Contents lists available at ScienceDirect BBA - Proteins and Proteomics journal homepage: www.elsevier.com/locate/bbapap Ubiquitin folds via a flip-twist-lock mechanism Manoj Mandal a , Atanu Das b, ⁎ , Chaitali Mukhopadhyay b, ⁎ a Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan b Department of Chemistry, University of Calcutta, 92 A.P.C. Road, Kolkata 700009, India ARTICLE INFO Keywords: Dewetting transition Hydrophobic collapse Folding nucleus Salt bridge Conformational ensembles Folding mechanism Molecular simulations ABSTRACT To perform specific functional activities, the majority of proteins should fold into their distinct three-dimen- sional conformations. However, the biologically active conformation of a protein is generally found to be marginally stable than the other conformations that the chain can adopt. How a protein finds its native con- formation from its post-synthesis unfolded structure in a complex conformational landscape is the unsolved question that still drives the protein folding community. Here, we report the folding mechanism of a globular protein, ubiquitin, from its chemically denatured state using all-atom molecular dynamics simulations. From the kinetic analysis of the simulated trajectories we show that the folding process can be described by the hydro- phobic collapse mechanism, initiated by the “dewetting transition”, and subsequently assisted by the origination of an N-terminal folding nucleus, and finally supported by a native salt-bridge interaction between K11 and E34. We show that ubiquitin folds via an intermediate. Finally, we confirm the presence of “biological water” and explain its role to the folding process. 1. Introduction Proteome integrity and hence, cellular health depends on – (1) the abundance of every protein in the cell, and (2) the correct balance between them. The cell must rigorously regulate the three steps that dictate the generation, function, and concentration of proteins in the cell – synthesis, folding, and degradation. The function of most cellular proteins is related to their three-dimensional native conformation [1]. These globular proteins fold into a specific, water-soluble, functional, biologically active structure. The foldability of a protein and the ther- modynamic stability of its native state under physiological conditions depend on its amino acid code, the single-letter building blocks of proteins. Proteins largely fold with astounding reliability; however, in a few instances, when this in-built folding potential fails to perform, or a protein undergoes partial unfolding, the protein gets folded or degraded by a refined mechanism assisted by chaperones and proteasomes, triggered by the cell [2]; failure of which leads to protein misfolding and formation of disease-causing toxic aggregates [3,4]. The linear amino acid sequence of the newly synthesized polypeptide chain can be up to several thousand amino acids in length. However, even a small polypeptide chain contains the possibility of sampling astronomically large number of conformations. Moreover, the preferred biologically active native state has a very minute edge in terms of the relative order of stability compared to the other conformations the chain can adopt under physiological conditions [5]. Naturally, this makes the folding process imperfect, which leads to protein misfolding, and finally off- pathway fibrillar aggregates. The scientific community initially found it intangibly problematic to explain how a molecule finds its folded structure from its vast number of possible unfolded configurations via a random-search process in a biologically relevant timescale. This even- tually gave the notion of a protein-folding pathway. The search for such a pathway is the motive for protein-folding studies since the idea of multiple parallel pathways to the folded native state has been spor- adically conceived. Multiple theories/models have been put forward to explain the folding process – helix-coil theory (chemical reaction model) [6], fra- mework model [7], hydrophobic collapse model [8], nucleation-con- densation model [9], energy landscape theory [10] etc. Likewise, various forces are believed to influence the folding phenomenon e.g. residual native interactions in the unfolded state [11], on- and off-pathway non- native contacts [12], van der Waals forces [13], hydrogen bonding [14], salt bridges [15], and water-mediated interactions [16]. Cutting- https://doi.org/10.1016/j.bbapap.2019.140299 Received 19 June 2019; Received in revised form 14 September 2019; Accepted 1 October 2019 Abbreviations: Q f , The fraction of intra-protein backbone-backbone contacts; Q hb , The fraction of intra-protein backbone-backbone hydrogen bonds; RMSD Core , Root mean square deviation of the hydrophobic core residues; R g Core , Radius of gyration of the hydrophobic core residues; SASA Core , Solvent accessible surface area of the hydrophobic core residues; D, Denatured; N ‡ , Near-native N-terminal with unstructured C-terminal; C ‡ , Partially structured C-terminal with completely formed N- terminal; F, Folded ⁎ Corresponding authors. E-mail addresses: atanucu08@gmail.com (A. Das), cmchem@caluniv.ac.in (C. Mukhopadhyay). BBA - Proteins and Proteomics 1868 (2020) 140299 Available online 30 October 2019 1570-9639/ © 2019 Elsevier B.V. All rights reserved. T