Jayant B. Udgaonkar

Direct evidence for a dry molten globule intermediate during the unfolding of a small protein

Little is known about how proteins begin to unfold. In particular, how and when water molecules penetrate into the protein interior during unfolding, thereby enabling the dissolution of specific structure, is poorly understood. The hypothesis that the native state expands initially into a dry molten globule, in which tight packing interactions are broken, but whose hydrophobic core has not expanded sufficiently to be able to absorb water molecules, has very little experimental support. Here, we report our analysis of the earliest observable events during the unfolding of single chain monellin (MNEI), a small plant protein. Far- and near-UV circular dichroism measurements of GdnHCl-induced unfolding indicate that a molten globule intermediate forms initially, before the major slow unfolding reaction commences. Steady-state fluorescence resonance energy transfer measurements show that the C-terminal end of the single helix of MNEI initially moves rapidly away from the single tryptophan residue that is close to the N-terminal end of the helix. The average end-to-end distance of the protein also expands during unfolding to the molten globule intermediate. At this time, water has yet to penetrate the protein core, according to the evidence from intrinsic tryptophan fluorescence and 8-anilino-1-naphthalenesulfonic acid fluorescence-monitored kinetic unfolding measurements. Our results therefore provide direct evidence for a dry molten globule intermediate at the initial stage of unfolding. Our results further suggest that the structural transition between the native and dry molten globule states could be an all-or-none transition, whereas further swelling of the globule appears to occur gradually.

Structure is lost incrementally during the unfolding of barstar

Coincidental equilibrium unfolding transitions observed by multiple structural probes are taken to justify the modeling of protein unfolding as a two-state, N [rlhar2 ] U, cooperative process. However, for many of the large number of proteins that undergo apparently two-state equilibrium unfolding reactions, folding intermediates are detected in kinetic experiments. The small protein barstar is one such protein. Here the two-state model for equilibrium unfolding has been critically evaluated in barstar by estimating the intramolecular distance distribution by time-resolved fluorescence resonance energy transfer (TR-FRET) methods, in which fluorescence decay kinetics are analyzed by the maximum entropy method (MEM). Using a mutant form of barstar containing only Trp 53 as the fluorescence donor and a thionitrobenzoic acid moiety attached to Cys 82 as the fluorescence acceptor, the distance between the donor and acceptor has been shown to increase incrementally with increasing denaturant concentration. Although other probes, such as circular dichroism and fluorescence intensity, suggest that the labeled protein undergoes two-state equilibrium unfolding, the TR-FRET probe clearly indicates multistate equilibrium unfolding. Native protein expands progressively through a continuum of native-like forms that achieve the dimensions of a molten globule, whose heterogeneity increases with increasing denaturant concentration and which appears to be separated from the unfolded ensemble by a free energy barrier.

Multiple Routes and Structural Heterogeneity in Protein Folding

Experimental studies show that many proteins fold along sequential pathways defined by folding intermediates. An intermediate may not always be a single population of molecules but may consist of subpopulations that differ in their average structure. These subpopulations are likely to fold via independent pathways. Parallel folding and unfolding pathways appear to arise because of structural heterogeneity. For some proteins, the folding pathways can effectively switch either because different subpopulations of an intermediate get populated under different folding conditions, or because intermediates on otherwise hidden pathways get stabilized, leading to their utilization becoming discernible, or because mutations stabilize different substructures. Therefore, the same protein may fold via different pathways in different folding conditions. Multiple folding pathways make folding robust, and evolution is likely to have selected for this robustness to ensure that a protein will fold under the varying conditions prevalent in different cellular contexts.

Salt-Induced Modulation of the Pathway of Amyloid Fibril Formation by the Mouse Prion Protein

To investigate how the heterogeneity inherent in the formation of worm-like amyloid fibrils by the mouse prion protein is modulated by a change in aggregation conditions, as well as to determine how heterogeneity in reaction leads to heterogeneity in structure, the amyloid fibril formation reaction of the protein at low pH was studied in the presence of various salts. It is shown that beta-rich oligomers of different sizes and structures are formed at low and high NaCl concentrations, as determined by Fourier transfer infrared (FTIR) spectroscopy and dynamic light scattering (DLS). The worm-like fibrils formed from the beta-rich oligomers at low and high NaCl concentrations also differ in their internal structure, as determined by FTIR measurements. The apparent rate constant for the formation of the worm-like amyloid fibrils shows a very steep sigmoidal dependence on NaCl concentration, suggesting that the effect occurs because of the binding of many ions. The effect of salt in modulating the kinetics of worm-like fibril formation occurs at ionic strengths below 200 mM, over different concentration ranges for different salts, and is shown to depend not only on the ionic strength but also on the nature of the anion. The ability of different anions to promote worm-like fibril formation does not follow the Hofmeister series but instead follows the electroselectivity series for anion binding. Hence, it appears that the effect of salt is because of the linkage of the aggregation reaction to anion binding to the protein. A comparison of the apparent rate constants measured from the changes in thioflavin T fluorescence, circular dichroism, and DLS, which occur during worm-like fibril formation, suggests that conformational conversion follows fibril elongation at low NaCl concentration and follows fibril formation at high NaCl concentration.

Evidence for stepwise formation of amyloid fibrils by the mouse prion protein.

The full-length mouse prion protein, moPrP, is shown to form worm-like amyloid fibrils at pH 2 in the presence of 0.15 M NaCl, in a slow process that is accelerated at higher temperatures. Upon reduction in pH to 2, native moPrP transforms into a mixture of soluble beta-rich oligomers and alpha-rich monomers, which exist in a slow, concentration-dependent equilibrium with each other. It is shown that only the beta-rich oligomers and not the alpha-rich monomers, can form worm-like amyloid fibrils. The mechanism of formation of the worm-like amyloid fibrils from the beta-rich oligomers has been studied with four different physical probes over a range of temperatures and over a range of protein concentrations. The observed rate of fibrillation is the same, whether measured by changes in ellipticity at 216 nm, in thioflavin fluorescence upon binding, or in the mean hydrodynamic radius. The observed rate is significantly slower when monitored by total scattering intensity, suggesting that lateral association of the worm-like fibrils occurs after they form. The activation energy for worm-like fibril formation was determined to be 129 kJ/mol. The observed rate of fibrillation increases with an increase in protein concentration, but saturates at protein concentrations above 50 microM. The dependence of the observed rate of fibrillation on protein concentration suggests that aggregate growth is rate-limiting at low protein concentration and that conformational change, which is independent of protein concentration, becomes rate-limiting at higher protein concentrations. Hence, fibril formation by moPrP occurs in at least two separate steps. Longer but fewer worm-like fibrils are seen to form at low protein concentration, and shorter but more worm-like fibrils are seen to form at higher protein concentrations. This observation suggests that the beta-rich oligomers grow progressively in size to form critical higher order-oligomers from which the worm-like amyloid fibrils then form.

Continuous dissolution of structure during the unfolding of a small protein

The unfolding kinetics of many small proteins appears to be first order, when measured by ensemble-averaging probes such as fluorescence and circular dichroism. For one such protein, monellin, it is shown here that hidden behind this deceptive simplicity is a complexity that becomes evident with the use of experimental probes that are able to discriminate between different conformations in an ensemble of structures. In this study, the unfolding of monellin has been probed by measurement of the changes in the distributions of 4 different intramolecular distances, using a multisite, time-resolved fluorescence resonance energy transfer methodology. During the course of unfolding, the protein molecules are seen to undergo slow and continuous, diffusive swelling. The swelling process can be modeled as the slow diffusive swelling of a Rouse-like chain with some additional noncovalent, intramolecular interactions. Here, we show that specific structure is lost during the swelling process gradually, and not in an all-or-none manner, during unfolding.

Understanding the Kinetic Roles of the Inducer Heparin and of Rod-like Protofibrils during Amyloid Fibril Formation by Tau Protein

Background: The kinetic role of heparin and of intermediates populated during Tau protein aggregation is not fully understood. Results: Heparin contributes to the initial steps of fibrillation, whereas protofibrillar intermediates accumulate transiently. Conclusion: Heparin is involved in nucleation, and the transient rod-like protofibrils are off-pathway species. Significance: Understanding the kinetic role of heparin and the protofibrils has implications for the development of therapies for tauopathies. The aggregation of the natively disordered protein, Tau, to form lesions called neurofibrillary tangles is a characteristic feature of several neurodegenerative tauopathies. The polyanion, heparin, is commonly used as an inducer in studies of Tau aggregation in vitro, but there is surprisingly no comprehensive model describing, quantitatively, all aspects of the heparin-induced aggregation reaction. In this study, rate constants and extents of fibril formation by the four repeat domain of Tau (Tau4RD) have been reproducibly determined over a full range of heparin and protein concentrations. The kinetic role of heparin in the nucleation-dependent fibril formation reaction is shown to be limited to participation in the initial rate-limiting steps; a single heparin molecule binds two Tau4RD molecules, forming an aggregation-competent protein dimer, which then serves as a building block for further fibril growth. Importantly, the minimal kinetic model that is proposed can quantitatively account for the characteristic bell-shaped dependence of the aggregation kinetics on the stoichiometry of protein to heparin. Very importantly, this study also identifies for the first time short and thin, rod-like protofibrils that are populated transiently, early during the time course of fibril formation. The identification of these protofibrils as bona fide off-pathway species has implications for the development of therapies for tauopathies based on driving fibril formation as a means of protecting the cell from smaller, putatively toxic aggregates.

Conformational Conversion May Precede or Follow Aggregate Elongation on Alternative Pathways of Amyloid Protofibril Formation

A major goal in the study of protein aggregation is to understand how the conformational heterogeneity characteristic of the process leads to structurally distinct amyloid fibrils. The small protein barstar is known to form amyloid protofibrils in multiple steps at low pH: a small oligomer, the A-form, first transforms into a larger spherical higher oligomeric intermediate (HOI), which then self-associates to form the elongated protofibril. To determine how the conformational conversion reaction during aggregation is coupled to the process of protofibril formation, cysteine-scanning mutagenesis was first used to identify specific residue positions in the protein sequence, which are important in defining the nature of the aggregation process. Two classes of mutant proteins, which are distinguished by their kinetics of aggregation at high protein concentration, have been identified: Class I mutant proteins undergo conformational conversion, as measured by an increase in thioflavin T binding ability and an increase in circular dichroism at 216 nm, significantly faster than Class II mutant proteins. At low protein concentration, the rates of conformational conversion are, however, identical for both classes of mutant proteins. At high protein concentration, the two classes of mutant proteins can be further distinguished on the basis of their rates of protofibril growth, as determined from dynamic light-scattering measurements. For Class I mutant proteins, protofibril elongation occurs at the same, or slightly faster, rate than conformational conversion. For Class II mutant proteins, protofibril elongation is significantly slower than conformational conversion. Dynamic light scattering measurements and atomic force microscopy imaging indicate that for the Class I mutant proteins, conformational conversion occurs concurrently with the self-association of prefibrillar HOIs into protofibrils. On the other hand, for the Class II mutant proteins, the prefibrillar HOI first undergoes conformational conversion, and the conformationally converted HOIs then self-associate to form protofibrils. The two classes of mutant proteins appear, therefore, to use structurally distinct pathways to form amyloid protofibrils. On one pathway, conformational conversion occurs along with, or after, elongation of the oligomers; on the other pathway, conformational conversion precedes elongation of the oligomers. Single mutations in the protein can cause aggregation to switch from one pathway to the other. Importantly, the protofibrils formed by the two classes of mutant proteins have significantly different diameters and different internal structures.

Structurally Distinct Amyloid Protofibrils Form on Separate Pathways of Aggregation of a Small Protein

Understanding the structural as well as mechanistic basis of the conformational polymorphism evident during amyloid protofibril and fibril formation by proteins is an important goal in the study of protein aggregation. In this report, we compare two separate routes to amyloid protofibril formation by the small protein barstar, one induced by the addition of trifluoroethanol (TFE) and the other by heat. The study reveals that the TFE-induced aggregation of barstar leads to protofibrils that differ from heat-induced protofibrils in their external dimensions and internal structures as well as in the mechanisms of their formation. Atomic force microscopy reveals that the TFE-induced protofibrils have about half the thickness of the heat-induced protofibrils. The thickness of the TFE-induced protofibrils (1.14 +/- 0.24) suggests that they form a beta-sheet monolayer, while the thickness of the heat-induced protofibrils (2.56 +/- 0.32) suggests that they are built up from a pair (bilayer) of beta-sheets. Fourier-transform infrared (FTIR) as well as circular dichroism (CD) spectroscopy shows that the heat-induced protofibrils are not pure beta-sheet structures but that they also contain other structures (alpha-helix and/or random coil). In contrast, the TFE-induced protofibrils contain more beta-sheet structures and less of other structures, if any. The FTIR and CD spectra also reveal that the two differently created protofibrils differ in the internal structures of their beta-sheets. The TFE-induced protofibrils differ from the heat-induced protofibrils also in the kinetics of their formation. For the heat-induced reaction, the kinetics are monophasic without any lag phase, while the kinetics of the formation of TFE-induced protofibrils are sigmoidal with an initial lag phase. It appears that the TFE-induced and the heat-induced reactions involve distinct pathways for the formation of amyloid protofibrils. The existence of alternative pathways leading to amyloid protofibrils of distinct structures has important implications in understanding the kinetic origin of amyloid polymorphism.

Exploring the cooperativity of the fast folding reaction of a small protein using pulsed thiol labeling and mass spectrometry

It has been difficult to obtain directly residue-specific information on side chain packing during a fast (ms) protein folding reaction. Such information is necessary to determine the extent to which structural changes in different parts of the protein molecule are coupled together in defining the cooperativity of the overall folding transition. In this study, structural changes occurring during the major fast folding reaction of the small protein barstar have been characterized at the level of individual residue side chains. A pulsed cysteine labeling methodology has been employed in conjunction with mass spectrometry. This provides, with ms temporal resolution, direct information on structure formation at 10 different locations in barstar during its folding. Cysteine residues located on the surface of native barstar, at four different positions, remain fully solvent-accessible throughout the folding process, indicating the absence of any ephemeral nonnative structure in which these four cysteine residues get transiently buried. For buried cysteine residues, the rates of the change in cysteine-thiol accessibility to rapid chemical labeling by the thiol reagent methyl methanethiosulfonate appear to be dependent upon the location of the cysteine residue in the protein and are different from the rate measured by the change in tryptophan fluorescence. But the rates vary over only a 3-fold range. Nevertheless, a comparison of the kinetics of the change in accessibility of the cysteine 3 thiol with those of the change in the fluorescence of tryptophan 53, as well as of their denaturant dependences, indicates that the major folding reaction comprises more than one step.