Fundamentals of unfolding, refolding and aggregation of food proteins Kerensa Broersen Promotoren: Prof. dr. R.J. Hamer Hoogleraar in de technologie van graaneiwitten, Wageningen Universiteit Prof. dr. ir. A.G.J. Voragen Hoogleraar in de levensmiddelenchemie, Wageningen Universiteit Co-promotor: Dr. H.H.J. de Jongh Projectleider, Wageningen Centre for Food Sciences Promotiecommissie: Prof. dr. P.L. Mateo-Alarcón Universidad de Granada, Spanje Prof. dr. I. Braakman Universiteit Utrecht Prof. dr. ir. W. Norde Rijksuniversiteit Groningen/Wageningen Universiteit Prof. dr. E. van der Linden Wageningen Universiteit Dr. H. Nieuwenhuijse Friesland Foods, Deventer Dit onderzoek is uitgevoerd binnen de onderzoeksschool VLAG. Fundamentals of unfolding, refolding and aggregation of food proteins Kerensa Broersen Proefschrift ter verkrijging van de graad van doctor op gezag van de rector magnificus van Wageningen Universiteit, prof. dr. M.J. Kropff, in het openbaar te verdedigen op vrijdag 23 september 2005 des namiddags om half twee in de Aula Broersen, K. Fundamentals of unfolding, refolding and aggregation of food proteins Thesis Wageningen University, The Netherlands 2005 – with summary in Dutch ISBN: 90-8504-250-x Abstract Broersen K Fundamentals of unfolding, refolding and aggregation of food proteins PhD thesis Wageningen University, The Netherlands, 2005 Key words Food, proteins, unfolding, refolding, aggregation, chemical engineering, glycosylation, electrostatics, sulfhydryl groups, structure, stability, thermodynamics, kinetics Protein functionality in food products and in the body strongly relies on the fact that proteins can adopt a number of different conformations. These conformations include folded and (partially) unfolded conformations and many more subtle structural variants in-between. The various conformations are separated by kinetic barriers and, as such, require an activation energy to traverse from the one thermodynamically stable state into another. Also at a molecular level these thermodynamically different conformations are structured in different ways. It was found that even very subtle differences in conformation can have dramatic consequences for the aggregation propensity of proteins. For example, thermodynamic and/or kinetic intermediately trapped protein structures have been found to be sometimes highly prone to intermolecular interactions, primarily through the partial exposure of functional groups. This led us to focus in this thesis on the process from folded to aggregation prone state as opposed to the aggregation process itself. The central aim of this thesis is to explore structural features of proteins of importance to the generation of aggregation prone protein molecules. The relevance of investigating structural in combination with thermodynamic and kinetic parameters in the context of aggregation may be clear. The approach selected involves chemical engineering in which amino groups or carboxyl groups are converted into a chemical group with different properties. The chemical modifications have been specifically selected for their previously reported effects on protein aggregation, and include glycosylation, introduction of sulfhydryl groups and charge modification. Subsequently, this led to a detailed description of the structural impact of the modifications on thermodynamic parameters related to aggregate formation. It was found that the various modifications applied interact with the aggregation process in a rather diverse manner. The accumulation of data from this work in combination with results from literature was used to significantly improve the understanding of factors relevant to aggregation and to develop a general hypothesis for aggregation propensity, the AGPRO model. This model can be used within the food and pharmaceutical industry to determine the aggregation propensity of proteins used in formulae and medication. In het wijde van deze wereld duw ik almaar het hoge gras opzij op zoek naar het antwoord CONTENTS ABSTRACT CHAPTER 1 Introduction 9 Part I Protein glycosylation CHAPTER 2 Glycoforms of β-lactoglobulin with improved thermostability 27 and preserved structural packing. CHAPTER 3 Glycosylation affects protein unfolding/refolding mechanism 47 and the formation of aggregation-prone intermediates. CHAPTER 4 Glucosylation of β-lactoglobulin lowers the heat capacity 73 change of unfolding: a unique way to affect protein thermodynamics. CHAPTER 5 The origins of the effect of glucosylation on the aggregation 91 mechanism of β-lactoglobulin. Part II Electrostatics and protein stability CHAPTER 6 Electrostatics controls fibril formation – part I: Charge 117 engineering of proteins affects unfolding. CHAPTER 7 Electrostatics controls fibril formation – part II: Net charge 141 can affect fibril morphology and gel properties of ovalbumin. Part III Disulphide bonds and protein aggregation CHAPTER 8 Sulfhydryl groups do not affect heat-induced aggregation rate 167 of ovalbumin but do affect aggregate morphology. CHAPTER 9 Discussion: Driving forces for protein aggregation in perspective – the AGgregation PROpensity (AGPRO) model. 183 SUMMARY/SAMENVATTING 219 Chapter 1 CHAPTER 1 INTRODUCTION Introduction In protein-rich food products, such as cheese and yoghurt, proteins do not only serve a role as nutrients but also as texturiser. Figure 1 shows a number of naturally protein-rich food products such as nuts, meat, beans, fish, dairy products and eggs. The recognition of the versatility and value of proteins in food products led to the exploration of large-scale industrial isolation procedures of food proteins from protein-rich by-products from for example cheese production. Today these isolates are widely applied in food products to control texture. Next to a natural or additive component of food products, proteins are also an important constituent of our body and its number of appearances and functions, which have been defined today, seems beyond limits. The consumption of protein-rich food products, either originating from plants or from animals (Figure 1), provides us with the essential amino acids to build proteins required to fulfil roles in the body such as enzymes or hormones. 9 Introduction Figure 1 Examples of protein-rich food products: eggs, nuts, yoghurt, meat, dairy desserts, kidney beans, cheese, tempeh and fish. The large variety of naturally occurring proteins provides the food industry with an extensive choice for protein sources to apply to obtain the textural, sensory or nutritional properties as desired. However, the current lack of understanding of protein behaviour and the high cost to evaluate a large number of protein sources for their suitability in food products has greatly frustrated the development process of new protein-containing food products. As structure and function of the diverse proteins have been found to be strongly correlated in the past (Morley et al 2004, De Sanctis et al 2004, Coombe & Kett 2005, Deignan et al 2004, Zemser et al 1998) it seems reasonable to select protein systems based on their structural properties. However, considering only the primary sequence or molecular weight of a protein provides insufficient insight to design functionality in food products. Examples of primary structure properties are the ratio of hydrophobic to hydrophilic residues, the number of disulphide bonds and free sulfhydryl groups and positive and negative charge (Chiti et al 2002, 2003, Linding et al 2004, DuBay et al 2004, Fernandez-Escamilla 2004, Hayakawa & Nakai 1985). 10 Chapter 1 Next to the primary sequence, the folding, comprised of interactions at a secondary and tertiary structural level, more importantly determines the actual extent of exposure of hydrophobic regions, disulphide bonds/sulfhydryl groups and apparent net charge (Chiti et al 2002). Below, the structures of ovalbumin, an egg white protein (Figure 2a) and β- lactoglobulin, a whey protein (Figure 2b) are depicted which will serve as an example to illustrate this. a b Figure 2 Ribbon drawings of the tertiary and secondary structure of a) chicken egg ovalbumin (1OVA: Protein Data Bank) and b) bovine β-lactoglobulin (1BEB: Protein Data Bank) as solved by X- ray crystallography. The black structures represent helices and the dark grey structures represent sheets and the light grey structures represent random coil and turns. Both proteins are important food ingredients but they cannot be simply exchanged without affecting the properties of the food product. These differences originate, apart from a significant difference in size, among others, also from significantly varying proportions of the different types of secondary structure elements. As Figure 2 shows, approximately 18% of the secondary structure of β-lactoglobulin attains a so-called α-helix conformation whereas for ovalbumin the relative proportion of α-helix is 30% (Tedford et al 1998). Moreover, ovalbumin and β-lactoglobulin have been found to expose significantly different thermodynamic behaviour (such as heat- or denaturant-resistance). These complexities already suggest why it has been difficult in the past to predict the functional properties of proteins in food products from a single property of the polypeptide chain. Furthermore, extensive knowledge on the relative significance of structural and thermodynamic properties on protein behaviour is not always available, limiting the choice and efficient use of proteins 11 Introduction as food ingredients and requiring time-consuming trial-and-error studies to determine the