Biology 2022, 11, 1764. https://doi.org/10.3390/biology11121764 www.mdpi.com/journal/biology Article A Structure-Based Mechanism for the Denaturing Action of Urea, Guanidinium Ion and Thiocyanate Ion Antonella Paladino 1, *, Nicole Balasco 2 , Luigi Vitagliano 1 and Giuseppe Graziano 3, * 1 Institute of Biostructures and Bioimaging, CNR, Via Pietro Castellino 111, 80131 Naples, Italy 2 Institute of Molecular Biology and Pathology, CNR c/o Department Chemistry, Sapienza University of Rome, P. le A. Moro 5, 00185 Rome, Italy 3 Department of Science and Technology, University of Sannio, Via Francesco de Sanctis snc, 82100 Benevento, Italy * Correspondence: antonella.paladino@cnr.it (A.P.); graziano@unisannio.it (G.G.) Simple Summary: The detailed characterization of urea binding sites in protein structures shows that urea can establish multiple types of interactions, in line with recent findings reported for guan- idinium and thiocyanate, thus confirming that promiscuity is a general property of protein dena- turants. Our analyses support a denaturing model based on protein-denaturant direct interactions to practically equal and independent sites. We also underscore insightful features that can inform on the milder denaturing power displayed by urea. Abstract: An exhaustive analysis of all the protein structures deposited in the Protein Data Bank, here performed, has allowed the identification of hundredths of protein-bound urea molecules and the structural characterization of such binding sites. It emerged that, even though urea molecules are largely involved in hydrogen bonds with both backbone and side chains, they are also able to make van der Waals contacts with nonpolar moieties. As similar findings have also been previously reported for guanidinium and thiocyanate, this observation suggests that promiscuity is a general property of protein denaturants. Present data provide strong support for a mechanism based on the protein-denaturant direct interactions with a denaturant binding model to equal and independent sites. In this general framework, our investigations also highlight some interesting insights into the different denaturing power of urea compared to guanidinium/thiocyanate. Keywords: urea; protein stability; urea-protein interactions; chemical denaturants 1. Introduction The elucidation of the physico-chemical factors that drive protein folding is a puz- zling and largely unsolved issue that has attracted the attention of the scientific commu- nity since the determination of the first three-dimensional structure of a globular protein, more than sixty years ago [1]. The difficulties encountered in this field are related to the observation that the folded structure of a protein is marginally more stable in comparison to the ensemble of its unfolded states [2,3]. Although the development of machine learn- ing algorithms—that in many cases provide reliable three-dimensional models of proteins starting from their sequences, represents a revolution in structural biology with unfore- seeable consequences—many fundamental aspects of protein folding remain obscure [4]. These include the definition of structure-stability relationships and the interpretation, at the molecular level, of the plethora of experimental data on the impact that chemical de- naturants have on protein folding. It is known for a long time that the conformational stability of globular proteins can be strongly affected by the addition to water of certain chemical species that prove to have Citation: Paladino, A.; Balasco, N.; Vitagliano, L.; Graziano, G. A Structure-Based Mechanism for the Denaturing Action of Urea, Guanidinium Ion and Thiocyanate Ion. Biology 2022, 11, 1764. https://doi.org/10.3390/ biology11121764 Academic Editor: Andrew Clayton Received: 31 October 2022 Accepted: 2 December 2022 Published: 5 December 2022 Publisher’s Note: MDPI stays neu- tral with regard to jurisdictional claims in published maps and institu- tional affiliations. Copyright: © 2022 by the authors. Li- censee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and con- ditions of the Creative Commons At- tribution (CC BY) license (https://cre- ativecommons.org/licenses/by/4.0/).