Saninha: mulher adúltera na Belle Époque

Ana Emília Ribeiro (1876-1951) responsabilizada pela sociedade brasileira pelo homicídio de seu esposo Euclides da Cunha, viveu como julgada e condenada, talvez por não ter se subjugado ao imaginário de mulher do projeto político dos primeiros anos da República Brasileira. Em sua trajetória, Saninha, como Ana Emília era conhecida, nos serve de exemplo para refletir sobre as duras penas sofridas por uma mulher que desrespeitasse as imposições da sociedade na Belle Époque carioca. Na luta pelo que acreditava ser o melhor para sua vida esta mulher foi rejeitada, exilada, exposta em sua intimidade publicamente por toda alta sociedade do período. Traída e abandonada por aquele a quem dedicou o seu grande amor, morreu solitária sem nunca ter se defendido publicamente

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⇋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

Polypeptide chain collapse and protein folding

Polypeptide chain collapse is an integral component of a protein folding reaction. In this review, experimental characterization of the interplay of polypeptide chain collapse, secondary structure formation, consolidation of the hydrophobic core and the development of tertiary interactions, is scrutinized. In particular, the polypeptide chain collapse reaction is examined in the context of the three phenomenological models of protein folding – the hydrophobic collapse model, the framework model and the nucleation condensation model – which describe different ways by which polypeptide chains are able to fold in biologically relevant time-scales

Surface expansion is independent of and occurs faster than core solvation during the unfolding of barstar

The denaturant-induced unfolding kinetics of the 89-residue protein, barstar, have been examined using fluorescence resonance energy transfer (FRET) at 25°C and pH 8.0. The core tryptophan, Trp53, in barstar serves as a fluorescence donor, and a thionitrobenzoic acid moiety (TNB) attached to a cysteine residue acts as an acceptor to form an efficient FRET pair. Four different single-cysteine containing mutants of barstar with cysteine residues at positions 25, 40, 62, and 82 were studied. The unfolding kinetics of the four mutant forms of barstar were monitored by measurement of the changes in the fluorescence intensity of Trp53 in the unlabeled and TNB-labeled proteins. The rate of change of fluorescence of the single-tryptophan residue, Trp53, in the unlabeled protein, where no FRET occurs, yields the rate of solvation of the core. This rate is similar for all four unlabeled proteins. The rate of the increase in the fluorescence of Trp53 in the labeled protein, where FRET from the tryptophan to the TNB label occurs, yields the rate of decrease in FRET efficiency during unfolding. The decrease in FRET efficiency for proteins labeled at either of the two buried positions (Cys40 or Cys82) occurs at a rate similar to the rate of core solvation. The decrease in FRET efficiency for the acceptor at Cys40 is also shown to be sensitive to the isomerization of the Tyr47-Pro48 cis bond. For the proteins where the label is at a solvent-exposed position (Cys25 and Cys62), the decrease in FRET efficiency occurs in two kinetic phases; 15-25% of the FRET efficiency decreases in the faster phase, and the remaining FRET efficiency decreases in a slower phase, the rate of which is the same as the rate of core solvation. These results clearly indicate that, during unfolding, the protein surface expands faster than, and independently of, water intrusion into the core

Differential salt-induced stabilization of structure in the initial folding intermediate ensemble of barstar

The effects of two salts, KCl and MgCl2, on the stability and folding kinetics of barstar have been studied at pH 8. Equilibrium urea unfolding curves were used to show that the free energy of unfolding, ΔGUN, of barstar increased from a value of 4.7 kcal mol−1 in the absence of salt to a value of 6.9 kcal mol−1 in the presence of 1 M KCl or 1 M MgCl2. For both salts, ΔGUN increases linearly with an increase in concentration of salt from 0 M to 1 M, suggesting that stabilization of the native state occurs primarily through a Hofmeister effect. Refolding kinetics were studied in detail in the presence of 1 M KCl as well as in the presence of 1 M MgCl2, and it is shown that the basic folding mechanism is not altered upon addition of salt. The major effects on the refolding kinetics can be attributed to the stabilization of the initial burst phase ensemble, IE, by salt. Stabilization of structure in IE by KCl causes the fluorescence properties of IE to change, so that there is an initial burst phase change in fluorescence at 320 nm, during refolding. The structure in IE is stabilized by MgCl2, but no burst phase change in fluorescence at 320 nm is observed during refolding. The fluorescence emission spectra of IE show that when refolding is initiated in 1 M KCl, the three tryptophan residues in IE are less solvent exposed than when folding is initiated in 1 M MgCl2. Stabilization of IE leads to an acceleration in the rate of the fast observable phase of folding by both salts, suggesting that structure of the transition state resembles that of IE. The stabilization of IE by salts can be accounted for largely by the same mechanism that accounts for the stabilization of the native state of the protein, namely through the Hofmeister effect. The salts do not affect the rates of the slower phases of folding, indicating that the late intermediate ensemble, IL, is not stabilized by salts. Stabilization of the native state results in deceleration of the fast unfolding rate, which has virtually no dependence on the concentration of KCl or MgCl2 at high concentrations. The observation that the salt-induced stabilization of structure in IE is accompanied by an acceleration in the fast folding rate, suggests that IE is likely to be a productive on-pathway intermediate

Folding of tryptophan mutants of barstar: evidence for an initialhydrophobic collapse on the folding pathway

1997X ABSTRACT: The contributions of the three tryptophan residues of barstar to the spectroscopic properties, stability, and folding of the protein have been studied by mutating two of the tryptophans, Trp38 and Trp44, individually as well as together, to phenylalanines, Phe. The three mutant proteins studied are shown to be similar to wt barstar in structure by activity measurements as well as by spectroscopic characterization. Fluorescence energy transfer between the tryptophans as well as quenching by their local structural environments complicates the analysis of the contributions of the individual tryptophans to the fluorescence of the wt protein, but it is demonstrated that Trp53, which is completely buried within the hydrophobic core, makes the dominant contribution to the fluorescence, while the fluorescence of Trp38 is largely quenched in the fully folded protein. GdnHCl- as well as temperature-induced equilibrium unfolding studies, using three different structural probes, indicate that W38FW44F, where both Trp38 and Trp44 have been removed, follows a two-state unfolding transition and is less stable than the wt barstar. The fluorescence-monitored folding and unfolding kinetics of W38FW44F have been studied in detail. W38FW44F folds 2-fold faster and unfolds 3-fold faster than wt barstar. A large fraction of the total fluorescence change that occurs during folding occurs in a burst phase within 4 ms after commencement of folding. A similar burst phase change in fluorescence, although to a smaller extent, is shown to occu

Folding subdomains of thioredoxin characterized by native-state hydrogen exchange

Native-state hydrogen exchange (HX) studies, used in conjunction with NMR spectroscopy, have been carried out on Escherichia coli thioredoxin (Trx) for characterizing two folding subdomains of the protein. The backbone amide protons of only the slowest-exchanging 24 amino acid residues, of a total of 108 amino acid residues, could be followed at pH 7. The free energy of the opening event that results in an amide hydrogen exchanging with solvent (ΔGop) was determined at each of the 24 amide hydrogen sites. The values of ΔGop for the amide hydrogens belonging to residues in the helices α1, α2, and α4 are consistent with them exchanging with the solvent only when the fully unfolded state is sampled transiently under native conditions. The denaturant-dependences of the values of ΔGop provide very little evidence that the protein samples partially unfolded forms, lower in energy than the unfolded state. The amide hydrogens belonging to the residues in the β strands, which form the core of the protein, appear to have higher values of ΔGop than amide hydrogens belonging to residues in the helices, suggesting that they might be more stable to exchange. This apparently higher stability to HX of the β strands might be either because they exchange out their amide hydrogens in a high energy intermediate preceding the globally unfolded state, or, more likely, because they form residual structure in the globally unfolded state. In either case, the central β strands—β3, β2, and β4—would appear to form a cooperatively folding subunit of the protein. The native-state HX methodology has made it possible to characterize the free energy landscape that Trx can sample under equilibrium native conditions

Stabilization of barstar by chemical modification of the buried cysteines

The internal packing of residues in the small monomeric protein barstar was severely perturbed by chemical modification of the two buried cysteine residues with the thiol reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) after prior unfolding of the protein using guanidine hydrochloride (GdnHCl). The modification produces mixed disulfides between 5-thio(2-nitrobenzoic acid) and the two Cys residues. To understand the effects of the modification of the individual cysteine residues, Cys40 and Cys82, the modification was also carried out on the two single Cys→Ala mutant forms of barstar, C40A and C82A, whose structures, activities, and stabilities were first shown to be similar to those of wt barstar. Equilibrium GdnHCl-induced denaturation studies on wt barstar show that the modification causes the midpoint of the denaturation curve to increase by 0.6 M and the stability to increase by 1.3 kcal mol<SUP>−1</SUP>. Both C40A and C82A also denature at higher concentrations of GdnHCl after modification. Modification of Cys40 has approximately the same stabilizing contribution as does modification of Cys82. The structures of the modified and unmodified proteins have been compared using circular dichroism (CD) spectroscopy, UV difference absorption spectroscopy, and fluorescence spectroscopy. It is shown that the 5-thio(2-nitrobenzoic acid) groups introduced by reaction with DTNB are buried in hydrophobic environments in the modified C40A and C82A mutant proteins, as well as in modified wt barstar. The far-UV CD spectra of the modified and unmodified proteins are similar, but the mean residue ellipticity at 220 nm of wt barstar is reduced by 30% upon modification. Such a decrease is not seen for either C40A or C82A. The barnase-inhibiting activities of the three modified proteins are shown to be similar to those of the corresponding unmodified proteins. Thus, the severe perturbations of the internal packing, which result in a significant increase in stability, do not appear to affect the overall fold of barstar