About the Structural Role of Disulfide Bridges in Serum Albumins: Evidence from Protein Simulated Unfolding Guillaume Paris, 1 Sebastian Kraszewski, 2 Christophe Ramseyer, 2 Mironel Enescu 1 1 Laboratoire Chrono Environnement UMR CNRS 6249, Faculte´des Sciences et Techniques, La Bouloie, Universite´de Franche-Comte´, 25030 Besanc ¸on cedex, France 2 Laboratoire de Nanome´decine, Imagerie et The´rapeutique EA 4662, Faculte´des Sciences etTechniques, La Bouloie, Universite´de Franche-Comte´, CHU Besanc ¸on, 25030 Besanc ¸on cedex, France Received 29 March 2012; revised 10 May 2012; accepted 17 May 2012 Published online 24 May 2012 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bip.22096 This article was originally published online as an accepted preprint. The ‘‘Published Online’’ date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley. com INTRODUCTION T he covalent SÀÀS bonds (disulfide bridges) formed between cysteine residues play a special role in stabi- lizing the native structure of many proteins. This is a fact clearly emphasized by reductive protein unfold- ing or oxidative protein refolding experiments where the cleaving or the reconstitution of the SÀÀS bridges are employed for controlling the protein structure. 1–4 In a simple mechanistic interpretation, a disulfide bridge is equivalent to a constraint introduced in the protein space phase that stabil- izes the folded with respect to the unfolded structure by sig- nificantly reducing the entropy of this latter one. 5 Experi- ments based on urea and thermal induced denaturation indi- cate that, in average, the conformation stability of the folded protein structure is reduced by about 3 kcal mol 21 when one SÀÀS bridge is broken. 6,7 However, the structural consequen- ces of removing the SÀÀS bridges were found to vary from one protein to another. For example, the reduction of one of the four SÀÀS bridges of ribonuclease A produces a species with a native like-structure while the reduction of two SÀÀS About the Structural Role of Disulfide Bridges in Serum Albumins: Evidence from Protein Simulated Unfolding Additional Supporting Information may be found in the online version of this article. Correspondence to: Mironel Enescu; e-mail: mironel.enescu@univ-fcomte.fr ABSTRACT: The role of the 17 disulfide (SÀÀS) bridges in preserving the native conformation of human serum albumin (HSA) is investigated by performing classical molecular dynamics (MD) simulations on protein structures with intact and, respectively, reduced SÀÀS bridges. The thermal unfolding simulations predict a clear destabilization of the protein secondary structure upon reduction of the SÀÀS bridges as well as a significant distortion of the tertiary structure that is revealed by the changes in the protein native contacts fraction. The effect of the SÀÀS bridges reduction on the protein compactness was tested by calculating Gibbs free energy profiles with respect to the protein gyration radius. The theoretical results obtained using the OPLS-AA and the AMBER ff03 force fields are in agreement with the available experimental data. Beyond the validation of the simulation method, the results here reported provide new insights into the mechanism of the protein reductive/ oxidative unfolding/folding processes. It is predicted that in the native conformation of the protein, the thiol (ÀÀSH) groups belonging to the same reduced SÀÀS bridge are located in potential wells that maintain them in contact. The ÀÀSH pairs can be dispatched by specific conformational transitions of the peptide chain located in the neighborhood of the cysteine residues. # 2012 Wiley Periodicals, Inc. Biopolymers 97: 889–898, 2012. Keywords: MD simulation; SÀÀS bridge; OPLS-AA; AMBER ff03; helicity; native contacts V V C 2012 Wiley Periodicals, Inc. Biopolymers Volume 97 / Number 11 889