Energetically significant networks of coupled interactions within an unfolded protein

Abstract

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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