Mutational Analysis Demonstrates that Specific Electrostatic Interactions can Play a Key Role in the Denatured State Ensemble of Proteins

Cho, Jae-Hyun; Raleigh, Daniel P.

doi:10.1016/j.jmb.2005.08.019

Cited by 69 publications

(134 citation statements)

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“…We hypothesize that nonnative electrostatic interactions in the DSE involving K12, and for example D8, are energetically linked to the formation of transient hydrophobic clusters in the DSE. Mutation of D8 has been shown to modulate the DSE, consistent with this hypothesis (24). K12 and D8 do not interact in the native state.…”

Section: Resultssupporting

confidence: 65%

“…1A) (21,(23)(24)(25)(26). Of particular interest is the observation of nonnative interactions in the DSE of NTL9 involving K12 (21,24). Replacement of K12 by Met increases the stability of the domain by 1.9 kcal mol −1 , a result which is thought to be dominated by DSE effects (21,24).…”

Section: Resultsmentioning

confidence: 99%

“…Of particular interest is the observation of nonnative interactions in the DSE of NTL9 involving K12 (21,24). Replacement of K12 by Met increases the stability of the domain by 1.9 kcal mol −1 , a result which is thought to be dominated by DSE effects (21,24). Significant interactions have been observed in the DSE of Significance Unfolded and partially unfolded proteins participate in a wide range of biological processes from pathological aggregation to the regulation of normal cellular activity.…”

Section: Resultsmentioning

confidence: 99%

“…pK a measurements report native state electrostatic energetics and are a sensitive probe of the effect of mutations (35). The pK a values are identical in the two proteins, indicating that the mutation does not perturb native state electrostatic interactions involving the acidic side chains (Table S4) (21,24 The thermodynamic studies indicate that K12 is coupled to a multitude of hydrophobic sites. A cartoon representative of how this might occur is depicted in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…It adopts a common α-β structure, folds in an apparent two-state manner as judged by extensive equilibrium and kinetic studies (Fig. 1A) (21,(23)(24)(25)(26). Of particular interest is the observation of nonnative interactions in the DSE of NTL9 involving K12 (21, 24).…”

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confidence: 99%

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Energetically significant networks of coupled interactions within an unfolded protein

Cho

Meng

Sato

et al. 2014

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

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Unfolded and partially unfolded proteins participate in a wide range of biological processes from pathological aggregation to the regulation of normal cellular activity. Unfolded states can be populated under strongly denaturing conditions, but the ensemble which is relevant for folding, stability, and aggregation is that populated under physiological conditions. Characterization of nonnative states is critical for the understanding of these processes, yet comparatively little is known about their energetics and their structural propensities under native conditions. The standard view is that energetically significant coupled interactions involving multiple residues are generally not present in the denatured state ensemble (DSE) or in intrinsically disordered proteins. Using the N-terminal domain of the ribosomal protein L9, a small α-β protein, as an experimental model system, we demonstrate that networks of energetically significant, coupled interactions can form in the DSE of globular proteins, and can involve residues that are distant in sequence and spatially well separated in the native structure. X-ray crystallography, NMR, dynamics studies, native state pK a measurements, and thermodynamic analysis of more than 25 mutants demonstrate that residues are energetically coupled in the DSE. Altering these interactions by mutation affects the stability of the domain. Mutations that alter the energetics of the DSE can impact the analysis of cooperativity and folding, and may play a role in determining the propensity to aggregate.protein folding | protein stability | ϕ-value analysis | double-mutant cycle T he properties of nonnative states of proteins have attracted considerable attention because they can play critical roles in cell signaling, translocation across membranes, temperaturesensitive phenotypes, and in a wide range of protein deposition diseases (1-14). The properties of unfolded states are also relevant to any discussion of intrinsically disordered proteins (IDPs) (1). The denatured state ensemble (DSE) can be populated under strongly denaturing conditions and studied at equilibrium; however, the physiologically relevant DSE is the state that is in equilibrium with the folded state under native conditions. This state is difficult to study directly and most studies of the DSE under native conditions have made use of indirect approaches. Nevertheless, experimental and theoretical work has shown that the DSE populated under physiological conditions is not a random coil, but can contain native and nonnative structure, stabilized by local and nonlocal interactions. However, very little is known about the energetics of these interactions, particularly ones involving residues distant in sequence, or about energetically coupled interactions involving multiple residues in the . These are a critical piece of the folding puzzle; they impact protein aggregation and folding, and have important implications for the properties of IDPs. Recent studies offer evidence for hydrophobic clusters, electrostatic interactio...

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Section: Resultssupporting

confidence: 65%