The Kinetic Pathway of Folding of Barnase

Khan, Farhan R.; Chuang, Jessica I.; Gianni, Stefano; Fersht, Alan R.

doi:10.1016/j.jmb.2003.08.024

Cited by 76 publications

(93 citation statements)

References 55 publications

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“…This is well below the detection limit of the equilibrium experiments. Therefore, as in similar studies with other proteins (31,40,41,42), the equilibrium data were analyzed according to a two-state model, yielding information about the global stability of the prion protein (free energy difference between the native and fully unfolded states). Nevertheless, the close correspondence of ⌬G UN 0,kin and ⌬G UN 0,eq provides additional evidence that the sequential three-state model used to analyze kinetic data accurately describes the folding of the wild-type huPrP-(90 -231) and pathogenic variants thereof.…”

Section: Resultsmentioning

confidence: 99%

The Effect of Disease-associated Mutations on the Folding Pathway of Human Prion Protein

Apetri

Surewicz

2004

Journal of Biological Chemistry

146

137

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Propagation of transmissible spongiform encephalopathies is believed to involve the conversion of cellular prion protein, PrP C , into a misfolded oligomeric form, PrPSc . An important step toward understanding the mechanism of this conversion is to elucidate the folding pathway(s) of the prion protein. We reported recently (Apetri, A. C., and Surewicz, W. K. (2002) J. Biol. Chem. 277, 44589 -44592) that the folding of wild-type prion protein can best be described by a three-state sequential model involving a partially folded intermediate. Here we have performed kinetic stopped-flow studies for a number of recombinant prion protein variants carrying mutations associated with familial forms of prion disease. Analysis of kinetic data clearly demonstrates the presence of partially structured intermediates on the refolding pathway of each PrP variant studied. In each case, the partially folded state is at least one order of magnitude more populated than the fully unfolded state. The present study also reveals that, for the majority of PrP variants tested, mutations linked to familial prion diseases result in a pronounced increase in the thermodynamic stability, and thus the population, of the folding intermediate. These data strongly suggest that partially structured intermediates of PrP may play a crucial role in prion protein conversion, serving as direct precursors of the pathogenic PrP Sc isoform.Prions are infectious pathogens that cause a group of fatal neurodegenerative diseases known as transmissible spongiform encephalopathies (1-4). These diseases can be sporadic, inherited, or acquired by infection. The human forms of prion disorders include Creutzfeldt-Jacob disease, GerstmannStraussler-Scheinker disease, fatal familial insomnia, and kuru, whereas the animal diseases encompass bovine spongiform encephalopathy in cattle, chronic wasting disorders in deer and elk, and scrapie in sheep. The histopathological marker of all prion diseases is the accumulation in the brain of an abnormal isoform of prion protein, PrP Sc . 1 This protein is derived by a post-translational mechanism from the normal (cellular) prion protein, PrP C . According to the protein-only hypothesis, PrP Sc is the sole component of the infectious prion agent (1, 5). It is also believed that when introduced into a normal host PrP Sc induces the conversion of endogenous PrP C into PrP Sc , and that this conversion is the central event in the propagation of the disease. Although the ultimate proof for the protein-only model is still missing (6), the central role of prion protein in the propagation of transmissible spongiform encephalopathies is documented by a wealth of biochemical data as well as animal studies (1-7). Furthermore, the notion that proteins can act as infectious agents is supported by studies on prion-like phenomena in yeast and fungi (8 -10).Human PrP C is a 209-residue glycoprotein that contains a single disulfide bond and is attached to the plasma membrane via a glycosylphosphatidylinositol anchor. NMR structural studies hav...

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Section: Resultsmentioning

confidence: 99%

The Effect of Disease-associated Mutations on the Folding Pathway of Human Prion Protein

Apetri

Surewicz

2004

Journal of Biological Chemistry

146

137

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“…Observed rate constants over the urea concentration range studied are described by the sum of Eqs. 1 and 3 (47,48).…”

Section: Methodsmentioning

confidence: 99%

Comparison of successive transition states for folding reveals alternative early folding pathways of two homologous proteins

Calosci

Celestine

Richter

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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The energy landscape theory provides a general framework for describing protein folding reactions. Because a large number of studies, however, have focused on two-state proteins with single well-defined folding pathways and without detectable intermediates, the extent to which free energy landscapes are shaped up by the native topology at the early stages of the folding process has not been fully characterized experimentally. To this end, we have investigated the folding mechanisms of two homologous threestate proteins, PTP-BL PDZ2 and PSD-95 PDZ3, and compared the early and late transition states on their folding pathways. Through a combination of ⌽ value analysis and molecular dynamics simulations we obtained atomic-level structures of the transition states of these homologous three-state proteins and found that the late transition states are much more structurally similar than the early ones. Our findings thus reveal that, while the native state topology defines essentially in a unique way the late stages of folding, it leaves significant freedom to the early events, a result that reflects the funneling of the free energy landscape toward the native state.energy landscape ͉ kinetics ͉ molecular dynamics ͉ phi analysis ͉ protein folding T he description of the folding process of a protein in terms of pathways on a free energy landscape has provided much insight into the mechanism of the folding reaction (1-4). Free energy landscapes of many proteins appear to resemble the shape of a funnel that guides the folding process toward the native state (5-7). According to this view, folding is considered a stochastic process so that a protein reaches its native conformation through folding pathways made up by an ensemble of different trajectories (6,(8)(9)(10). It has been difficult, however, to establish experimentally the extent to which these trajectories can differ from each other, in particular in the wider region of the free energy funnel corresponding to the initial stages of the folding process, where a considerable heterogeneity may be expected. Many proteins exhibit single folding pathways composed of families of closely related trajectories, but parallel folding pathways have also been observed (11), as well as significant changes of pathways and transition state structures upon circular permutation (12, 13) or solvent conditions (14-16). In addition, different transition state structures were characterized in two homologous proteins with a highly symmetric native structure (17).To obtain a glimpse of the width of the free energy landscape at the early stages of the folding reaction, we compared the folding pathways of two homologous three-state proteins. The study of homologous proteins represents a powerful approach to obtain insight into the process of protein folding (18-22), especially when combined with structural information on intermediate events (17,(23)(24)(25). The transition states of two-state proteins were compared in a series of studies (17, 23, 24, 26).Here we present a vivid illustration of...

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“…(37)(38)(39). They are all proteins from organisms that grow at moderate temperatures, so they do not suffice for clarifying the mechanism of adaptation to high temperature.…”

Section: Temperature Dependence Of ∆G(h 2 O)mentioning

confidence: 99%

Kinetically Robust Monomeric Protein from a Hyperthermophile

et al. 2004

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Equilibrium and kinetic studies were carried out under denaturation conditions to clarify the energetic features of the high stability of a monomeric protein, ribonuclease HII, from a hyperthermophile, Thermococcus kodakaraensis (Tk-RNase HII). Guanidine hydrochloride (GdnHCl)-induced unfolding and refolding were measured with circular dichroism at 220 nm, and heat-induced denaturation was studied with differential scanning calorimetry. Both GdnHCl-and heat-induced denaturation are very reversible. It was difficult to obtain the equilibrated unfolding curve of Tk-RNase HII below 40°C, because of the remarkably slow unfolding. The two-state unfolding and refolding reactions attained equilibrium at 50°C after 2 weeks. The Gibbs energy change of GdnHCl-induced unfolding (∆G(H 2 O)) at 50°C was 43.6 kJ mol -1 . The denaturation temperature in the DSC measurement shifted as a function of the scan rate; the denaturation temperature at a scan rate of 90°C h -1 was higher than at a scan rate of 5°C h -1 . The unfolding and refolding kinetics of Tk-RNase HII were approximated as a first-order reaction. The ln k u and ln k r values depended linearly on the denaturant concentration between 10 and 50°C. The ∆G(H 2 O) value obtained from the rate constant in water using the two-state model at 50°C, 44.5 kJ mol -1 , was coincident with that from the equilibrium study, 43.6 kJ mol -1 , suggesting the two-state folding of TkRNase HII. The values for the rate constant in water of the unfolding for Tk-RNase HII were much smaller than those of E. coli RNase HI and Thermus thermophilus RNase HI, which has a denaturation temperature similar to that of Tk-RNase HII. In contrast, little difference was observed in the refolding rates among these proteins. These results indicate that the stabilization mechanism of monomeric protein from a hyperthermophile, Tk-RNase HII, with reversible two-state folding is characterized by remarkably slow unfolding.Microorganisms can be classified into psychrophiles, mesophiles, thermophiles, or hyperthermophiles on the basis of the differences in their optimal growth temperatures. The stabilities of proteins from these microorganisms exhibit different properties. Proteins from hyperthermophiles generally reveal greater stability than those from any other microorganisms. Therefore, proteins from hyperthermophiles are expected to provide unique information about protein folding, stability, and function (1-7).The stability of proteins in solution is evaluated by the Gibbs energy changes (∆G) upon denaturation, when they are reversible under experimental conditions. The value of ∆G is obtained from equilibrium and kinetic experiments. Studies concerning the stability of proteins from hyperthermophiles have focused primarily on the equilibrium aspects (8-13). The results suggested that extremely high stability of these proteins can be achieved by increasing the number of ionic interactions and the extent of hydrophobic surface burial. Several reports have recently appeared in which the equilibrium...

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The Kinetic Pathway of Folding of Barnase

Cited by 76 publications

References 55 publications

The Effect of Disease-associated Mutations on the Folding Pathway of Human Prion Protein

The Effect of Disease-associated Mutations on the Folding Pathway of Human Prion Protein

Comparison of successive transition states for folding reveals alternative early folding pathways of two homologous proteins

Kinetically Robust Monomeric Protein from a Hyperthermophile

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