Stabilization of beta-lactoglobulin by polyols and sugars against temperature-induced denaturation involves diverse and specific structural regions of the protein
Introduction
Functional properties of food proteins have a great impact on quality and organoleptic attributes of foods, and are related to their structural features. The structural features of food proteins may undergo transient or permanent modification during processing as related to mechanical, chemical, and thermal stress. Thus, the processes aimed at achieving a given structural modification (and therefore a particular property), have to be fine-tuned by taking into account both kinetic and thermodynamic aspects. Food composition also must be taken into account, as the physicochemical properties of food systems (concentration, pH, presence of co-solutes, and so on) affect the thermodynamic and/or kinetic stability even of systems as simple as a single protein in solution.
Polyols – also referred to as osmolytes – are among the co-solutes with the highest impact the thermodynamic stability of protein in solution. Polyols exert multiple effect on proteins as they: (i) increase structural stability towards thermal (Back, Oakenfull, & Smith, 1979) or chemical (Sola-Penna, Ferreira-Pereira, dos Passos Lemos, & Meyer-Ferwandes, 1997) denaturation; (ii) protect protein against loss of structure/activity during freezing (Carpenter & Crowe, 1988) and drying processes (Colaço et al., 1992, Sun and Davidson, 1998). However, despite the practical relevance of these issues and the large number of studies, the exact molecular mechanism of the structural stabilization of proteins in solution by polyols is still not well understood, and stabilizers are often used on quite empirical basis.
Among the proposed hypotheses, the concept of preferential exclusion has gained widespread acceptance. This hypothesis implies that polyols/water and water/proteins interaction are more favorites than polyols/protein interaction. Water molecules are preferentially attracted towards the surface of the protein, leading to the exclusion of other solutes from the vicinity of the protein. These effects would result in a preferential hydration of the protein, responsible of the stabilizing effect (see Lee and Timasheff (1981) and references therein). Whether this “preventive” action is direct or indirect – that is, involving a change in the properties of the solvent water in the presence of polyols – yet remains a matter of debate.
The issue is complicated further by the fact that many studies do not express results in comparable ways. Also very often putative equilibrium effects are considered without paying attention to the common occurrence in proteins of structural regions of different kinetic and/or thermodynamic stability, and to irreversible effects ensuing from unfolding (such as aggregation) with the associated kinetic aspects. Indeed, the peculiar mechanism and the different kinetics of individual steps in protein unfolding – and of events affecting specific structural regions – are often considered only marginally. These aspects often are of outmost relevance, as the rate and extent of the formation of partially unfolded structures often dictates the outcome of the denaturing process itself in processes that are of pathological relevance (as exemplified by the formation of amyloids (Azinas et al., 2011, Colombo et al., 2011, Ricagno et al., 2010, Santambrogio et al., 2010) and in many processes that rely on partial protein denaturation for practical purposes, as exemplified by countless food-related household and industrial manipulations of protein-based single- and multiple-phase mixtures.
Bovine milk betalactoglobulin (BLG) is a relatively small lipocalin abundant in whey, is a common food ingredient, and is a food allergen. BLG has been shown to unfold through a series of sequential reversible and irreversible steps (Cairoli et al., 1994, Eberini et al., 2012, Iametti et al., 1996, Roefs and De Kruif, 1994). Each of the individual steps in BLG unfolding shows a distinctive dependence on the nature and intensity of physical unfolding treatments (Barbiroli et al., 2011, Fessas et al., 2001), on pH, on the concentration of chemical denaturants (Eberini et al., 2012), as well as on the protein concentration and aggregation state (Fessas et al., 2001, Iametti et al., 1995, Iametti et al., 1998). Individual steps of BLG unfolding also have been shown to depend on the presence of protein-bound natural ligands (Barbiroli et al. 2011).
Partially unfolded BLG has been used to entrap ligands of possible physiological or pharmacological interest (Barbiroli et al., 2010, Lozinsky et al., 2006), and partially unfolded forms of BLG have been shown to participate in ordered and disordered polymerization events (Cairoli et al., 1994, Rasmussen et al., 2007, Roefs and De Kruif, 1994). Also, BLG partially unfolds – despite the inherent stability of its structure – when contacting the surface of hydrophobic nanoparticles in the absence of chemical and physical denaturing agents (Miriani et al., 2014). Finally, BLG has been shown to undergo partial unfolding – as observed for many other proteins (Miriani, Keerati-U-Rai, Corredig, Iametti, & Bonomi, 2011) – when used to stabilize oil-in-water emulsions (Marengo et al., 2016), a common use for whey proteins in the food industry. Given the relevance of BLG as a food allergen, it has to be noted that unfolding intermediates of BLG display altered immunoreactivity and altered sensitivity to proteases (Marengo et al., 2016, Miriani et al., 2014). These features have been exploited to lower the potential allergenicity of BLG (Iametti et al., 2002, Iametti et al., 2003).
The wealth of details available on individual steps of BLG unfolding makes this protein well suited for analyzing the molecular determinants of the effects of polyols on protein stability. However, given the ease of formation of covalent adducts between BLG and carbohydrates or their derivatives upon thermal treatment in the presence of reducing sugars (Liu et al., 2012, Meltretter et al., 2013) this study was limited to non-reducing sugars (sucrose and trehalose) and to bona fide polyols (sorbitol and glycerol). Aside from contributing to understanding the molecular basis of the protective effects, these studies are also of practical interest, as whey and whey proteins are used in combination with other ingredients (including large amounts of various sugars), in a broad array of food products and food processes also outside the dairy sector.
Section snippets
Proteins and chemicals
All reagents used were in the highest purity commercially available, and were purchased from Sigma-Aldrich (Milan, Italy) unless otherwise specified. To avoid the presence of the partially denatured BLG species found in commercial preparations and to remove naturally occurring bound fatty acids, the protein was purified from unprocessed milk whey, and made lipid-free through passage on a hydroxyl-alkoxypropyl-Dextran column according to procedures reported elsewhere (Barbiroli et al., 2011).
A methodological foreword
The effects of different polyols on the protein structure and stability were studied on lipid-free BLG, because hydrophobic binding sites in as-purified BLG are partially saturated by hydrophobic ligands (typically, various types of fatty acids), that affect the overall stability of the protein (Barbiroli et al., 2011). Complete removal of BLG-bound hydrophobic species ensures that stability studies are carried out on a homogeneous protein population.
The structure of BLG (Brownlow et al., 1997,
Conclusions
Although all the co-solutes but glycerol have similar effects on the overall stability of the protein, they affect in quite dissimilar fashion the temperature stability of the two main structural regions in BLG. Even in the absence of possible cooperative effects between the movement of the C-terminus alpha-helix and the modifications occurring in the beta-barrel main body of the protein, it is clear that the protective effects towards both regions have an entropic origin, and involve solvent
Acknowledgements
This study has been partially supported by DeFENS funds (Linea A, to A.B.). Dr Marengo is the grateful recipient of a post-graduate fellowship from the University of Milan.
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