Copper(II) coordination properties of GxG peptides: Key role of side chains of central residues on coordination of formed systems; combined potentiometric and ITC studies Joanna Makowska a,⇑ , Dariusz Wyrzykowski a , Boguslaw Pilarski b , Damian Neubauer c , El _ zbieta Kamysz a , Aleksandra Tesmar a , Lech Chmurzyn´ ski a a Faculty of Chemistry, University of Gdan´sk, ul. Wita Stwosza 63, 80-308 Gdan´sk, Poland b Cerko Sp. z o.o. Sp.k, Al. Zwycie˛stwa 96/98, 81-451 Gdynia, Poland c Faculty of Pharmacy, Medical University of Gdan´sk, Al. Gen. J. Hallera 107, 80-416 Gdan´sk, Poland article info Article history: Received 19 April 2018 Received in revised form 27 August 2018 Accepted 28 August 2018 Available online 29 August 2018 abstract Isothermal titration calorimetry (ITC) and potentiometric titration (PT) methods were used to study the interactions of copper(II) ions with GAG, GDG, GKG, and GHG peptides. The calorimetric measurements were run in a MES buffer with the pH value of 6.0 at 298.15 K. Based on the results of PT data supported by theoretical calculations, the dissociation constants were calculated for the investigated peptides. The quantification of the proton competition with the metal for the peptide and incorporation it into the ITC data analysis enabled to obtain the pH-independent parameters, namely the binding constants (K) and the free energy of binding (DG). Furthermore, the relationship between the proposed coordination modes of the considered peptides and the thermodynamic parameters (K, DG and DH) has been discussed. Ó 2018 Elsevier Ltd. 1. Introduction The ionization state of amino acid residues determines the net charge on a protein, and this is important to the structure, function, stability and solubility of the protein. Consequently, scientists have a strong interest in the pK values of the ionizable groups of pro- teins and in the factors that perturb them [1]. Intrinsic backbone conformational preferences of amino acids determine the local structures of unfolded protein chains; this intrinsic preferences might guide the folding processes at early stages of the protein folding [2]. Combined analyses of data for GxG model peptides (x represents a set of different amino acid residues) are crucial for this subject. It has been found that all types of classical turn-like and turn-supporting conformations can be transiently formed by GxG-tripeptides in aqueous solutions [3]. In view of the relevance of turns for initiation of folding processes and for local order in intrinsically disordered proteins and peptides, further information regarding the existence of these metastable turns is warranted. In the past, it was reported that GxG peptides with long, bulky and predominantly hydrophobic side chains can sample different types of beta2 and gamma2 turn-supporting conformations [4]. Moreover, short peptides that consist of no more than 20 amino acid residues with molecular weights less than 3.5 kDa are signal- ing molecules involved in the regulation of homeostasis at differ- ent levels of the organization of living matter. I. P. Ashmarin identified endogenous regulatory peptides as a part of an ‘‘intricate system of specialized signaling molecules and information carriers between the cells of the body” [5]. The design of drugs based on short peptides, peptide biotechnology, is a topical area of modern molecular biology and pharmacology. Based on the results of long term research, it was concluded that the activity of short peptides is selective or tissue specific [6,7,8]. Moreover peptides are also valuable and selective ligands for metal binding. The ability of peptides to form complexes is very often exploited in medicine for the treatment of diseases related to the excess of metal ions in the organisms. Because copper is an essential element involved in important biological functions, numerous studies have been carried out providing critical informa- tion on the different Cu 2+ coordination modes and the reactivity of the resulting copper(II) peptide complexes [9–14]. Native proteins mainly coordinate Cu 2+ through the amino acid side chains forming single species. The authors expect that presented in this paper results will contribute to the better understanding of the coordination mode of copper(II) in the GxG complexes with free terminal groups. https://doi.org/10.1016/j.jct.2018.08.040 0021-9614/Ó 2018 Elsevier Ltd. ⇑ Corresponding author. E-mail address: joanna.makowska@ug.edu.pl (J. Makowska). J. Chem. Thermodynamics 128 (2019) 336–343 Contents lists available at ScienceDirect J. Chem. Thermodynamics journal homepage: www.elsevier.com/locate/jct