Evidence for a molten globule-like transition state in protein folding from determination of activation volumes

Vidugiris, Gediminas; Markley, John L.; Royer, Catherine A.

doi:10.1021/bi00015a001

Cited by 171 publications

(189 citation statements)

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“…Analysis of the curves of fluorescence intensity vs. pressure p was performed using the analysis program BIOEQS (Royer et al, 1991 ;Royer & Beechem, 1992;Royer, 1993) fitting the intensity data in terms of a two-state transition from the native to the fully unfolded state (F + U) to yield a free energy AG, and volume change AV, for the transitions, as previously described (Vidugiris et al, 1995). AG = -RTlnK,, = -RTIn((I(f)-I ( p ) ) / ( Z ( p ) -I ( u ) ) ) (1) and where I ( f ) , I ( u ) , and I ( p ) correspond to the fluorescence intensities of the native and unfolded states and that at pressure p .…”

Section: Discussionmentioning

confidence: 99%

“…Snase is a small, single domain monomeric protein with no disulfide bridges that folds in a two-state manner in equilibrium unfolding assays (Shortle & Meeker, 1986;Carra & Privalov, 1995). In fact, although the kinetics of Snase folding are complex at atmospheric pressure (Chen et al, 199 1, 1992: Su et al, 1996, this complexity disappears with pressure, presumably because the intermediate species occupy larger system volumes than either the folded or unfolded states and thus are destabilized by pressure (Vidugiris et al, 1995(Vidugiris et al, , 1996. Particularly useful to our studies is the fact that Snase is relatively unstable, such that with a AV,, of -77 k 8 mL/mol, at pH 5.5, the midpoint of the folding transition is 1.5 kbar, well within the pressure range available in our pressure apparatus ( I atm, 2.5 kbar).…”

Section: Kj F T Y and Ca Royermentioning

confidence: 99%

“…Moreover they found that the volume change was independent of pressure. Negative values of AVu have also been determined for lysozyme (Li et al, 1976;Samarasinghe et al, 1992), chymotrypsinogen (Hawley, 1971;Li et al, 1976;Wong & Heremans, 1988), myoglobin (Marden et'al., 1986), ovalbumin (Suzuki et al, 1963), ribonuclease A (Takeda et al, 1995), and staphylococcal nuclease and a series of mutants of this protein (Eftink et al, 1991;Royer et al, 1993;Vidugiris et al, 1995;Eftink & Ramsay, 1996;Frye et al, 1996;Frye & Royer, 1997).…”

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Probing the contribution of internal cavities to the volume change of protein unfolding under pressure

Frye¹,

Royer²

1998

Protein Science

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The structural origin of the decrease in system volume upon protein denaturation by pressure has remained a puzzle for decades. This negative volume change upon unfolding is assumed to arise globally from more intimate interactions between the polypeptide chain and water, including electrostriction of buried charges that become exposed upon unfolding, hydration of the polypeptide backbone and amino acid side chains and elimination of packing defects and internal void volumes upon unfolding of the chain. However, the relative signs and magnitudes of each of these contributing factors have not been experimentally determined. Our laboratory has probed the fundamental basis for the volume change upon unfolding of staphylococcal nuclease (Snase) using variable solution conditions and point mutants of Snase (Royer CA et al., 1993, Biochemistry 325222-5232; Frye KJ et al., 1996, Biochemistry 35:10234-10239). Our prior results indicate that for Snase, neither electrostriction nor polar or nonpolar hydration contributes significantly to the value of the volume change of unfolding. In the present work, we investigate the pressure induced unfolding of three point mutants of Snase in which internal cavity size is altered. The experimentally determined volume changes of unfolding for the mutants suggest that loss of internal void volume upon unfolding represents the major contributing factor to the value of the volume change of Snase unfolding.Keywords: high pressure; protein folding; staphylococcal nuclease; volume change Understanding protein folding remains one of the most challenging problems in modem molecular biophysics. Considerable progress has been made in the last decade in the characterization of the fundamental thermodynamic and kinetic aspects of the process, as well as in the structural characterization of the various states, native, intermediate, and unfolded, implicated in the transitions. From a thermodynamic point of view, the fundamental parameter in protein temperature stability, the heat capacity change at constant pressure, which is responsible for the temperature dependence of the entropy, enthalpy, and free energy of folding, has been parameterized and can now be modeled adequately, in terms of the changes in exposure of hydrophobic and hydrophilic moieties of particular proteins based on their sequences and structures (Privalov & Gill, 1988;Murphy & Freire, 1992).Athorough understanding of the thermodynamics of protein folding requires, in addition to the temperature effects, a fundamental comprehension of pressure effects as well. However, in terms of the behavior of proteins under pressure, our level of understanding is much less advanced than in the field of temperature denaturation. (For reviews of pressure effects on proteins see Jaenicke, 1981; HerReprint requests to: Catherine A. Royer, Centre de Biochimie Structurale, INSERM U414, Facultt de Pharmacie, 15 ave. Ch. Flahault, 34060 Montpellier CEDEX, France; e-mail: royer@tome.cbs.univ-montpl.fr. emans, 1982;Weber & Drickamer, 1983;Si...

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Section: Discussionmentioning

confidence: 99%

Section: Kj F T Y and Ca Royermentioning

confidence: 99%

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Probing the contribution of internal cavities to the volume change of protein unfolding under pressure

Frye¹,

Royer²

1998

Protein Science

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“…but also the final stage of cluster formation (folding of a protein) because of the transient increase in the molecular surface at the point at which water molecules are excluded from the interior of the cluster (protein core). Measurements of the pressure dependence of the rates of folding and unfolding of staphylococcal nuclease support this view: there is a considerable increase in volume of the protein solvent system at the transition state (Vidugiris et al, 1995). A barrier consisting of an expanded water-excluding state (which might resemble Fig.…”

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confidence: 91%

A desolvation barrier to hydrophobic cluster formation may contribute to the rate‐limiting step in protein folding

1997

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To gain insight into the free energy changes accompanying protein hydrophobic core formation, we have used computer simulations to study the formation of small clusters of nonpolar solutes in water. A barrier to association is observed at the largest solute separation that does not allow substantial solvent penetration. The barrier reflects an effective increase in the size of the cavity occupied by the expanded but water-excluding cluster relative to both the close-packed cluster and the fully solvated separated solutes; a similar effect may contribute to the barrier to protein foldinghnfolding. Importantly for the simulation of protein folding without explicit solvent, we find that the interactions between nonpolar solutes of varying size and number can be approximated by a linear function of the molecular surface, but not the solvent-accessible surface of the solutes. Comparison of the free energy of cluster formation to that of dimer formation suggests that the assumption of pair additivity implicit in current protein database derived potentials may be in error. Keywords: hydrophobic interaction; potential of mean force; protein foldingThe hydrophobic interaction is extremely important in the folding and stabilization of proteins (Kauzmann, 1959;Dill, 1990), but is relatively poorly understood. In spite of this poor understanding, computer modeling of protein folding has developed substantially over the past IO years. One approach is to simulate protein folding in the presence of explicitly modeled water molecules, which, depending upon the accuracy of the water model, should at least in part reproduce the hydrophobic interaction. Molecular dynamics simulations with an explicit solvent model have provided valuable insights into possible foldinghnfolding trajectories (Daggett & Levitt, 1994;Karplus & Sali, 1995). However, because of the complexity of the processes being simulated and the absence of an obvious reaction coordinate, most studies have focused on qualitative features of the trajectories and not sought to calculate free energy changes (the study of Brooks and Boczko [I9951 is an exception). A second approach avoids the complexity and high computational demands of simulations with hundreds of explicit water molecules by deriving effective potentials from the distribution of amino acid residues in known protein structures (reviewed in Jones & Thornton, 1996). Although not modeled explicitly, the hydrophobic interaction dominates such "knowledge-based' potentials. These relationships are not true interaction potentials in a rigorous sense, but they have been extremely useful in a wide variety of applications, including protein fold recognition (Jones &

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“…To prove this hypothesis, a high pressure and particularly the pressure-jump technique (43)(44)(45)(46) was applied to study the activation energy parameters and structural changes involved in mature amyloid fibril disassembly by using the recombinant prion protein (PrP) 3 increase in pressure forced PrP amyloid fibrils to change irreversibly to a new, less cytotoxic state that was still partly fibrillar but lacked the typical structural features of amyloids. The analysis of the relaxation kinetics toward this new state gave information about the reaction mechanism and the transition state ensemble.…”

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Amyloid Features and Neuronal Toxicity of Mature Prion Fibrils Are Highly Sensitive to High Pressure

Moustaine

Perrier

et al. 2011

Journal of Biological Chemistry

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Prion proteins (PrP) can aggregate into toxic and possibly infectious amyloid fibrils. This particular macrostructure confers on them an extreme and still unexplained stability. To provide mechanistic insights into this self-assembly process, we used high pressure as a thermodynamic tool for perturbing the structure of mature amyloid fibrils that were prepared from recombinant full-length mouse PrP. Application of high pressure led to irreversible loss of several specific amyloid features, such as thioflavin T and 8-anilino-1-naphthalene sulfonate binding, alteration of the characteristic proteinase K digestion pattern, and a significant decrease in the ␤-sheet structure and cytotoxicity of amyloid fibrils. Partial disaggregation of the mature fibrils into monomeric soluble PrP was observed. The remaining amyloid fibrils underwent a change in secondary structure that led to morphologically different fibrils composed of a reduced number of proto-filaments. The kinetics of these reactions was studied by recording the pressure-induced dissociation of thioflavin T from the amyloid fibrils. Analysis of the pressure and temperature dependence of the relaxation rates revealed partly unstructured and hydrated kinetic transition states and highlighted the importance of collapsing and hydrating inter-and intramolecular cavities to overcome the high free energy barrier that stabilizes amyloid fibrils.Amyloid fibrils are filamentous polypeptide aggregates that are associated with devastating disorders such as Alzheimer and Parkinson diseases, type II diabetes, and prion (proteinaceous infectious particle) diseases, including Creutzfeldt-Jakob and mad cow disease (1, 2). There is increasing evidence that under appropriate conditions the amyloid state is accessible also to many other proteins that are not related to diseases, suggesting that the amyloid state is a generic structural feature, which might be adopted by any polypeptide chain (3-6).The development of in vitro model systems together with various biophysical and biochemical techniques (7-9) has improved the knowledge on the physicochemical basis of amyloid formation as well as on their structural and biochemical properties. It is now well recognized that a common property of amyloid fibrils is the extensive stacking of intermolecular ␤-strands that are arranged perpendicularly to the fibril axis and stabilized by a dense network of non-covalent interactions (10 -13). These fibrils consist of a variable number and arrangement of thin assemblies called proto-filaments that give rise to different fibril morphologies of diameters between 5 and 30 nanometers, both in in vitro preparations or in tissues (14 -22). Fibrils and their precursors are generally cytotoxic (23, 24) and are, thus, thought to be responsible for the neurodegeneration that is associated with many amyloid diseases.Yet the fundamental parameters that govern the protein aggregation process and dictate fibril stability/clearance are not well known. As the study of protein folding has been greatly advanc...

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Evidence for a molten globule-like transition state in protein folding from determination of activation volumes

Cited by 171 publications

References 17 publications

Probing the contribution of internal cavities to the volume change of protein unfolding under pressure

Probing the contribution of internal cavities to the volume change of protein unfolding under pressure

A desolvation barrier to hydrophobic cluster formation may contribute to the rate‐limiting step in protein folding

Amyloid Features and Neuronal Toxicity of Mature Prion Fibrils Are Highly Sensitive to High Pressure

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