Energetic Evidence for Formation of a pH-dependent Hydrophobic Cluster in the Denatured State of Thermus thermophilus Ribonuclease H

Guzmán-Casado, Mercedes; Parody-Morreale, Antonio; Robic, Srebrenka; Marqusee, Susan; Sanchez-Ruiz, José M.

doi:10.1016/s0022-2836(03)00513-8

Cited by 77 publications

(83 citation statements)

References 47 publications

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“…DSC experiments, calculation of absolute heat capacities, and variablebarrier analyses were carried out as described (13,59,60). Electrostatic calculations based on the Tanford-Kirkwood model were carried out as we have previously described in detail (19,39).…”

Section: See Supporting Information (Si) Materials and Methods For A mentioning

confidence: 99%

Large-scale modulation of thermodynamic protein folding barriers linked to electrostatics

Halskau

Pérez-Jiménez

Ibarra‐Molero

et al. 2008

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

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natural selection ͉ homologous proteins ͉ structural pK shifts ͉ conformational ensembles ͉ differential scanning calorimetry C onventionally, the folding and function of single-domain proteins are described as two-state processes in analogy to elementary chemical reactions. The protein molecule is assumed to reside in either of two states: folded or active and unfolded or inactive, which interconvert by crossing a high free-energy barrier with transition-state-like kinetics. However, proteins can in principle exist in many different conformations or microstates because of their thousands of degrees of freedom. Such inherent complexity is best described by using low-dimensional freeenergy surfaces, which are obtained by projecting the solventaveraged energy as a function of atomic coordinates onto a few order parameters [the energy landscape approach (1)]. A freeenergy surface description includes the two-state folding model as a particular scenario, but is more general. In this approach barriers, basins of attraction, and even finer topographical details of the surface (i.e., roughness) emerge from detailed balancing between conformational entropy and the energy from stabilizing interactions (see refs. 2 and 3 for some specific examples). Therefore, surfaces that exhibit marginal barriers or are even completely barrierless appear as interesting alternatives to the two-state folding picture (1). These predictions have been confirmed experimentally in recent years (4-6).If the folding barriers are comparable to the thermal energy (RT), ensembles of conformations with an intermediate degree of structure become significantly populated and interconvert with diffusive dynamics. This realization opens a realm of possibilities for the experimental study of protein folding reactions and mechanisms (recently reviewed in ref. 7). Moreover, it also has important implications for protein function because the conformational fluctuations associated with marginal folding barriers could be exploited to modulate activity and/or synchronize the action of enzymes in sequential reactions (8). Examples of how this could be achieved have been recently explored for protein binding with the fly-cast model (9, 10) and related analyses (11), as well as for the optimization of allosteric coupling (12).Another important consequence of shallow free-energy surfaces is that their shape can be resolved in equilibrium experiments sensitive to the energy fluctuations associated with protein conformation, such as differential scanning calorimetry (DSC). Building on this idea, Muñoz and Sanchez-Ruiz have recently developed a phenomenological variable-barrier model for the analysis of DSC data (13). In this analysis, the DSC thermogram is fitted to an idealized (i.e., a Landau quartic polynomial) one-dimensional free-energy surface from which it is possible to estimate the barrier height and the population of conformational ensembles with an intermediate degree of structure (13). Although these folding barriers are intrinsically ''thermodynamic,'' ...

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Section: See Supporting Information (Si) Materials and Methods For A mentioning

confidence: 99%

Large-scale modulation of thermodynamic protein folding barriers linked to electrostatics

Halskau

Pérez-Jiménez

Ibarra‐Molero

et al. 2008

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

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“…This is an important observation since it shows that DSE electrostatic interactions can make large contributions to protein stability. Significant DSE electrostatic interactions have also been observed in RNase H and ribonuclease Sa [29][30][31]. The case of RNase H is particularly interesting because it provides an example of the possible functional consequences of DSE structure.…”

Section: Electrostatic Interactions Are Found In the Denatured State mentioning

confidence: 99%

“…Calorimetric studies show that the difference is due to DSE effects. The reduced ΔC p o for thermophilic RNase H is due a pHdependent hydrophobic cluster in the DSE [29]. The detailed studies of the Pace group on ribonuclease Sa has shown that mutation of a single surface exposed Asp significantly stabilized the protein by disrupting non-native DSE electrostatic interactions.…”

Section: Electrostatic Interactions Are Found In the Denatured State mentioning

confidence: 99%

Electrostatic interactions in the denatured state ensemble: Their effect upon protein folding and protein stability

Cho¹,

Sato²,

Horng³

et al. 2008

Archives of Biochemistry and Biophysics

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It is now recognized that the denatured state ensemble (DSE) of proteins can contain significant amounts of structure, particularly under native conditions. Well-studied examples include small units of hydrogen bonded secondary structure, particularly helices or turns as well hydrophobic clusters. Other types of interactions are less well characterized and it has often been assumed that electrostatic interactions play at most a minor role in the DSE. However, recent studies have shown that both favorable and unfavorable electrostatic interactions can be formed in the DSE. These can include surprisingly specific non-native interactions that can even persist in the transition state for protein folding. DSE electrostatic interactions can be energetically significant and their modulation either by mutation or by varying solution conditions can have a major impact upon protein stability. pH dependent stability studies have shown that electrostatic interactions can contribute up to 4 kcal mol −1 to the stability of the DSE.

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“…Given that unfolded proteins are ensembles of conformations of varying structure (ref. 25 and references therein), it is reasonable to assume that the enthalpy fluctuations remaining in the unfolded state after native baseline subtraction arise from the coupled structural reorganizations of the nonnative polypeptide chain and surrounding solvent. Because these fluctuations are relevant to protein-folding reactions, an enthalpy scale so defined should be as close to a true structural scale as a phenomenological approach permits.…”

Section: A Critical Assessment Of the Phenomenological Variable-barriermentioning

confidence: 99%

Exploring protein-folding ensembles: A variable-barrier model for the analysis of equilibrium unfolding experiments

Muñoz

Sanchez-Ruiz²

2004

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

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Recent theoretical and experimental results point to the existence of small barriers to protein folding. These barriers can even be absent altogether, resulting in a continuous folding transition (i.e., downhill folding). With small barriers, the detailed properties of folding ensembles may become accessible to equilibrium experiments. However, further progress is hampered because folding experiments are interpreted with chemical models (e.g., the twostate model), which assume the existence of well defined macrostates separated by arbitrarily high barriers. Here we introduce a phenomenological model based on the classical Landau theory for critical transitions. In this physical model the height of the thermodynamic free energy barrier and the general properties of the folding ensemble are directly obtained from the experimental data. From the analysis of differential scanning calorimetry data alone, our model identifies the presence of a significant (>35 kJ͞mol) barrier for the two-state protein thioredoxin and the absence of a barrier for BBL, a previously characterized downhill folding protein. These results illustrate the potential of our approach for extracting the general features of protein ensembles from equilibrium folding experiments.experimental analysis ͉ free energy barrier ͉ downhill folding ͉ two-state folding ͉ phenomenological model I n contrast to the situation in many fields of modern physics, experimental results in protein folding are seldom directly interpretable by analytical theory or computer simulations. The interpretation typically involves a simple phenomenological model with which experimental results are analyzed, and the outcome of such ad hoc analysis is used to extract conclusions regarding experiments. A paradigm of this principle is the two-state model, in which protein-folding reactions are analyzed in terms of a chemical equilibrium between two independent species, native (N) and unfolded (U):[1]In Eq. 1, species with intermediate degree of structure are ignored, and the transition from one state to the other is of the first order. The use of a two-state model and its obvious generalization (i.e., a series of chemical equilibria between n structurally defined macrostates) is deeply rooted in the tradition of describing chemical transformations of small molecules as reaction schemes. Despite the obvious limitations of comparing protein folding with simple chemical reactions, the two-state protein-folding model enjoys tremendous popularity. One of the reasons is that this model seems to accommodate the folding behavior of a large set of single-domain proteins (1). Two-state folding could also provide proteins with a significant biological advantage by conferring upon them kinetic stability in vivo (see ref. 2 for a recent discussion). However, to make sure that the two-state character of proteins is not a self-fulfilling prophecy (3), it is important to analyze experimental data with a procedure that does not make assumptions about the existence of a free energy barrier. A more ...

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Energetic Evidence for Formation of a pH-dependent Hydrophobic Cluster in the Denatured State of Thermus thermophilus Ribonuclease H

Cited by 77 publications

References 47 publications

Large-scale modulation of thermodynamic protein folding barriers linked to electrostatics

Large-scale modulation of thermodynamic protein folding barriers linked to electrostatics

Electrostatic interactions in the denatured state ensemble: Their effect upon protein folding and protein stability

Exploring protein-folding ensembles: A variable-barrier model for the analysis of equilibrium unfolding experiments

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