgaonkar, Jayant B} } @article {120166, title = {Differentiating between the sequence of structural events on alternative pathways of folding of a heterodimeric protein.}, journal = {Protein Sci}, year = {2022}, month = {2022 Nov 16}, pages = {e4513}, abstract = {

Distinguishing between competing pathways of folding of a protein, on the basis of how they differ in their progress of structure acquisition, remains an important challenge in protein folding studies. A previous study had shown that the heterodimeric protein, double chain monellin (dcMN) switches between alternative folding pathways upon a change in guanidine hydrochloride (GdnHCl) concentration. In the current study, the folding of dcMN has been characterized by the pulsed hydrogen exchange (HX) labelling methodology used in conjunction with mass spectrometry. Quantification of the extent to which folding intermediates accumulate and then disappear with time of folding at both low and high GdnHCl concentrations, where the folding pathways are known to be different, shows that the folding mechanism is describable by a triangular three-state mechanism. Structural characterization of the productive folding intermediates populated on the alternative pathways has enabled the pathways to be differentiated on the basis of the progress of structure acquisition that occurs on them. The intermediates on the two pathways differ in the extent to which the α-helix and the rest of the β-sheet have acquired structure that is protective against HX. The major difference is, however, that β2 has not acquired any protective structure in the intermediate formed on one pathway, but it has acquired significant protective structure in the intermediate formed on the alternative pathway. Hence the sequence of structural events is different on the two alternative pathways. This article is protected by copyright. All rights reserved.

}, issn = {1469-896X}, doi = {10.1002/pro.4513}, author = {Bhattacharjee, Rupam and Udgaonkar, Jayant B} } @article {110446, title = {Elongation of Fibrils Formed by a Tau Fragment is Inhibited by a Transient Dimeric Intermediate.}, journal = {J Phys Chem B}, year = {2022}, month = {2022 May 03}, abstract = {

The formation and propagation of aggregates of the tau protein in the brain are associated with the tauopathy group of neurodegenerative diseases. Different tauopathies have been shown to be associated with structurally distinct aggregates of tau. However, the mechanism by which different structural folds arise remains poorly understood. In this study of fibril formation by the fragment tau-K18 of tau, it is shown that the Lys 280 {\textrightarrow} Glu mutation in the variant tau-K18 K280E forms fibrils that are morphologically distinct from those formed by wild-type (wt) tau-K18. The mutant fibrils appear to have two protofilaments twisted around each other, whereas the wt fibrils are straight and appear to have a single protofilament. Modeling the kinetics of seeded aggregation, using a simple Michaelis-Menten-like mechanism, reveals that the two morphologically distinct fibrils are elongated with different catalytic efficiencies. Surprisingly, when the elongation of monomeric tau-K18 is seeded with tau-K18 K280E fibrils, it is seen to be inhibited at high monomer concentrations. Such inhibition is not seen when elongation is seeded with tau-K18 fibrils. The mechanism of inhibition is shown to be describable as uncompetitive inhibition, in which a transient dimeric form of tau-K18 acts as an uncompetitive inhibitor. Importantly, a dimeric form of tau-K18 is seen to be populated to a detectable extent early during aggregation. A covalently linked tau dimer, with an inter-molecular disulphide linkage, is shown to be capable of acting as an inhibitor. In summary, a quantitative kinetic approach has provided an understanding of how the formation of distinct structural folds of tau fibrils can be modulated by mutation and how the elongation of one fibril type, but not the other, is inhibited by a transiently formed dimer.

}, issn = {1520-5207}, doi = {10.1021/acs.jpcb.1c10752}, author = {Kumar, Harish and Udgaonkar, Jayant B} } @article {118432, title = {Evolutionarily conserved proline residues impede the misfolding of the mouse prion protein by destabilizing an aggregation-competent partially unfolded form }, journal = {J Mol Biol.}, year = {2022}, month = {10/2022}, abstract = {

The misfolding of the prion protein has been linked to several neurodegenerative diseases. Despite extensive studies, the mechanism of the misfolding process remains poorly understood. The present study structurally delineates the role of the conserved proline residues present in the structured C-terminal domain of the mouse prion protein (moPrP) in the misfolding process. It is shown that mutation of these Pro residues to Ala leads to destabilization of the native (N) state, and also to rapid misfolding. Using hydrogen-deuterium exchange (HDX) studies coupled with mass spectrometry (MS), it has been shown that the N state of moPrP is in rapid equilibrium with a partially unfolded form (PUF2*) at pH 4. It has been shown that the Pro to Ala mutations make PUF2* energetically more accessible from the N state by stabilizing it relative to the unfolded (U) state. The apparent rate constant of misfolding is found to be linearly proportional to the extent to which PUF2* is populated in equilibrium with the N state, strongly indicating that misfolding commences from PUF2*. It has also been shown that the Pro residues restrict the boundary of the structural core of the misfolded oligomers. Overall, this study highlights how the conserved proline residues control misfolding of the prion protein by modulating the stability of the partially unfolded form from which misfolding commences.

}, doi = {10.1016/j.jmb.2022.167854 }, author = {Pal, S. and Udgaonkar, Jayant B} } @article {108058, title = {Heterogeneity in Protein Folding and Unfolding Reactions.}, journal = {Chem Rev}, year = {2022}, month = {2022 Mar 11}, abstract = {

Proteins have dynamic structures that undergo chain motions on time scales spanning from picoseconds to seconds. Resolving the resultant conformational heterogeneity is essential for gaining accurate insight into fundamental mechanistic aspects of the protein folding reaction. The use of high-resolution structural probes, sensitive to population distributions, has begun to enable the resolution of site-specific conformational heterogeneity at different stages of the folding reaction. Different states populated during protein folding, including the unfolded state, collapsed intermediate states, and even the native state, are found to possess significant conformational heterogeneity. Heterogeneity in protein folding and unfolding reactions originates from the reduced cooperativity of various kinds of physicochemical interactions between various structural elements of a protein, and between a protein and solvent. Heterogeneity may arise because of functional or evolutionary constraints. Conformational substates within the unfolded state and the collapsed intermediates that exchange at rates slower than the subsequent folding steps give rise to heterogeneity on the protein folding pathways. Multiple folding pathways are likely to represent distinct sequences of structure formation. Insight into the nature of the energy barriers separating different conformational states populated during (un)folding can also be obtained by resolving heterogeneity.

}, issn = {1520-6890}, doi = {10.1021/acs.chemrev.1c00704}, author = {Bhatia, Sandhya and Udgaonkar, Jayant B} } @article {103407, title = {Native State Hydrogen Exchange-Mass Spectrometry Methods to Probe Protein Folding and Unfolding.}, journal = {Methods Mol Biol}, volume = {2376}, year = {2022}, month = {2022}, pages = {143-159}, abstract = {

Native state hydrogen exchange (HX) methods provide high-resolution structural data on the rare and transient opening motions in proteins under native conditions. Mass spectrometry-based HX methods (HX-MS) have gained popularity because of their ability to delineate population distributions, which allow a direct determination of the mechanism of inter conversion of the partially folded states under native conditions. Various technological advancements have provided further impetus to the development of HX-MS-based experiments to study protein folding. Classical HX-MS studies use proteolytic digestion to produce fragments of the protein subsequent to HX in solution, in order to obtain structural data. New chemical fragmentation methods, which achieve the same result as proteolysis and cause minimal change to the HX pattern in the protein, provide an attractive alternative to proteolysis. Moreover, when used in conjunction with proteolysis, chemical fragmentation methods have significantly increased the structural resolution afforded by HX-MS studies, even bringing them at par with the single amino acid resolution observed in NMR-based measurements. Experiments based on one such chemical fragmentation method, electron transfer dissociation (ETD), are described in this chapter. The ETD HX-MS method is introduced using data from a protein which is inherently resistant to proteolytic digestion as example of how such an experiment can provide high-resolution structural data on the folding-unfolding transitions of the protein under native conditions.

}, issn = {1940-6029}, doi = {10.1007/978-1-0716-1716-8_8}, author = {Malhotra, Pooja and Udgaonkar, Jayant B} } @article {115046, title = {α-Synuclein aggregation intermediates form fibril polymorphs with distinct prion-like properties.}, journal = {J Mol Biol}, year = {2022}, month = {2022 Jul 27}, pages = {167761}, abstract = {

α-Synuclein (α-Syn) amyloids in synucleinopathies are suggested to be structurally and functionally diverse, reminiscent of prion-like strains. But how the aggregation of the same precursor protein results in the formation of fibril polymorphs remains elusive. Here, we demonstrate the structure-function relationship of two polymorphs, pre-matured fibrils (PMFs) and helix-matured fibrils (HMFs), based on α-Syn aggregation intermediates. These polymorphs display the structural differences as demonstrated by solid-state NMR and mass spectrometry studies and also possess different cellular activities such as seeding, internalization, and cell-to-cell transfer of aggregates. HMFs with a compact core structure exhibit low seeding potency but readily internalize and transfer from one cell to another. The less structured PMFs lack transcellular transfer ability but induce abundant α-Syn pathology and trigger the formation of aggresomes in cells. Overall, the study highlights that the conformational heterogeneity in the aggregation pathway may lead to fibril polymorphs with distinct prion-like behavior.

}, issn = {1089-8638}, doi = {10.1016/j.jmb.2022.167761}, author = {Mehra, Surabhi and Ahlawat, Sahil and Kumar, Harish and Datta, Debalina and Navalkar, Ambuja and Singh, Nitu and Patel, Komal and Gadhe, Laxmikant and Kadu, Pradeep and Kumar, Rakesh and Jha, Narendra N and Sakunthala, Arunima and Sawner, Ajay S and Padinhateeri, Ranjith and Udgaonkar, Jayant B and Agarwal, Vipin and Maji, Samir K} } @article {99937, title = {Destabilization of polar interactions in the prion protein triggers misfolding and oligomerization.}, journal = {Protein Sci}, year = {2021}, month = {2021 Sep 23}, abstract = {

The prion protein (PrP) misfolds and oligomerizes at pH 4 in the presence of physiological salt concentrations. Low pH and salt cause structural perturbations in the monomeric prion protein that leads to misfolding and oligomerization. However, the changes in stability within different regions of the PrP prior to oligomerization are poorly understood. In this study, we have characterized the local stability in PrP at high resolution using amide temperature coefficients (T ) measured by NMR spectroscopy. The local stability of PrP was investigated under native as well as oligomerizing conditions. We have also studied the rapidly oligomerizing PrP variant (Q216R) and the protective PrP variant (A6). We report that at low pH, salt destabilizes PrP at several polar residues, and the hydrogen bonds in helices α2 and α3 are weakened. In addition, salt changes the curvature of the α3 helix, which likely disrupts α2-α3 contacts and leads to oligomerization. These results are corroborated by the T values of rapidly oligomerizing Q216R-PrP. The poly-alanine substitution in A6-PrP stabilizes α2, which prevents oligomerization. Altogether, these results highlight the importance of native polar interactions in determining the stability of PrP and reveal the structural disruptions in PrP that lead to misfolding and oligomerization. This article is protected by copyright. All rights reserved.

}, issn = {1469-896X}, doi = {10.1002/pro.4188}, author = {Bhate, Suhas H and Udgaonkar, Jayant B and Das, Ranabir} } @article {88406, title = {The Lys 280 ➔ Gln mutation mimicking disease-linked acetylation of Lys 280 in tau extends the structural core of fibrils and modulates their catalytic properties.}, journal = {Protein Sci}, year = {2021}, month = {2021 Jan 25}, abstract = {

Amyloid fibrillar aggregates isolated from the brains of patients with neurodegenerative diseases invariably have post-translational modifications (PTMs). The roles that PTMs play in modulating the structures and polymorphism of amyloid aggregates, and hence their ability to catalyze the conversion of monomeric protein to their fibrillar structure is, however, poorly understood. This is particularly true in the case of tau aggregates, where specific folds of fibrillar tau have been implicated in specific tauopathies. Several PTMs, including acetylation at Lys 280, increase aggregation of tau in the brain, and increase neurodegeneration. In this study, tau-K18 K280Q, in which the Lys 280 ➔ Gln mutation is used to mimic acetylation at Lys 280, is shown, using HX-MS measurements, to form fibrils with a structural core that is longer than that of tau-K18 fibrils. Measurements of critical concentrations show that the binding affinity of monomeric tau-K18 for its fibrillar counterpart is only marginally more than that of monomeric tau-K18 K280Q for its fibrillar counterpart. Quantitative analysis of the kinetics of seeded aggregation, using a simple Michaelis-Menten-like model, in which the monomer first binds and then undergoes conformational conversion to β-strand, shows that the fibrils of tau-K18 K280Q convert monomeric protein more slowly than do fibrils of tau-K18. In contrast, monomeric tau-K18 K280Q is converted faster to fibrils than is monomeric tau-K18. Thus, the effect of Lys 280 acetylation on tau aggregate propagation in brain cells is expected to depend on the amount of acetylated tau present, and on whether the propagating seed is acetylated at Lys 280 or not. This article is protected by copyright. All rights reserved.

}, issn = {1469-896X}, doi = {10.1002/pro.4030}, author = {Kumar, Harish and Udgaonkar, Jayant B} } @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 {91471, title = {Resolving Site-Specific Heterogeneity of the Unfolded State under Folding Conditions.}, journal = {J Phys Chem Lett}, year = {2021}, month = {2021 Mar 25}, pages = {3295-3302}, abstract = {

Understanding the properties of the unfolded state under folding conditions is of fundamental importance for gaining mechanistic insight into folding as well as misfolding reactions. Toward achieving this objective, the folding reaction of a small protein, monellin, has been resolved structurally and temporally, with the use of the multisite time-resolved FRET methodology. The present study establishes that the initial polypeptide chain collapse is not only heterogeneous but also structurally asymmetric and nonuniform. The population-averaged size for the segments spanning parts of the β-sheet decreases much more than that for the α-helix. Multisite measurements enabled specific and nonspecific components of the initial chain collapse to be discerned. The expanded and compact intermediate subensembles have the properties of a nonspecifically collapsed (hence, random-coil-like) and specifically collapsed (hence, globular) polymer, respectively. During subsequent folding, both the subensembles underwent contraction to varying extents at the four monitored segments, which was close to gradual in nature. The expanded intermediate subensemble exhibited an additional very slow contraction, suggestive of the presence of non-native interactions that result in a higher effective viscosity slowing down intrachain motions under folding conditions.

}, issn = {1948-7185}, doi = {10.1021/acs.jpclett.1c00098}, author = {Bhatia, Sandhya and Krishnamoorthy, Guruswamy and Udgaonkar, Jayant B} } @article {99938, title = {Structural Characterization of the Cooperativity of Unfolding of a Heterodimeric Protein Using Hydrogen Exchange-Mass Spectrometry.}, journal = {J Mol Biol}, year = {2021}, month = {2021 Sep 23}, pages = {167268}, abstract = {

Little is known about how the sequence of structural changes in one chain of a heterodimeric protein is coupled to those in the other chain during protein folding and unfolding reactions, and whether individual secondary structural changes in the two chains occur in one or many coordinated steps. Here, the unfolding mechanism of a small heterodimeric protein, double chain monellin, has been characterized using hydrogen exchange-mass spectrometry. Transient structure opening, which enables HX, was found to be describable by a five state N <-> I <-> I <-> I <-> U mechanism. Structural changes occur gradually in the first three steps, and cooperatively in the last step. β strands 2, 4 and 5, as well as the α-helix undergo transient unfolding during all three non-cooperative steps, while β1 and the two loops on both sides of the helix undergo transient unfolding during the first two steps. In the absence of GdnHCl, only β3 in chain A of the protein unfolds during the last cooperative step, while in the presence of 1 M GdnHCl, not only β3, but also β2 in chain B unfolds cooperatively. Hence, the extent of cooperative structural change and size of the cooperative unfolding unit increase when the protein is destabilized by denaturant. The naturally evolved two-chain variant of monellin folds and unfolds in a more cooperative manner than does a single chain variant created artificially, suggesting that increasing folding cooperativity, even at the cost of decreasing stability, may be a driving force in the evolution of proteins.

}, issn = {1089-8638}, doi = {10.1016/j.jmb.2021.167268}, author = {Bhattacharjee, Rupam and Udgaonkar, Jayant B} } @article {70226, title = {Binding-induced folding under unfolding conditions: Switching between induced fit and conformational selection mechanisms.}, journal = {J Biol Chem}, volume = {294}, year = {2019}, month = {2019 Nov 08}, pages = {16942-16952}, abstract = {

The chemistry of protein-ligand binding is the basis of virtually every biological process. Ligand binding can be essential for a protein to function in the cell by stabilizing or altering the conformation of a protein, particularly for partially or completely unstructured proteins. However, the mechanisms by which ligand binding impacts disordered proteins or influences the role of disorder in protein folding is not clear. To gain insight into this question, the mechanism of folding induced by the binding of a Pro-rich peptide ligand to the SH3 domain of phosphatidylinositol 3-kinase unfolded in the presence of urea has been studied using kinetic methods. Under strongly denaturing conditions, folding was found to follow a conformational selection (CS) mechanism. However, under mildly denaturing conditions, a ligand concentration-dependent switch in the mechanism was observed. The folding mechanism switched from being predominantly a CS mechanism at low ligand concentrations to being predominantly an induced fit (IF) mechanism at high ligand concentrations. The switch in the mechanism manifests itself as an increase in the reaction flux along the IF pathway at high ligand concentrations. The results indicate that, in the case of intrinsically disordered proteins too, the folding mechanism is determined by the concentration of the ligand that induces structure formation.

}, issn = {1083-351X}, doi = {10.1074/jbc.RA119.009742}, author = {Sen, Sreemantee and Udgaonkar, Jayant B} } @article {50995, title = {A five-residue motif for the design of domain swapping in proteins.}, journal = {Nat Commun}, volume = {10}, year = {2019}, month = {2019 01 28}, pages = {452}, abstract = {

Domain swapping is the process by which identical monomeric proteins exchange structural elements to generate dimers/oligomers. Although engineered domain swapping is a compelling strategy for protein assembly, its application has been limited due to the lack of simple and reliable design approaches. Here, we demonstrate that the hydrophobic five-residue {\textquoteright}cystatin motif{\textquoteright} (QVVAG) from the domain-swapping protein Stefin B, when engineered into a solvent-exposed, tight surface loop between two β-strands prevents the loop from folding back upon itself, and drives domain swapping in non-domain-swapping proteins. High-resolution structural studies demonstrate that engineering the QVVAG stretch independently into various surface loops of four structurally distinct non-domain-swapping proteins enabled the design of different modes of domain swapping in these proteins, including single, double and open-ended domain swapping. These results suggest that the introduction of the QVVAG motif can be used as a mutational approach for engineering domain swapping in diverse β-hairpin proteins.

}, issn = {2041-1723}, doi = {10.1038/s41467-019-08295-x}, author = {Nandwani, Neha and Surana, Parag and Negi, Hitendra and Mascarenhas, Nahren M and Udgaonkar, Jayant B and Das, Ranabir and Gosavi, Shachi} } @article {68511, title = {Introducing the Mechanical Forces in Biochemistry Special Issue.}, journal = {Biochemistry}, volume = {58}, year = {2019}, month = {2019 Nov 26}, pages = {4655-4656}, issn = {1520-4995}, doi = {10.1021/acs.biochem.9b00999}, author = {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 {62759, title = {Observation of Continuous Contraction and a Metastable Misfolded State during the Collapse and Folding of a Small Protein.}, journal = {J Mol Biol}, year = {2019}, month = {2019 Jul 19}, abstract = {

To obtain proper insight into how structure develops during a protein folding reaction, it is necessary to understand the nature and mechanism of the polypeptide chain collapse reaction, which marks the initiation of folding. Here, the time-resolved fluorescence resonance energy transfer technique, in which the decay of the fluorescence light intensity with time is used to determine the time evolution of the distribution of intra-molecular distances, has been utilized to study the folding of the small protein, monellin. It is seen that when folding begins, about one-third of the protein molecules collapse into a molten globule state (I), from which they relax by continuous further contraction to transit to the native state. The larger fraction gets trapped into a metastable misfolded state. Exit from this metastable state occurs via collapse to the lower free energy I state. This exit is slow, on a time scale of seconds, because of activation energy barriers. The trapped misfolded molecules as well as the I molecules contract continuously and slowly as structure develops. A phenomenological model of Markovian evolution of the polymer chain undergoing folding, incorporating these features, has been developed, which fits well the experimentally observed time evolution of distance distributions. The observation that the "wrong turn" to the misfolded state has not been eliminated by evolution belies the common belief that the folding of functional protein sequences is very different from that of a random heteropolymer, and the former have been selected by evolution to fold quickly.

}, issn = {1089-8638}, doi = {10.1016/j.jmb.2019.07.024}, author = {Bhatia, Sandhya and Krishnamoorthy, G and Dhar, Deepak and Udgaonkar, Jayant B} } @article {49278, title = {Ruggedness in the Free Energy Landscape Dictates Misfolding of the Prion Protein.}, journal = {J Mol Biol}, year = {2019}, month = {2019 Jan 03}, abstract = {

Experimental determination of the key features of the free energy landscapes of proteins which dictate their adeptness to fold correctly, or propensity to misfold and aggregate, and which are modulated upon a change from physiological to aggregation-prone conditions, is a difficult challenge. In this study, sub-millisecond kinetic measurements of the folding and unfolding of the mouse prion protein (moPrP), reveal how the free energy landscape becomes more complex upon a shift from physiological (pH 7) to aggregation-prone (pH 4) conditions. Folding and unfolding utilize the same single pathway at pH 7, but at pH 4, folding occurs on a pathway distinct from the unfolding pathway. Moreover, the kinetics of both folding and unfolding at pH 4 depend not only on the final conditions, but also on the conditions under which the processes are initiated. Unfolding can be made to switch to occur on the folding pathway by varying the initial conditions. Folding and unfolding pathways appear to occupy different regions of the free energy landscape, which are separated by large free energy barriers that change with a change in the initial conditions. These barriers direct unfolding of the native protein to proceed via an aggregation-prone intermediate previously identified to initiate the misfolding of moPrP at low pH, thus identifying a plausible mechanism by which the ruggedness of the free energy landscape of a protein may modulate its aggregation propensity.

}, issn = {1089-8638}, doi = {10.1016/j.jmb.2018.12.009}, author = {Moulick, Roumita and Goluguri, Rama Reddy 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 {43845, title = {The Osmolyte TMAO Modulates Protein Folding Cooperativity by Altering Global Protein Stability.}, journal = {Biochemistry}, year = {2018}, month = {2018 Sep 24}, abstract = {

The folding of many globular proteins from the unfolded (U) to the native (N) state appears to be describable by a two-state N <-> U model, which has led to the general belief that protein folding occurs in a highly cooperative manner. One reason for the widespread belief in "two-state folding" is that protein folding reactions are invariably studied by ensemble averaging probes and not by probes that can distinguish as well as quantify the multiple conformations that may be present. Consequently, how cooperativity is affected by protein stability, protein sequence, and solvent conditions is poorly understood. In this study, hydrogen exchange coupled to mass spectrometry (HX-MS) of the PI3K SH3 domain was carried out in the presence of a stabilizing osmolyte, trimethylamine N-oxide (TMAO). By showing that HX occurs under the EX1 regime even in the presence of 2 M TMAO, we were able to examine the temporal evolution of the populations of the different conformations present together. A strong link between protein folding cooperativity and protein stability is revealed. Increasing stability is accompanied by an increase in the ruggedness of the free energy landscape as well as diminished cooperativity; the number of amide sites simultaneously opening up their structure decreases with an increase in TMAO concentration. A comparison of the effect of TMAO to that of urea on the intrinsic dynamics of the PI3K SH3 domain indicates that TMAO counteracts the effect of urea not only on protein stability but also on protein folding cooperativity.

}, issn = {1520-4995}, doi = {10.1021/acs.biochem.8b00698}, author = {Jethva, Prashant N and Udgaonkar, Jayant B} } @article {29630, title = {Site-specific time-resolved FRET reveals local variations in the unfolding mechanism in an apparently two-state protein unfolding transition.}, journal = {Phys Chem Chem Phys}, volume = {20}, year = {2018}, month = {2018 Jan 31}, pages = {3216-3232}, abstract = {

Protein folding/unfolding transitions between the native (N) and unfolded (U) states are usually describable as two-state, only because of the dominant presence of the N and/or U states, because of which high energy intermediates remain undetected. Delineation of the cooperativity underlying these transitions, and characterization of high energy intermediates that are populated sparsely, have been difficult challenges, especially under equilibrium conditions, and require the use of a sensitive probe that reports on both the structures and population distributions of the partially unfolded intermediates. In this study, the use of multisite time-resolved FRET to monitor structural change in five specific segments of the small protein monellin, has brought out local deviations from two-state behavior during unfolding. It is shown that in some segments of the protein structure, denaturant-induced unfolding proceeds first by gradual expansion of the N state, then by an all-or-none transition from the N state ensemble to the U state ensemble, followed finally by expansion of the U state. Segments encompassing the sole helix appear, however, to unfold completely through a gradual transition from the N to U states. Finally, it is shown that equilibrium unfolding of monellin is not only heterogeneous, but that the degree of non-cooperativity differs between the sole α-helix and different parts of the β-sheet.

}, issn = {1463-9084}, doi = {10.1039/c7cp06214a}, author = {Bhatia, Sandhya and Krishnamoorthy, G and Udgaonkar, Jayant B} } @article {35145, title = {Structural mechanisms of oligomer and amyloid fibril formation by the prion protein.}, journal = {Chem Commun (Camb)}, year = {2018}, month = {2018 May 23}, abstract = {

Misfolding and aggregation of the prion protein is responsible for multiple neurodegenerative diseases. Works from several laboratories on folding of both the WT and multiple pathogenic mutant variants of the prion protein have identified several structurally dissimilar intermediates, which might be potential precursors to misfolding and aggregation. The misfolded aggregates themselves are morphologically distinct, critically dependent on the solution conditions under which they are prepared, but always β-sheet rich. Despite the lack of an atomic resolution structure of the infectious pathogenic agent in prion diseases, several low resolution models have identified the β-sheet rich core of the aggregates formed in vitro, to lie in the α2-α3 subdomain of the prion protein, albeit with local stabilities that vary with the type of aggregate. This feature article describes recent advances in the investigation of in vitro prion protein aggregation using multiple spectroscopic probes, with particular focus on (1) identifying aggregation-prone conformations of the monomeric protein, (2) conditions which trigger misfolding and oligomerization, (3) the mechanism of misfolding and aggregation, and (4) the structure of the misfolded intermediates and final aggregates.

}, issn = {1364-548X}, doi = {10.1039/c8cc03053g}, author = {Sengupta, Ishita and Udgaonkar, Jayant B} } @article {20316, title = {Amino-acid composition after loop deletion drives domain swapping.}, journal = {Protein Sci}, volume = {26}, year = {2017}, month = {10/2017}, chapter = {1994}, abstract = {

Rational engineering of a protein to enable domain swapping requires an understanding of the sequence, structural and energetic factors that favor the domain-swapped oligomer over the monomer. While it is known that the deletion of loops between β-strands can promote domain swapping, the spliced sequence at the position of the loop deletion is thought to have a minimal role to play in such domain swapping. Here, two loop-deletion mutants of the non-domain-swapping protein monellin, frame-shifted by a single residue, were designed. Although the spliced sequence in the two mutants differed by only one residue at the site of the deletion, only one of them (YEIKG) promoted domain swapping. The mutant containing the spliced sequence YENKG was entirely monomeric. This new understanding that the domain swapping propensity after loop deletion may depend critically on the chemical composition of the shortened loop will facilitate the rational design of domain swapping. This article is protected by copyright. All rights reserved.

}, issn = {1469-896X}, doi = {10.1002/pro.3237}, author = {Nandwani, Neha and Surana, Parag and Udgaonkar, Jayant B and Das, Ranabir and Gosavi, Shachi} } @article {22020, title = {Chemical Denaturants Smoothen Ruggedness on the Free Energy Landscape of Protein Folding.}, journal = {Biochemistry}, volume = {56}, year = {2017}, month = {2017 Aug 08}, pages = {4053-4063}, abstract = {

To characterize experimentally the ruggedness of the free energy landscape of protein folding is challenging, because the distributed small free energy barriers are usually dominated by one, or a few, large activation free energy barriers. This study delineates changes in the roughness of the free energy landscape by making use of the observation that a decrease in ruggedness is accompanied invariably by an increase in folding cooperativity. Hydrogen exchange (HX) coupled to mass spectrometry was used to detect transient sampling of local energy minima and the global unfolded state on the free energy landscape of the small protein single-chain monellin. Under native conditions, local noncooperative openings result in interconversions between Boltzmann-distributed intermediate states, populated on an extremely rugged "uphill" energy landscape. The cooperativity of these interconversions was increased by selectively destabilizing the native state via mutations, and further by the addition of a chemical denaturant. The perturbation of stability alone resulted in seven backbone amide sites exchanging cooperatively. The size of the cooperatively exchanging and/or unfolding unit did not depend on the extent of protein destabilization. Only upon the addition of a denaturant to a destabilized mutant variant did seven additional backbone amide sites exchange cooperatively. Segmentwise analysis of the HX kinetics of the mutant variants further confirmed that the observed increase in cooperativity was due to the smoothing of the ruggedness of the free energy landscape of folding of the protein by the chemical denaturant.

}, keywords = {Amino Acid Substitution, Deuterium Exchange Measurement, Energy Transfer, Guanidine, Indicators and Reagents, Kinetics, Menispermaceae, Models, Molecular, Mutagenesis, Site-Directed, Mutation, Plant Proteins, Protein Conformation, Protein Denaturation, Protein Folding, Protein Stability, Thermodynamics}, issn = {1520-4995}, doi = {10.1021/acs.biochem.7b00367}, author = {Malhotra, Pooja and Jethva, Prashant N and Udgaonkar, Jayant B} } @article {20329, title = {A Dry Transition State More Compact Than the Native State Is Stabilized by Non-Native Interactions during the Unfolding of a Small Protein.}, journal = {Biochemistry}, year = {2017}, month = {2017 Jul 11}, abstract = {

Defining the role of non-native interactions in directing the course of protein folding or unfolding reactions has been a difficult challenge. In particular, the extent to which such interactions play a productive role by stabilizing the structures of transition states (TSs) found on the folding and unfolding pathways of proteins is not known. On the contrary, it is thought that the TSs are expanded forms of the N state stabilized by native interactions, and it is not known whether non-native interactions can modulate TS structure. In this study of the unfolding of the SH3 domain of PI3 kinase using a microsecond mixing methodology, partial non-native structure formation is shown to occur initially during unfolding. The TS of this partial "folding during unfolding" reaction is more compact than the N state: the apparent rate constant of Trp53 burial during this reaction decreases with an increase in denaturant concentration. Kinetic studies of the unfolding of mutant variants suggest that the unusually compact TS is stabilized by interactions not present in N and that these non-native interactions are hydrophobic in nature. It was determined that mutation could be used to tune the degree of compaction in the TS.

}, issn = {1520-4995}, doi = {10.1021/acs.biochem.7b00388}, author = {Sen, Sreemantee and Goluguri, Rama Reddy and Udgaonkar, Jayant B} } @article {20954, title = {Expression and purification of single cysteine-containing mutant variants of the mouse prion protein by oxidative refolding.}, journal = {Protein Expr Purif}, year = {2017}, month = {2017 Jul 20}, abstract = {

The folding and aggregation of proteins has been studied extensively, using multiple probes. To facilitate such experiments, introduction of spectroscopically-active moieties in to the protein of interest is often necessary. This is commonly achieved by specifically labelling cysteine residues in the protein, which are either present naturally or introduced artificially by site-directed mutagenesis. In the case of the recombinant prion protein, which is normally expressed in inclusion bodies, the presence of the native disulfide bond complicates the correct refolding of single cysteine-containing mutant variants of the protein. To overcome this major bottleneck, a simple purification strategy for single tryptophan, single cysteine-containing mutant variants of the mouse prion protein is presented, with yields comparable to that of the wild type protein. The protein(s) obtained by this method are correctly folded, with a single reduced cysteine, and the native disulfide bond between residues C178 and C213 intact. The β-sheet rich oligomers formed from these mutant variant protein(s) are identical to the wild type protein oligomer. The current strategy facilitates sample preparation for a number of high resolution spectroscopic measurements for the prion protein, which specifically require thiol labelling.

}, issn = {1096-0279}, doi = {10.1016/j.pep.2017.07.014}, author = {Sengupta, Ishita and Udgaonkar, Jayant B} } @article {28252, title = {The G126V Mutation in the Mouse Prion Protein Hinders Nucleation-Dependent Fibril Formation by Slowing Initial Fibril Growth and by Increasing the Critical Concentration.}, journal = {Biochemistry}, volume = {56}, year = {2017}, month = {2017 11 07}, pages = {5931-5942}, abstract = {

The middle disordered hydrophobic region of the prion protein plays a critical role in conformational conversion of the protein, with pathogenic as well as protective mutations being localized to this region. In particular, it has been shown that the G127V mutation in this region of the human prion protein (huPrP) is protective against the spread of prion disease, but the mechanism of protection remains unknown. In this study, quantitative analyses of the kinetics of fibril formation by wild-type mouse prion protein (moPrP) and G126V moPrP (equivalent to G127V huPrP) reveal important differences: the critical concentration is higher, the lag phase is longer, and the initial effective rate constant of fibril growth is slower for the mutant variant. The study offers a simple biophysical explanation for why the G127V mutation in huPrP would be protective in humans: the \~{}5-fold increase in critical concentration caused by the mutation likely results in the critical concentration (below which fibril formation cannot occur) being higher that the concentration of the protein present in and on cells in vivo.

}, keywords = {Animals, Humans, Hydrophobic and Hydrophilic Interactions, Kinetics, Mice, Models, Theoretical, Mutation, Missense, Polymerization, Prion Proteins, PrP 27-30 Protein}, issn = {1520-4995}, doi = {10.1021/acs.biochem.7b00894}, author = {Sabareesan, Ambadi Thody and Udgaonkar, Jayant B} } @article {12889, title = {Identification and structural characterization of the precursor conformation of the prion protein which directly initiates misfolding and oligomerization.}, journal = {J Mol Biol}, year = {2017}, month = {2017 Jan 29}, abstract = {

To identify and structurally characterize the precursor conformation of the prion protein (PrP), from which misfolding and aggregation directly commence, has been a long-standing goal. Misfolding converts the α-helical, non-pathogenic functional form of PrP to pathogenic, β-structured oligomeric and amyloidogenic forms, which are the cause of prion diseases. Susceptibility to sporadic prion disease correlates well with the propensity of PrP to misfold to cytotoxic, proteinase-K resistant oligomeric conformations, at low pH. In this study, mutagenesis at the hydrophobic core of the mouse prion protein has been shown to stabilize a monomeric, unfolding intermediate (I), which is populated significantly at equilibrium at low pH. Importantly, the rate of formation of β-structured oligomers at low pH is found to correlate well with the extent to which this intermediate is populated. The misfolding process is limited by dimerization of I, indicating that I is the monomeric precursor conformation which directly initiates misfolding. Structural and thermodynamic characterization by native state hydrogen-deuterium exchange-mass spectrometry studies indicate that the precursor conformation is a partially unfolded form of PrP which forms under misfolding-prone solvent conditions.

}, issn = {1089-8638}, doi = {10.1016/j.jmb.2017.01.019}, author = {Moulick, Roumita 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 {17517, title = {Salt-mediated oligomerization of the mouse prion protein monitored by real time NMR.}, journal = {J Mol Biol}, year = {2017}, month = {2017 May 11}, abstract = {

The prion protein forms {\ss}-rich soluble oligomers in vitro at pH4 in the presence of physiological concentrations of salt. In the absence of salt, oligomerization and misfolding does not take place in an experimentally tractable timescale. While it is well established that a lowering of pH facilitates misfolding and oligomerization of this protein, the role of salt remains poorly understood. Here, solution-state NMR was used to probe perturbations in the monomeric mouse prion protein moPrP) structure immediately upon salt-addition, prior to the commencement of the oligomerization reaction. The weak binding of salt at multiple sites dispersed all over the monomeric protein causes a weak non-specific perturbation of structure throughout the protein. The only significant perturbation occurs in the loop between helix 2 and 3 in and around the partially buried K193-E195 salt bridge. The disruption of this key electrostatic interaction is the earliest detectable change in the monomer before any major conformational change occurs and appears to constitute the trigger for the commencement of misfolding and oligomerization. Subsequently, the kinetics of monomer loss, due to oligomerization, was monitored at the individual residue level. The oligomerization reaction was found to be rate-limited by association and not conformational change, with an average reaction order of 2.6, across residues. Not surprisingly, salt accelerated the oligomerization kinetics, in a non-specific manner, by electrostatic screening of the highly charged monomers at acidic pH. Together, these results allowed a demarcation of the specific and non-specific effects of salt on prion protein misfolding and oligomerization.

}, issn = {1089-8638}, doi = {10.1016/j.jmb.2017.05.006}, author = {Sengupta, Ishita and Bhate, Suhas H and Das, Ranabir and Udgaonkar, Jayant B} } @article {20350, title = {Stepwise Assembly of β-Sheet Structure during the Folding of an SH3 Domain Revealed by a Pulsed Hydrogen Exchange Mass Spectrometry Study.}, journal = {Biochemistry}, year = {2017}, month = {2017 Jul 14}, abstract = {

Dissecting temporally the sequence of secondary structural changes, and determining how these specific changes modulate conformational heterogeneity, remain major goals of protein folding studies. In this study, the folding of the SH3 domain of PI3 kinase has been characterized using pulsed hydrogen exchange mass spectrometry (HX-MS). The folding could be described as a four-state process, U <-> IVE <-> IE <-> N, where IVE and IE are structurally heterogeneous intermediate ensembles. Compared to U, early intermediate IVE has a marginally increased level of protection against HX of amides along the entire length of the polypeptide. Sequential assembly into β-sheet structure has been resolved temporally. Three of the five β-strands acquire nativelike structure before the rate-limiting step. β-Strands 2 and 5 acquire nativelike structure in IVE, while β-strand 4 does so in IE. β-Strand 1 acquires nativelike structure only during the last step of the folding process. Hence, the HX-MS study has resolved the order of assembly of the β-strands for the formation of the two β-sheets, which previous studies utilizing Φ-value analysis of several different SH3 domains had been unable to accomplish. Moreover, it is shown that structural heterogeneity decreases in a stepwise manner during the three stages of folding.

}, issn = {1520-4995}, doi = {10.1021/acs.biochem.7b00374}, author = {Aghera, Nilesh and Udgaonkar, Jayant B} } @article {3549, title = {Editorial overview: Folding and binding: Dynamic conformational heterogeneity is pivotal to cell life.}, journal = {Curr Opin Struct Biol}, volume = {36}, year = {2016}, month = {2016 Feb}, pages = {iv-vi}, issn = {1879-033X}, doi = {10.1016/j.sbi.2016.01.012}, author = {Nussinov, Ruth and Udgaonkar, Jayant B} } @article {7040, title = {How cooperative are protein folding and unfolding transitions?}, journal = {Protein Sci}, year = {2016}, month = {2016 Aug 13}, abstract = {

A thermodynamically and kinetically simple picture of protein folding envisages only two states, native (N) and unfolded (U), separated by a single activation free energy barrier, and interconverting by cooperative two-state transitions. The folding/unfolding transitions of many proteins occur, however, in multiple discrete steps associated with the formation of intermediates, which is indicative of reduced cooperativity. Furthermore, much advancement in experimental and computational approaches has demonstrated entirely non-cooperative (gradual) transitions via a continuum of states and a multitude of small energetic barriers between the N and U states of some proteins. These findings have been instrumental towards providing a structural rationale for cooperative versus non-cooperative transitions, based on the coupling between interaction networks in proteins. The cooperativity inherent in a folding/unfolding reaction appears to be context dependent, and can be tuned via experimental conditions which change the stabilities of N and U. The evolution of cooperativity in protein folding transitions is linked closely to the evolution of function as well as the aggregation propensity of the protein. A large activation energy barrier in a fully cooperative transition can provide the kinetic control required to prevent the accumulation of partially unfolded forms, which may promote aggregation. Nevertheless, increasing evidence for barrier-less {\textquoteright}downhill{\textquoteright} folding, as well as for continuous {\textquoteright}uphill{\textquoteright} unfolding transitions, indicate that gradual non-cooperative processes may be ubiquitous features on the free energy landscape of protein folding. This article is protected by copyright. All rights reserved.

}, issn = {1469-896X}, doi = {10.1002/pro.3015}, author = {Malhotra, Pooja 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 {3540, title = {The Pathogenic A116V Mutation Enhances Ion-Selective Channel Formation by Prion Protein in Membranes.}, journal = {Biophys J}, volume = {110}, year = {2016}, month = {2016 Apr 26}, pages = {1766-76}, abstract = {

Prion diseases are a group of fatal neurodegenerative disorders that afflict mammals. Misfolded and aggregated forms of the prion protein (PrP(Sc)) have been associated with many prion diseases. A transmembrane form of PrP favored by the pathogenic mutation A116V is associated with Gerstmann-Str{\"a}ussler-Scheinker syndrome, but no accumulation of PrP(Sc) is detected. However, the role of the transmembrane form of PrP in pathological processes leading to neuronal death remains unclear. This study reports that the full-length mouse PrP (moPrP) significantly increases the permeability of living cells to K(+), and forms K(+)- and Ca(2+)-selective channels in lipid membranes. Importantly, the pathogenic mutation A116V greatly increases the channel-forming capability of moPrP. The channels thus formed are impermeable to sodium and chloride ions, and are blocked by blockers of voltage-gated ion channels. Hydrogen-deuterium exchange studies coupled with mass spectrometry (HDX-MS) show that upon interaction with lipid, the central hydrophobic region (109-132) of the protein is protected against exchange, making it a good candidate for inserting into the membrane and lining the channel. HDX-MS also shows a dramatic increase in the protein-lipid stoichiometry for A116V moPrP, providing a rationale for its increased channel-forming capability. The results suggest that ion channel formation may be a possible mechanism of PrP-mediated neurodegeneration by the transmembrane forms of PrP.

}, issn = {1542-0086}, doi = {10.1016/j.bpj.2016.03.017}, author = {Sabareesan, Ambadi Thody and Singh, Jogender and Roy, Samrat and Udgaonkar, Jayant B and Mathew, M K} } @article {1169, title = {The pathogenic mutation T182A converts the prion protein into a molten globule-like conformation whose misfolding to oligomers but not to fibrils is drastically accelerated.}, journal = {Biochemistry}, volume = {55}, year = {2016}, month = {2016}, pages = {459-69}, abstract = {

Delineation of the effects of pathogenic mutations linked with familial prion diseases, on the structure and misfolding of prion protein (PrP), will be useful in understanding the molecular mechanism of PrP misfolding. Here, it has been shown that the pathogenic mutation T182A causes a drastic reduction in the apparent cooperativity and enthalpy of unfolding of the mouse prion protein (moPrP) under misfolding-prone conditions by converting the protein into a molten globule (MG)-like conformation. Hydrogen-deuterium exchange studies in conjunction with mass spectrometry indicate that the T182A mutation disrupts the core of the protein, thereby increasing overall structural dynamics. T182A moPrP is shown to misfold to oligomers very much faster than does wt moPrP, but to misfold to fibrils at a rate similar to that of wt moPrP. This observation suggests that oligomers are unlikely to play a productive role on the direct pathway of aggregation from monomer to fibrils. The observation that fully folded T182A moPrP has a MG-like structure, and that it misfolds to oligomers much faster than does wt moPrP, suggests that a MG-like intermediate, whose structure resembles that of fully folded T182A moPrP, might be populated early on the pathway of misfolding of wt moPrP to oligomers.

}, issn = {1520-4995}, doi = {10.1021/acs.biochem.5b01266}, author = {Singh, Jogender and Udgaonkar, Jayant B} } @article {7079, title = {Pathogenic mutations within the disordered palindromic region of the prion protein induce structure therein and accelerate the formation of misfolded oligomers.}, journal = {J Mol Biol}, year = {2016}, month = {2016 Aug 18}, abstract = {

Little is understood about how the intrinsically disordered N-terminal region (NTR) of the prion protein modulates its misfolding and aggregation, which lead to prion disease. In this study, two pathogenic mutations, G113 V and A116 V in the palindromic region of the NTR, are shown to have no effect on the structure, stability or dynamics of native mouse prion protein (moPrP), but to nevertheless accelerate misfolding and oligomerization. For wt moPrP, misfolding and oligomerization appear to occur concurrently, while for both mutant variants, oligomerization is shown to precede misfolding. Kinetic hydrogen deuterium exchange-mass spectrometry experiments show that sequence segment 89-132 from the NTR becomes structured, albeit weakly, during the oligomerization of both mutant variants. Importantly, this structure formation occurs prior to structural conversion in the CTD, and appears to be the reason why the formation of misfolded oligomers is accelerated by the pathogenic mutations.

}, issn = {1089-8638}, doi = {10.1016/j.jmb.2016.08.015}, author = {Sabareesan, A T and Udgaonkar, Jayant B} } @article {5241, title = {Secondary Structural Change Can Occur Diffusely and Not Modularly during Protein Folding and Unfolding Reactions.}, journal = {J Am Chem Soc}, volume = {138}, year = {2016}, month = {2016 May 11}, pages = {5866-78}, abstract = {

A major goal of protein folding studies is to understand the structural basis of the coupling between stabilizing interactions, which leads to cooperative conformational change. The goal is challenging because of the difficulty in simultaneously measuring global cooperativity by determining population distributions of the conformations present, and the structures of these conformations. Here, hydrogen exchange (HX) into the small protein monellin was carried out under conditions where structure-opening is rate limiting for most backbone amide sites. Detection by mass spectrometry allowed characterization of not only segment-specific structure-opening rates but also the cooperativity of unfolding of the different secondary structural segments of the protein. The segment-specific pattern of HX reveals that the backbone hydrogen-bonding network disassembles in a structurally diffuse, asynchronous manner. A comparison of the site-specific transient opening rates of secondary and tertiary structure in the protein provides a structural rationale for the observation that unfolding is hierarchical and describable by exponential kinetics, despite being diffuse. Since unfolding was studied in native conditions, the sequence of events during folding in the same conditions will be the reverse of the sequence of events observed during unfolding. Hence, the formation of secondary structural units during folding would also occur in a non-cooperative, diffuse, and asynchronous manner.

}, issn = {1520-5126}, doi = {10.1021/jacs.6b03356}, author = {Malhotra, Pooja and Udgaonkar, Jayant B} } @article {3550, title = {Unraveling the Molecular Mechanism of pH-Induced Misfolding and Oligomerization of the Prion Protein.}, journal = {J Mol Biol}, volume = {428}, year = {2016}, month = {2016 Mar 27}, pages = {1345-55}, abstract = {

The misfolding of the prion protein (PrP) to aggregated forms is linked to several neurodegenerative diseases. Misfolded oligomeric forms of PrP are associated with neurotoxicity and/or infectivity, but the molecular mechanism by which they form is still poorly understood. A reduction in pH is known to be a key factor that triggers misfolded oligomer formation by PrP, but the residues whose protonation is linked with misfolding remain unidentified. The structural consequences of the protonation of these residues also remain to be determined. In the current study, amino acid residues whose protonation is critical for PrP misfolding and oligomerization have been identified using site-directed mutagenesis and misfolding/oligomerization assays. It is shown that the protonation of either H186 or D201, which mimics the effects of pathogenic mutations (H186R and D201N) at both residue sites, is critically linked to the stability, misfolding and oligomerization of PrP. Hydrogen-deuterium exchange studies coupled with mass spectrometry show that the protonation of either H186 or D201 leads to the same common structural change: increased structural dynamics in helix 1 and that in the loop between helix 1 and β-strand 2. It is shown that the protonation of either of these residues is sufficient for accelerating misfolded oligomer formation, most likely because the protonation of either residue causes the same structural perturbation. Hence, the increased structural dynamics in helix 1 and that in the loop between helix 1 and β-strand 2 appear to play an early critical role in acid-induced misfolding of PrP.

}, issn = {1089-8638}, doi = {10.1016/j.jmb.2016.01.030}, author = {Singh, Jogender and Udgaonkar, Jayant B} } @article {878, title = {Formation of a CH-π contact in the core of native barstar during folding.}, journal = {J Phys Chem B}, volume = {119}, year = {2015}, month = {2015 Feb 19}, pages = {2928-32}, abstract = {

An important part of the protein folding process is the consolidation of the protein core through the formation of specific, directional contacts after the initial hydrophobic collapse. Here, we simultaneously monitor formation of core contacts and assembly of secondary structure through salt-induced folding by using resonance Raman spectroscopy. Unfolded barstar at pH 12 was refolded by gradual addition of sodium sulfate salt. Altered spectral characteristics of the Trp53 residue suggest that the core of the protein attains a CH-π interaction at a low concentration of the salt, with an increase in the packing density. Further increase in salt concentration produces a reduction in the solvent accessibility of the core. These data provide evidence that the core of the protein becomes rigid upon the addition of 0.6 M sodium sulfate. This is the first time that the formation of a CH-π interaction has been directly monitored during the folding of a protein.

}, issn = {1520-5207}, doi = {10.1021/jp512036p}, author = {Mil{\'a}n-Garc{\'e}s, Erix A and Thaore, Pallavi and Udgaonkar, Jayant B and Puranik, Mrinalini} } @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} } @article {162, title = {Partially Unfolded Forms of the Prion Protein Populated under Misfolding-promoting Conditions: CHARACTERIZATION BY HYDROGEN EXCHANGE MASS SPECTROMETRY AND NMR.}, journal = {J Biol Chem}, volume = {290}, year = {2015}, month = {2015 Oct 16}, pages = {25227-40}, abstract = {

The susceptibility of the cellular prion protein (PrP(C)) to convert to an alternative misfolded conformation (PrP(Sc)), which is the key event in the pathogenesis of prion diseases, is indicative of a conformationally flexible native (N) state. In the present study, hydrogen-deuterium exchange (HDX) in conjunction with mass spectrometry and nuclear magnetic resonance spectroscopy were used for the structural and energetic characterization of the N state of the full-length mouse prion protein, moPrP(23-231), under conditions that favor misfolding. The kinetics of HDX of 34 backbone amide hydrogens in the N state were determined at pH 4. In contrast to the results of previous HDX studies on the human and Syrian hamster prion proteins at a higher pH, various segments of moPrP were found to undergo different extents of subglobal unfolding events at pH 4, a pH at which the protein is known to be primed to misfold to a β-rich conformation. No residual structure around the disulfide bond was observed for the unfolded state at pH 4. The N state of the prion protein was observed to be at equilibrium with at least two partially unfolded forms (PUFs). These PUFs, which are accessed by stochastic fluctuations of the N state, have altered surface area exposure relative to the N state. One of these PUFs resembles a conformation previously implicated to be an initial intermediate in the conversion of monomeric protein into misfolded oligomer at pH 4.

}, issn = {1083-351X}, doi = {10.1074/jbc.M115.677575}, author = {Moulick, Roumita and Das, Ranabir and Udgaonkar, Jayant B} } @article {1027, title = {Rise of the Helix from a Collapsed Globule during the Folding of Monellin.}, journal = {Biochemistry}, volume = {54}, year = {2015}, month = {2015 Sep 1}, pages = {5356-65}, abstract = {

Early kinetic intermediates observed during the folding of many proteins are invariably compact and appear to possess some secondary structure. Consequently, it has been difficult to understand whether compaction drives secondary structure formation or secondary structure formation facilitates compaction during folding. In this study of the folding of single-chain monellin, it is shown that a kinetic molten globule (MG) is populated at 2 ms of folding. Far-UV circular dichroism (CD) measurements show that the kinetic MG is devoid of any helical structure even under the most stabilizing folding conditions. Multisite fluorescence resonance energy transfer (FRET) measurements show that the kinetic MG is compact with different segments having contracted to different extents. It is shown that the sequence segment that goes on to form the sole helix in the native protein is fully collapsed in the kinetic MG. This segment expands to accommodate the helix as the kinetic MG folds further to the native state, while other segments of the protein contract. Helix formation starting from the kinetic MG is shown to occur in multiple kinetic steps, whether measured by far-UV CD or by FRET.

}, keywords = {Amino Acid Substitution, Circular Dichroism, Cysteine, Fluorescence Resonance Energy Transfer, Hydrophobic and Hydrophilic Interactions, Kinetics, Models, Molecular, Mutagenesis, Site-Directed, Plant Proteins, Protein Conformation, Protein Folding, Protein Stability, Protein Structure, Secondary, Spectrometry, Fluorescence}, issn = {1520-4995}, doi = {10.1021/acs.biochem.5b00730}, author = {Goluguri, Rama Reddy and Udgaonkar, Jayant B} } @article {1014, title = {Structural effects of multiple pathogenic mutations suggest a model for the initiation of misfolding of the prion protein.}, journal = {Angew Chem Int Ed Engl}, volume = {54}, year = {2015}, month = {2015 Jun 22}, pages = {7529-33}, abstract = {

A molecular understanding of the prion diseases requires delineation of the origin of misfolding of the prion protein (PrP). An understanding of how different disease-linked mutations affect the structure and dynamics of native monomeric PrP can provide a clue about how misfolding commences. In this study, hydrogen-deuterium exchange mass spectrometry was used to show that several disease-linked mutant variants, which are thermodynamically destabilized, share a common structural perturbation in their native states: helix 1 is destabilized to an extent that correlates well with the destabilization of the native protein. The mutant variants misfold and form oligomers faster than does the wild-type protein, at rates that increase exponentially with the extent to which helix 1 is destabilized in the native protein. It appears, therefore, that the loss of helix 1 structure marks the beginning of PrP misfolding and oligomerization.

}, issn = {1521-3773}, doi = {10.1002/anie.201501011}, author = {Singh, Jogender and Udgaonkar, Jayant B} } @article {1013, title = {Tuning Cooperativity on the Free Energy Landscape of Protein Folding.}, journal = {Biochemistry}, volume = {54}, year = {2015}, month = {2015 Jun 9}, pages = {3431-41}, abstract = {

Understanding the origin of the cooperativity seemingly inherent in a folding or unfolding reaction has been a major challenge. In particular, the relationship between folding cooperativity and stability is poorly understood. In this study, native state hydrogen exchange in conjunction with mass spectrometry has been used to explore the free energy landscape accessible to the small protein monellin, when the stability of the protein is varied. Mass distributions obtained in the EX1 limit of exchange have allowed a direct distinction between correlated (cooperative) and uncorrelated (noncooperative) structure-opening processes. Under conditions where the native protein is maximally stable, a continuum of partially unfolded states is gradually sampled before the globally unfolded state is transiently sampled. Under conditions that stabilize the unfolded state of the protein, the slowest structure-opening reactions leading to complete unfolding become cooperative. The present study provides experimental evidence for a gradual uphill unfolding transition on a very slow time scale, in the presence of a large free energy difference between the native and unfolded states. The results suggest that the cooperativity that manifests itself in protein folding and unfolding reactions carried out in the presence of denaturant might merely be a consequence of the effect of the denaturant on the unfolded state and transition state stabilities.

}, keywords = {Models, Chemical, Plant Proteins, Protein Folding, Protein Structure, Tertiary}, issn = {1520-4995}, doi = {10.1021/acs.biochem.5b00247}, author = {Malhotra, Pooja and Udgaonkar, Jayant B} }