**1. Introduction**

Proteins under biological conditions exhibit marginal structural stability [1], and they unfold and refold repeatably in vivo [2]. Consequently, many of the biological processes that are facilitated by protein macromolecules are modulated by the properties and energetic character of the denatured state. Indeed, numerous efforts have shown that denatured state effects, such as residual structure [3], excluded volume [4], and intrinsic conformational propensities [5], have key roles in molecular recognition [6], allosteric signaling [7], folding [8,9], and stability [10]. A molecular-level understanding of how proteins are utilized for biological work thus requires characterization of the native, as well as the myriad of non-native, conformational states that exist in solution for a protein, the latter of which is referred to as its denatured state ensemble (DSE).

Despite its importance in understanding protein function, the probability and structural character of the full spectrum of states sampled by proteins are not known. Numerous studies have used short peptides as experimental models from which to probe the characteristics of the DSE [11–13]. The use of short peptides is advantageous because, being too short to fold, they offer access to unfolded states under otherwise folding conditions. Moreover, in the absence of folding, conformational preferences are simplified and locally driven by factors such as hydration [14] and steric hindrance [15]. These studies find that peptides

**Citation:** Paiz, E.A.; Lewis, K.A.; Whitten, S.T. Structural and Energetic Characterization of the Denatured State from the Perspectives of Peptides, the Coil Library, and Intrinsically Disordered Proteins. *Molecules* **2021**, *26*, 634. https:// doi.org/10.3390/molecules26030634

Academic Editor: Marilisa Leone Received: 1 January 2021 Accepted: 23 January 2021 Published: 26 January 2021

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have strong preferences for the polyproline II (PPII) backbone conformation, even at nonproline positions [12,16,17], suggesting that PPII structures are dominant components of the DSE. The PPII conformation is characterized by an extended left-handed helical turn with the amide hydrogen and the carboxyl oxygen of each peptide backbone projecting into solution, presumably making favorable contact with the solvent [18–20]. In addition, the PPII conformation appears to facilitate favorable intrachain n→π\* interactions, which should be a stabilizing factor [21]. Short peptides also exhibit conformational preferences for other backbone structures. At cold temperatures, alanine residues have intrinsic αhelix-forming tendencies (i.e., even in the absence of favorable side chain interactions) that are stabilized predominantly by peptide hydrogen bonds [22]. Elevated temperatures have been observed to promote low levels of β-strand [16] or β-turn [23], though the amino acid preferences for forming strand [24] or reverse turn structures [25,26] are thought to be highly context-dependent.

The protein coil library [27] also has been used as a structural model for the DSE [28–30]. These libraries are constructed from the segments of protein structure in the Protein Data Bank (PDB) that are found outside the α-helix and β-strand domains. Some libraries further omit additional conformationally restricted positions, such as those in reverse turns, or preceding prolines, or immediately flanking a region of secondary structure [29]. The underlying assumption when using a coil library as a DSE model is that site-specific effects on the intrinsic conformational preferences of the amino acids are minimized by averaging over many environments, and also by removing the regular and repetitive interactions associated with folded structures. Overall, coil libraries exhibit structural trends that are in good agreement with the results from peptide structural studies [29,31]. For example, chemical shifts and three-bond *J* couplings (<sup>3</sup> *JHNα*) measured in peptides by NMR spectroscopy can be reproduced from structural models made from the protein coil library [32–34]. Notably, and similar to the results obtained from peptides, strong preferences for PPII that vary by amino acid type are found in structural surveys of the protein coil library [28–30].

Intrinsically disordered proteins (IDPs) offer another experimental system from which to assess structural preferences in unfolded states under nondenaturing conditions [35]. While chemically denatured proteins are known to adopt macromolecular sizes that depend weakly on sequence details other than chain length [36], IDPs in water exhibit strong sequence-dependent influences on structural size [37]. Computer simulations show that steric effects on disordered structure cannot account for the hydrodynamic size dependence on sequence observed in IDPs [38]. Additionally, temperature changes are found to induce large shifts in the hydrodynamic size for disordered proteins in water [39–41] that can exceed the change in size associated with the heat denaturation of folded proteins of the same chain length [42]. The implication of these findings, albeit expected, is that monomeric disordered protein structure is both under thermodynamic control and highly sensitive to the primary sequence.

In this review, we show that the sequence dependence of IDP hydrodynamic size can be described from the amino acid-specific biases for PPII in the denatured state. Because PPII-rich structures are extended [43], the magnitude of a PPII preference in the denatured state can affect its mean hydrodynamic size [44,45]. Specifically, experiments that evaluate how IDP hydrodynamic size changes with compositional changes in the protein give an independent measure of PPII bias, and further reveal amino acid-specific preferences for PPII that are in good quantitative agreement with PPII bias determined experimentally in peptides [37]. Good agreement is also found when the IDP results are compared to PPII bias in the protein coil library. Moreover, the analysis of heat effects on IDP hydrodynamic size indicates the PPII bias is driven by a significant and favorable enthalpy, and is partially offset by an unfavorable entropy [37], which, again, agrees quantitatively with the peptide results [46]. Across these three models (i.e., peptides, the coil library, and IDPs), the data indicate that the structural and energetic character of the DSE at normal temperatures follows the predictions of a PPII-dominant ensemble. At cold temperatures, both peptides

and IDPs reveal the DSE can shift in population toward the α-helix backbone conformation. To demonstrate these conclusions, the following sections review results obtained from numerous spectroscopic and calorimetric studies on short peptides [11–13,16,17,46], surveys of structures in the protein coil library [28–30], and the more recently acquired sequenceand temperature-based analysis of IDP hydrodynamic sizes [37], showing that these three experimental systems used for characterizing unfolded proteins under folding conditions convey a surprisingly consistent structural and energetic view of the DSE.

α
