Journal of Molecular Graphics and Modelling 30 (2011) 24–30 Contents lists available at ScienceDirect Journal of Molecular Graphics and Modelling journal homepage: www.elsevier.com/locate/JMGM Simulation of urea-induced protein unfolding: A lesson from bovine -lactoglobulin Ivano Eberini a,∗ , Andrew Emerson b,1 , Cristina Sensi a,1 , Laura Ragona c , Piero Ricchiuto a , Alessandro Pedretti d , Elisabetta Gianazza a , Anna Tramontano e a Dipartimento di Scienze Farmacologiche, Università degli Studi di Milano, via Giuseppe Balzaretti 9, 20133 Milano, Italy b CINECA High Performance Systems, via Magnanelli 6/3, 40033 Casalecchio di Reno, Italy c Laboratorio NMR, Istituto per lo Studio delle Macromolecole, CNR, Via Bassini 15, 20133, Milano, Italy d Dipartimento di Scienze Farmaceutiche “Pietro Pratesi”, Università degli Studi di Milano, via Mangiagalli 25, 20133 Milano, Italy e Dipartimento di Fisica, Università degli Studi di Roma “La Sapienza”, P.le Aldo Moro 5, 00185 Roma, Italy article info Article history: Received 24 March 2011 Received in revised form 1 June 2011 Accepted 2 June 2011 Available online 13 June 2011 Keywords: Molecular dynamics Nuclear magnetic resonance Protein structure Osmolyte abstract To investigate the molecular mechanisms involved in the very initial stages of protein unfolding, we carried out one long (1 s) simulation of bovine -lactoglobulin (BLG) together with three (500 ns) sup- porting MD runs, in which the unfolding conditions were produced by adding the osmolyte urea to the simulated systems and/or by increasing the thermal energy raising the temperature from 300 to 350 K. BLG was chosen, since it is a well-characterized model protein, for which structural and folding proper- ties have been widely investigated by X-ray and NMR. MD trajectories were analyzed not only in terms of standard progress variables, such as backbone H-bonds, gyration radius width, secondary structure elements, but also through the scrutiny of interactions and dynamical behavior of specific key residues previously pointed out and investigated by NMR and belonging to a well known hydrophobic cluster. MD trajectories simulated in different unfolding conditions suggest that urea destabilizes BLG struc- ture weakening protein::protein hydrophobic interactions and the hydrogen bond network. The early unfolding events, better observed at higher temperature, affect both secondary and tertiary structure of the protein. © 2011 Elsevier Inc. All rights reserved. 1. Introduction Molecular dynamics (MD) is a useful approach for understand- ing molecular behavior at an atomic resolution. The continued increase in available computational power, and the availability of newer and more accurate force fields, are opening the way for new applications of this computational tool, e.g. in the field of structural biology [1]. In spite of the above improvements, protein folding is a process too slow and too complex to be accurately described via MD. Monitoring unfolding, both with experimental and with theoretical procedures, however, may provide a shortcut for inves- tigating the key patterns for protein stability [2], allowing crucial interactions stabilizing the folded form to be identified. High tem- peratures or high concentrations of some osmolytes, e.g. urea or ∗ Corresponding author at: Gruppo di Studio per la Proteomica e la Struttura delle Proteine, Dipartimento di Scienze Farmacologiche, Università degli Studi di Milano, via Giuseppe Balzaretti 9, I-20133 Milano, Italia. Tel.: +39 02 503 18256/18362; fax: +39 02 503 18284. E-mail address: ivano.eberini@gmail.com (I. Eberini). 1 These authors equally contributed to this work. guanidinium chloride, allow for, and enhance the rate of, protein unfolding. While urea is non-ionic, guanidinium chloride solutions contain a large number of charges, making the setup of a realistic force field difficult [3]. In the last few years a number of papers in which the mechanism of protein unfolding induced by urea has been investigated by MD simulations have appeared in the lit- erature [2,4–12]. Different simulation setups have been used, for example using classical MD or replica exchange MD, and with a wide range of temperatures and simulated times. It is however difficult to compare the results produced by different groups and, sometimes, to discriminate between the effects of the osmolyte and of the temperature. During this investigation we run MD simulations of long duration (up to 1 s) for a model protein, bovine -lactoglobulin (BLG), in urea. The aim of this work was to test whether MD could sample at least the initial stages of protein unfolding. BLG is a 162 amino acid -barrel protein belonging to the lipocalin family [13], consisting of nine antiparallel -sheets and one terminal -helical segment as determined by X-ray at neutral pH [14,15] and by NMR at acidic pH [16–19]. The folding properties of BLG have been widely investigated through different spectroscopic techniques such as circular dichroism, fluorescence and NMR [20–25] and by 1093-3263/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.jmgm.2011.06.004