Metal ions modulate thermal aggregation of beta-lactoglobulin: A joint chemical and physical characterization Giovanna Navarra a , Anna Tinti b , Michele Di Foggia b , Maurizio Leone a , Valeria Militello a , Armida Torreggiani c, ⁎ a Dipartimento di Fisica–Chimica, Università di Palermo, Via Archirafi 36, 90123 Palermo, Italy b Dipartimento di Scienze Biomediche e Neuromotorie, Università di Bologna, Via Belmeloro 8/2, 40126 Bologna, Italy c Istituto I.S.O.F., Consiglio Nazionale delle Ricerche, Via P. Gobetti 101, 40129 Bologna, Italy abstract article info Article history: Received 5 February 2014 Received in revised form 2 April 2014 Accepted 2 April 2014 Available online 13 April 2014 Keywords: Beta-lactoglobulin Copper and zinc ions Raman spectroscopy Infrared spectroscopy Dynamic light scattering Molecular basis of the role played by Cu 2+ and Zn 2+ ions during the thermal aggregation processes of beta- lactoglobulin (BLG) was studied by using a joint application of different techniques. In particular, Raman spectroscopy was very useful in identifying the different effects caused by the two metals at molecular level (i.e. changes in His protonation state, disulfides bridge conformation, and micro-environment of aromatic residues), evidencing the primary importance of the protein charge distribution during the aggregation process. Both metal ions are able to act on this factor and favor the protein aggregation, but Zn 2+ is able to alter the natural conformational state of BLG, causing a slight unfolding, whereas Cu 2+ ions play a role only during the thermal treatment. Thus, Zn 2+ ions favor the formation of bigger aggregates and branched fibril-like structures, whereas for Cu 2+ ions a greater number of cross-beta structures during thermal incubation and finally, fibrillar structures. The aggregation process occurs in two phases, as suggested by the measurements on the time evolution of the BLG aggregates: the first one is characterized by a partial unfolding of the protein and aggregate growth, forming oligomers and protofibrils, whereas the second one is characterized by further supramolecular assembly, leading to the formation of fibrils. © 2014 Elsevier Inc. All rights reserved. 1. Introduction A large number of proteins may form under appropriate conditions a variety of aggregated structures whose morphology resembles those of fibrils that can be accumulated in biological environments under pathological conditions [1–4]. The fibril formation seems to be driven by an appropriate destabilization of the native state [1,5], and typical of the early phases of fibril formation is the conversion of α-helices into β-sheets. However, also proteins containing a large fraction of β-sheets can be transformed in vitro into fibrillar structures through a process generally involving destabilization and consequent auto-assembly of partially unfolded intermediates. Fibrils are the final state of aggregated and re-organized protofibrils. The formation of amorphous aggregates, fibrils or gels may be initiated by thermal or chemically induced protein unfolding [6,7]. The formation of amorphous aggregates, fibrils or gels may be initi- ated by thermal or chemically induced protein unfolding [6,7]. Bovine β-lactoglobulin (BLG), a small globular protein of bovine milk, is made of 162 residues forming two antiparallel β-sheets [8]. Its known three-dimensional structure (Fig. 1) contains one free thiol group and it is stabilized by two disulfide bonds, Cys66–Cys160 and Cys106–Cys119, of which the first one generates a conformational re- straint that is reported to inhibit the formation of misfolded aggregation of BLG [9]. BLG is one of the most popular model proteins in the study of protein folding and conformation in vitro. Refolding of denaturated BLG proceeds through a non-native α-helical intermediate, making this protein a useful tool to investigate α–β transitions, important for example in the confor- mational transition of prion proteins. In addition, this protein can form ei- ther amorphous aggregates or amyloid fibrils upon changing the working parameter [10]. BLG, being the major whey protein in milk, is also impor- tant in food technological applications where the control of the protein denaturation/aggregation during heating is of outstanding importance for the acceptance of the final quality of the products and for avoiding allergenic problems [11]. Thus, we have selected BLG because of its double interest: it is a model beta-protein in aggregation processes and a thermal marker in industrial processes involved in milk treatment. Although the mechanism of BLG heat-aggregation has been exten- sively studied, it is not still completely understood and controlled [7, 12–14]. As a consequence of BLG aggregation, either amorphous aggre- gates or amyloid fibrils can be formed, depending on the experimental conditions [12,15]. Journal of Inorganic Biochemistry 137 (2014) 64–73 ⁎ Corresponding author at: Istituto ISOF (CNR), Via P. Gobetti n° 101, 40129 Bologna, Italy. Tel.: +39 051 6399821; fax: +39 051 6399844. E-mail address: armida.torreggiani@isof.cnr.it (A. Torreggiani). http://dx.doi.org/10.1016/j.jinorgbio.2014.04.003 0162-0134/© 2014 Elsevier Inc. All rights reserved. Contents lists available at ScienceDirect Journal of Inorganic Biochemistry journal homepage: www.elsevier.com/locate/jinorgbio