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Review
. 2020 May 15;21(10):3484.
doi: 10.3390/ijms21103484.

Understanding the Binding Induced Folding of Intrinsically Disordered Proteins by Protein Engineering: Caveats and Pitfalls

Francesca Malagrinò  1 , Lorenzo Visconti  1 , Livia Pagano  1 , Angelo Toto  1 , Francesca Troilo  1 , Stefano Gianni  1
Affiliations
Review

Understanding the Binding Induced Folding of Intrinsically Disordered Proteins by Protein Engineering: Caveats and Pitfalls

Francesca Malagrinò et al. Int J Mol Sci. .

Abstract

Many proteins lack a well-defined three-dimensional structure in isolation. These proteins, typically denoted as intrinsically disordered proteins (IDPs), may display a characteristic disorder-to-order transition when binding their physiological partner(s). From an experimental perspective, it is of great importance to establish the general grounds to understand how such folding processes may be explored. Here we discuss the caveats and the pitfalls arising when applying to IDPs one of the key techniques to characterize the folding of globular proteins, the Φ value analysis. This method is based on measurements of the free energy changes of transition and native states upon conservative, non-disrupting, mutations. On the basis of available data, we reinforce the validity of Φ value analysis in the study of IDPs and suggest future experiments to further validate this powerful experimental method.

Keywords: IDP; disorder-to order transition; folding kinetics; protein engineering; Φ value analysis.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Different Φ value profiles and related free energy diagrams for a two-state folding reaction of a wild-type (black line) and mutated (orange line) protein. The mutation is highlighted on the structure with an orange circle. ΔGN, ΔGTS and ΔGD refer to the changes in stability upon mutation of the native, transition and denatured states respectively. (a) Φ value = 0. Mutation does not affect the structure and the stability of the transition state. The mutated region appears unstructured in the transition state likewise in the denatured state (ΔGTS = 0) (b) Φ value = 1. The mutation insists in a native-like structured region of the transition state, with ΔGTS tending to ΔGN. In both cases, the free energy of the denatured state does not change upon mutation (ΔGD = 0). (c) The mutation affects the stability of the denatured state. This effect may reflect in an unusual Φ value, i.e., >1 or <0.
Scheme 1
Scheme 1
Schematic representation of the overall reaction mechanism involving the binding between an IDP and its ligand.
Figure 2
Figure 2
Conformational selection (Folding before binding) and Induced-fit (Folding after binding) models and related free energy diagrams. The ligand (L) and the intrinsically disordered protein (folded, IDPfold, and unfolded IDPunf) are colored in black and orange respectively. A plausible energy diagram arising in different kinetic conditions is depicted in panels (a–d), in all cases the bimolecular binding reaction is highlighted with dashed lines. The implications of different scenarios are discussed in the text.
Figure 3
Figure 3
Linear Free Energy Relationship (LFER) plot in protein folding. The data refer to the Φ value analysis of the SH3 domain from Grb2 (81). The structure of the native state, obtained by NMR, and the transition state, calculated by using restrained molecular dynamic simulations, are reported on the left and on the right of the plot, respectively. As explained in the text, linearity in LFER plots is a hallmark of the nucleation-condensation mechanism and represents a common feature of globular proteins. By following this scenario, the transition state is a distorted version of the native state, as nicely exemplified by the data reported in the figure.
Figure 4
Figure 4
Additional tests for the Φ value analysis. (a) exemplifies an energy diagram summarizing the double mutant cycle approach. In analogy to classical protein folding, the reactant and product are referred to as the D (denatured) and N (native) states respectively. Analogously, in binding, these could represent the free and bound state. (b) Φ−Φ plot of two homologous PDZ domains (data from [61]). The correlation between the observed values is a signature of conservation of the folding mechanism and provides per se an additional validation of the Φ value analysis.
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