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

Observation of multistate kinetics during the slow folding and unfolding of barstar

The kinetics of the slow folding and unfolding reactions of barstar, a bacterial ribonuclease inhibitor protein, have been studied at 23(±1)°C, pH 8, by the use of tryptophan fluorescence, far-UV circular dichroism (CD), near-UV CD, and transient mixing 1H nuclear magnetic resonance (NMR) spectroscopic measurements in the 0-4 M range of guanidine hydrochloride (GdnHCl) concentration. The denaturant dependences of the rates of folding and unfolding processes, and of the initial and final values of optical signals associated with these kinetic processes, have been determined for each of the four probes of measurement. Values determined for rates as well as amplitudes are shown to be very much probe dependent. Significant differences in the intensities and rates of appearance and disappearance of several resolved resonances in the real-time one-dimensional NMR spectra have been noted. The NMR spectra also show increasing dispersion of chemical shifts during the slow phase of refolding. The denaturant dependences of rates display characteristic folding chevrons with distinct rollovers under strongly native as well as strongly unfolding conditions. Analyses of the data and comparison of the results obtained with different probes of measurement appear to indicate the accumulation of a myriad of intermediates on parallel folding and unfolding pathways, and suggest the existence of an ensemble of transition states. The energetic stabilities of the intermediates estimated from kinetic data suggest that they are approximately half as stable as the fully folded protein. The slowness of the folding and unfolding processes (τ=10-333 s) and values of 20.5(±1.4) and 18(±0.5) kcal mol−1 for the activation energies of the slow refolding and unfolding reactions suggest that proline isomerization is involved in these reactions, and that the intermediates accumulate and are therefore detectable because the slow proline isomerization reaction serves as a kinetic trap during folding

Stopped-flow NMR measurement of hydrogen exchange rates in reduced horse cytochrome c under strongly destabilizing conditions

A procedure to measure exchange rates of fast exchanging protein amide hydrogens by time-resolved NMR spectroscopy following in situ initiation of the reaction by diluting a native protein solution into an exchanging deuterated buffer is described. The method has been used to measure exchange rates of a small set of amide hydrogens of reduced cytochrome c, maintained in a strictly anaerobic atmosphere, in the presence of an otherwise inaccessible range of guanidinium deuterochloride concentrations. The results for the measured protons indicate that hydrogen exchange in the unfolding transition region of cytochrome c reach the EX2 limit, but emphasize the difficulty in interpretation of the exchange mechanism in protein hydrogen exchange studies. Comparison of free energies of structure opening for the measured hydrogens with the global unfolding free energy monitored by far-UV CD measurements has indicated the presence of at least one partially unfolded equilibrium species of reduced cytochrome c. The results provide the first report of measurement of free energy of opening of structure to exchange in the 0-2-kcal/mol range

Folding of horse cytochrome c in the reduced state

Equilibrium and kinetic folding studies of horse cytochrome c in the reduced state have been carried out under strictly anaerobic conditions at neutral pH, 10°C, in the entire range of aqueous solubility of guanidinium hydrochloride (GdnHCl). Equilibrium unfolding transitions observed by Soret heme absorbance, excitation energy transfer from the lone tryptophan residue to the ferrous heme, and far-UV circular dichroism (CD) are all biphasic and superimposable, implying no accumulation of structural intermediates. The thermodynamic parameters obtained by two-state analysis of these transitions yielded ΔG(H2O)=18.8(±1.45) kcal mol−1, and Cm=5.1(±0.15) M GdnHCl, indicating unusual stability of reduced cytochrome c. These results have been used in conjunction with the redox potential of native cytochrome c and the known stability of oxidized cytochrome c to estimate a value of −164 mV as the redox potential of the unfolded protein. Stopped-flow kinetics of folding and unfolding have been recorded by Soret heme absorbance, and tryptophan fluorescence as observables. The refolding kinetics are monophasic in the transition region, but become biphasic as moderate to strongly native-like conditions are approached. There also is a burst folding reaction unobservable in the stopped-flow time window. Analyses of the two observable rates and their amplitudes indicate that the faster of the two rates corresponds to apparent two-state folding (U↔N) of 80-90% of unfolded molecules with a time constant in the range 190-550 μs estimated by linear extrapolation and model calculations. The remaining 10-20% of the population folds to an off-pathway intermediate, I, which is required to unfold first to the initial unfolded state, U, in order to refold correctly to the native state, N (I↔U↔N). The slower of the two observable rates, which has a positive slope in the linear functional dependence on the denaturant concentration indicating that an unfolding process under native-like conditions indeed exists, originates from the unfolding of I to U, which rate-limits the overall folding of these 10-20% of molecules. Both fast and slow rates are independent of protein concentration and pH of the refolding milieu, suggesting that the off-pathway intermediate is not a protein aggregate or trapped by heme misligation. The nature or type of unfolded-state heme ligation does not interfere with refolding. Equilibrium pH titration of the unfolded state yielded coupled ionization of the two non-native histidine ligands, H26 and H33, with a pKa value of 5.85. A substantial fraction of the unfolded population persists as the six-coordinate form even at low pH, suggesting ligation of the two methionine residues, M65 and M80. These results have been used along with the known ligand-binding properties of unfolded cytochrome c to propose a model for heme ligation dynamics. In contrast to refolding kinetics, the unfolding kinetics of reduced cytochrome c recorded by observation of Soret absorbance and tryptophan fluorescence are all slow, simple, and single-exponential. In the presence of 6.8 M GdnHCl, the unfolding time constant is ~300(±125) ms. There is no burst unfolding reaction. Simulations of the observed folding-unfolding kinetics by numerical solutions of the rate equations corresponding to the three-state I↔U↔N scheme have yielded the microscopic rate constants

Solution NMR Structure and Backbone Dynamics of Partially Disordered <i>Arabidopsis thaliana</i> Phloem Protein 16-1, a Putative mRNA Transporter

Although RNA-binding proteins in plant phloem are believed to perform long-distance systemic transport of RNA in the phloem conduit, the structure of none of them is known. <i>Arabidopsis thaliana</i> phloem protein 16-1 (<i>At</i>PP16-1) is such a putative mRNA transporter whose structure and backbone dynamics have been studied at pH 4.1 and 25 °C by high-resolution nuclear magnetic resonance spectroscopy. Results obtained using basic optical spectroscopic tools show that the protein is unstable with little secondary structure near the physiological pH of the phloem sap. Fluorescence-monitored titrations reveal that <i>At</i>PP16-1 binds not only <i>A</i>. <i>thaliana</i> RNA (<i>K</i>_diss ∼ 67 nM) but also sheared DNA and model dodecamer DNA, though the affinity for DNA is ∼15-fold lower. In the solution structure of the protein, secondary structural elements are formed by residues 3–9 (β1), 56–62 (β2), 133–135 (β3), and 96–110 (α-helix). Most of the rest of the chain segments are disordered. The N-terminally disordered regions (residues 10–55) form a small lobe, which conjoins the rest of the molecule via a deep and large irregular cleft that could have functional implications. The average order parameter extracted by model-free analysis of ¹⁵N relaxation and {¹H}–¹⁵N heteronuclear NOE data is 0.66, suggesting less restricted backbone motion. The average conformational entropy of the backbone NH vectors is −0.31 cal mol^–1 K^–1. These results also suggest structural disorder in <i>At</i>PP16-1

Dispersion Forces and the Molecular Origin of Internal Friction in Protein

Internal friction in macromolecules is one of the curious phenomena that control conformational changes and reaction rates. It is held here that dispersion interactions and London–van der Waals forces between nonbonded atoms are major contributors to internal friction. To demonstrate this, the flipping motion of aromatic rings of F10 and Y97 amino acid residues of cytochrome <i>c</i> has been studied in glycerol/water mixtures by cross relaxation-suppressed exchange nuclear magnetic resonance spectroscopy. The ring-flip rate is highly overdamped by glycerol, but this is not due to the effect of protein–solvent interactions on the Brownian dynamics of the protein, because glycerol cannot penetrate into the protein to slow the internal collective motions. Sound velocity in the protein under matching solvent conditions shows that glycerol exerts its effect by rather smothering the protein interior to produce reduced molecular compressibility and root-mean-square volume fluctuation (δ<i>V</i>_RMS), implying an increased number of dispersion interactions of nonbonded atoms. Hence, δ<i>V</i>_RMS can be used as a proxy for internal friction. By using the ansatz that internal friction is related to nonbonded interactions by the equation <i>f</i>(<i>n</i>) = <i>f</i>₀ + <i>f</i>₁<i>n</i> + <i>f</i>₂<i>n</i>² + ..., where the variable <i>n</i> is the extent of nonbonded interactions with <i>f</i>_<i>i</i> coefficients, the barrier to aromatic ring rotation is found to be flat. Also interesting is the appearance of a turnover region in the δ<i>V</i>_RMS dependence of the ring-flip rate, suggesting anomalous internal diffusion. We conclude that cohesive forces among nonbonded atoms are major contributors to the molecular origin of internal friction