water interfaces

Current Opinion in Colloid & Interface Science 18 (2013) 257–271

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Protein folding at emulsion oil/water interfaces Jia li Zhai a, b, Li Day b, Mare-Isabel Aguilar a, Tim J. Wooster c,⁎ a b c

Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC 3800, Australia CSIRO Animal, Food and Health Sciences, 671 Sneydes Road, Werribee, VIC 3030, Australia Nestle Research Centre, CH-1000 Lausanne 26, Switzerland

a r t i c l e

i n f o

Article history: Received 1 February 2013 Received in revised form 4 March 2013 Accepted 12 March 2013 Available online 26 March 2013

a b s t r a c t It has long been known that proteins change their conformation upon adsorption to emulsion oil/water interfaces. However, it is only recently that details of the specifics of these structural changes have emerged. The development of synchrotron radiation circular dichroism (SRCD), combined with advances in FTIR spectroscopy, has allowed the secondary and tertiary structure of proteins adsorbed at emulsion oil/water interfaces to be studied. SRCD in particular has provided quantitative information and has enabled new insights into the mechanisms and forces driving protein structure re-arrangement to be achieved. The extent of conformational re-arrangement of proteins at emulsion interfaces is influenced by several factors including; the inherit flexibility of the protein, the distribution of hydrophobic/hydrophilic domains within the protein sequence and the hydrophobicity of the oil phase. In general, proteins lose much of their tertiary structure upon adsorption to the oil/water interface and have considerable amounts of non-native secondary structure. Two key conformations have been identified in the structure of proteins at interfaces, intermolecular β-sheet and α-helix. The preferred conformation appears to be the α-helix which is the most compact amphipathic conformation at the oil/water interface. The polarity of the oil phase can have a considerable influence on the degree of protein conformational re-arrangement because it acts as a solvent for hydrophobic amino acids. The new conformation of proteins at interfaces also means that proteins undergo less heat induced re-arrangement at interfaces than in solution. Different conformations of proteins at interfaces impact on emulsification capability, emulsion stability and protein/emulsion digestion. Hence advances in the understanding of protein conformation at interfaces can help to identify suitable proteins and conditions for the preparation of emulsion based food products. © 2013 Elsevier Ltd. All rights reserved.

1. Introduction Protein adsorption at oil/water interfaces of emulsions is an important process in the manufacture of many emulsion-based products in food and pharmaceutical industries. The amphiphilic nature of proteins makes them a logical choice as emulsifiers/stabilisers of emulsions. In particular, milk proteins such as sodium caseinate and whey protein isolate have been widely used in food emulsions [1–4]. Studies over the past decades have shown that the properties of proteins adsorbed at oil/water interfaces play a key role in determining the physicochemical properties of emulsions, their stability, and how they interact with the body (taste, texture and digestion) [5,6•,7,8••,9•]. It is not surprising, therefore, that characterisation of protein interfacial structure and emulsion properties has received widespread interest in the field of food colloid and interface science [5,7,10–14]. Proteins help to stabilise emulsions through a combination of mechanisms [5,7,10–14]. First, during emulsification they adsorb to the interface forming a membrane which stabilises the droplet against re-coalescence ⁎ Corresponding author. Tel.: +41 217858714. E-mail address: [email protected] (T.J. Wooster). 1359-0294/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cocis.2013.03.002

by resisting tangential shear [5,7,10–14]. Then, depending on the structure the protein adopts at an interface, proteins can help to stabilise droplets against flocculation [5,7,10–14]. Globular proteins such as β-lactoglobulin (β-Lg) and α-lactalbumin (α-La) which adopt thin dense interfaces primarily impart flocculation stability via electrostatic repulsion [7,15]. In comparison, proteins which form thick disperse interfaces (i.e. caseins) impart flocculation stability by both steric and electrostatic repulsion [7,15]. Whether a protein creates a thin dense interface or an open disperse interface is to a large part determined by its conformation before and/or after adsorption [15]. Extensive effort has been made to understand the spatial distribution of proteins upon adsorption to oil/water interfaces [16•,17,18••,19] and how these relate to emulsion stability [1,14]. What is less well understood are the conformational changes that proteins undergo upon adsorption to emulsion interfaces and how this impacts on emulsion stability. It has long been known that, upon adsorption, proteins adopt a conformation that is different from their native structure in solution [20–22]. Logically, the process of adsorption necessitates conformational re-arrangement to enable hydrophobic amino acids within the core of globular proteins to interact with the oil phase. The first evidence

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that pointed to proteins adopting different conformations at emulsion oil/water interfaces came from indirect measurements. (Micro)calorimetry experiments showed that the denaturation peak of proteins was diminished or disappeared upon adsorption to oil/water interfaces [23]. Early digestion experiments showed that the proteolysis of many proteins was altered upon adsorption to emulsion interfaces [24,25••,26]. However, it is only in the last 10 or so years that quantitative information about the secondary and tertiary structure of proteins at interfaces has become possible [18••,27••,28••,29••]. This review highlights recent advances in measurement and understanding of protein folding at oil/water interfaces. There are a number of excellent articles and reviews on the properties of proteins adsorbed at air/water interfaces [14,30•,31] and at solid surfaces [32,33], therefore it was not our intention to review these topics further. Instead, we have chosen to focus on protein folding at emulsion oil/water interfaces, in part due to the increasing amount of new experimental data and our own particular interest in this area. Apart from reviewing the current understanding and latest data on the conformation of proteins at emulsion interfaces, we also attempt to provide discussion on a number of trends/topics that have been put forward to describe protein (un)folding at emulsion interfaces. Finally we try to highlight how/if understanding protein conformation at emulsion oil/water interfaces might be relevant to the emulsion formulation and functionality.

2. Molecular forces and structures of proteins in solution Proteins fold into their unique structure via a defined folding pathway from an initially unfolded chain of amino acids (the primary structure) into a well-defined three-dimensional (3D) structure (the tertiary structure) [34–37]. This defined 3D structure, i.e. the native structure of a protein, corresponds to the most easily accessible, and not the global, free energy minimum of that protein [36]. The native structure of a protein is a consequence of a delicate balance between all attractive and opposing molecular forces, including electrostatic forces, ion-pairing, van der Waals interactions, hydrogen bonding, the hydrophobic effect and the conformational entropy [34–36]. The hydrophobic effect is the dominant driving force in protein folding [34,38]. The hydrophobic effect, in the context of protein folding, refers to the tendency of the non-polar regions of a protein to come together so that the thermodynamically unfavourable contact with water is decreased. Conformational entropy is the dominant force opposing protein folding, and is related to the number of possible conformations that the polypeptide chain can adopt in solution [34,39]. There is local entropy (translational, rotational, vibrational) and non-local entropy (the excluded volume effect) [34]. The hydrophobic effect drives the initial step of the folding of a protein, resulting in a relatively compact state [34]. After this initial collapse of the chain, many segments of the amino acid side chains and the backbone become extremely compact and have limited volume of space to occupy [40]. While the hydrophobic effect leads to the compactness of a globular protein with the hydrophobic core, significant steric constraints and hydrogen bonding are largely responsible for the unique internal organisation comprised of a combination of secondary structures. The two dominant forces, i.e. the hydrophobic effect and the conformational entropy, eliminate a considerable amount of conformational possibilities and together with all the other interactions lead to the defined native structure of a protein. The three major secondary structures are the α-helix, the β-sheet and the β-turn (Fig. 1). The α-helix is a coiled structure stabilised by hydrogen bonding between the CO group of each amino acid and the NH group of the amino acid that is situated n + 4 residues along the sequence (Fig. 1A). The β-sheet is a pleated structure formed by hydrogen bonding between the NH group and the CO group on adjacent β-strands (Fig. 1B). Hydrogen bonding between the backbone amide and carbonyl groups plays an important role in the formation of the α-helix and the β-sheet (Fig. 1C) [34,41].

Fig. 1. A ball-and-stick representation of secondary structures. A) α-helix; B) anti-parallel β-sheet; and C) parallel β-sheet. The dashed, green lines depict hydrogen bonds between the backbone NH and CO groups. Adapted from Berg et al. [158].

One way of understanding the dominant forces in protein folding is to induce different conformations of a protein (e.g. denaturation) by application of external stress (i.e. heat/cold or chemical denaturants). The free energy stabilising the 3D structure of a protein (Eq. (1)) is surprisingly low, typically ranging from 20 to 50 kJ/mol or from 0.1 to 0.2 kT per residue [37]. ΔGfold ¼ ΔHb −TΔSchain −TΔSsolv

ð1Þ

where: ΔHb is the sum of all intermolecular interactions, ΔSsolv is the configurational energy of the solvent and ΔSchain is the configurational entropy of the protein chain. The unfolding mechanism associated with each denaturing condition can be explained by the relative contribution of each of the molecular forces involved in protein folding. Upon heating the gain in configurational entropy (melting of water structures around hydrophobic residues and backbone configurational entropy) is such that the hydrogen bonding and hydrophobic interactions are disrupted [34,42,43]. Addition of chemicals such as urea and guanidinium hydrochloride causes protein denaturation due to their effect on the interaction of the solvent phase with polar and non-polar amino acid residues [43]. 3. Techniques for measuring protein folding at emulsion interfaces 3.1. Fourier transform infrared spectroscopy Fourier transform infrared (FTIR) spectroscopy is an established tool to measure protein secondary structure. FTIR measures the infrared absorption of proteins in the amide I region (1580–1720 cm − 1) and amide II region (1510–1580 cm − 1), which are sensitive to protein secondary structure motifs. The frequency of the amide I and II absorptions is influenced by the strength of any hydrogen bonds involving the CO and NH groups of proteins. The α-helix and the β-sheet are associated with characteristic hydrogen bonding patterns (Fig. 1) and therefore give rise to characteristic FTIR absorptions (Fig. 2A and Table 1). The subsequent data analysis of FTIR spectra thus reveals information about protein secondary structure in a certain environment. Generally, the α-helix absorbs from 1648 to 1660 and from 1545 to 1551 cm − 1 in the amide I and II regions respectively; the β-sheet absorbs from 1625 to 1640 and from 1521 to 1525 cm−1 [44,45]. In comparison to far-UV circular dichroism (CD) spectroscopy, the major strength of FTIR lies in its ability to allow measurements of protein secondary structure in a variety of environments

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A

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B Hydrophilic 355nm

Hydrophobic 320nm

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Fig. 2. Examples of different spectroscopic techniques that can give information on different elements of a proteins structure. FTIR spectra (A) in the amide II region consist of a summation of peaks (coloured lines), and often a 1st derivative is taken to better identify the key absorptions (black lines). The peak in the fluorescence emission spectra of tryptophan (B) changes depending on whether it is exposed to water or the interior of the protein. The different secondary structure motifs have characteristic absorption spectra in the Far-UV CD (C). Each aromatic amino acid absorbs in characteristic regions of the Near-UV CD (D). A — reproduced with permission from Lee et al. [47•], C — from Kelly et al. [50], and D — redrawn from Kelly et al. [50].

such as aqueous solution, hydrated films and turbid emulsions. The light scattering arising from emulsions does not cause interference with the FTIR signal and FTIR has thus been used to measure the conformation of proteins adsorbed at oil/water interfaces [27••,46•,47•,48]. Unique assignments of individual secondary structures are often difficult because

Table 1 Amide I band frequencies (2nd derivative) and assignments to various secondary structure motifs for proteins in H2O and D2O. Table complied primarily from Jackson et al. [44] and Kong et al. [45]. Frequency 1610–1628 1625–1642 1640–1658 1648–1660 1660–1670 1667–1696

Main peaks H2O (D2O) cm−1 cm−1 cm−1 cm−1 cm−1 cm−1

1667–1696 cm−1 1667–1696 cm−1

1648 cm−1 (1645 cm−1) 1656 cm−1 (1653 cm−1) 1663 cm−1

1667–1685 cm−1 1691 & 1696 cm−1

Structural motif Aggregated strands β-sheet unordered α-Helix 310 Helix Anti-parallel/intermolecular β-sheet/β-turn Anti-parallel β-sheet β-turn

a complex protein structure generally produces an absorption profile of many overlapping peaks and shoulders (Fig. 2A) [44,45]. 3.2. Far UV circular dichroism spectroscopy Circular dichroism (CD) spectroscopy is a spectropolarimetry technique used to measure protein secondary and tertiary structure in solution. A CD instrument measures the difference in the absorbance of left- and right-handed polarised light by an optically active substance. Proteins are optically active molecules that can generate CD signals due to the presence of peptide bonds and aromatic amino acids [49,50]. In the far-UV region (170–260 nm), the peptide bond is the major chemical entity contributing to the CD signal and each type of secondary structure gives rise to a CD spectrum of a characteristic shape and magnitude (Fig. 2C) [50]. For example, an α-helix gives two negative minima at 222 nm and 208 nm and a positive peak at 190 nm (Fig. 2C). A β-sheet gives a single negative minimum at around 218 nm and much smaller negative ellipticity in comparison to an α-helix (Fig. 2C). Proteins with a large amount of disordered structure give very low ellipticity above 210 nm and a negative band at around 195 nm

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(Fig. 2C). Far UV CD spectroscopy can not only qualitatively examine secondary structure but also quantitatively estimate the secondary structural content of a protein. At its simplest, the CD spectrum of a protein can be assumed to be a linear combination of the spectra of all secondary structure components, weighted by their fraction [51]. CD spectra from specific polypeptides (i.e. poly-L-proline) were originally used for such analyses [51]. However, the analysis of far-UV CD spectra has advanced considerably with various mathematical methods (SELCON [51,52], CONTIN [53] and CDSSTR [54]) and reference data sets based on real proteins in solution now routinely being used [51,55,56]. Synchrotron radiation circular dichroism (SRCD) spectroscopy has emerged as a powerful tool which takes advantage of the strong intensity of the light available from synchrotron sources. SRCD produces high signal-to-noise spectra, extending the wavelength region that can be measured down to 160 nm for protein in solution (Fig. 2C) and most importantly enables improved penetration of light into highly absorbing and turbid samples [57 •,58]. For example, SRCD has been employed to study the secondary structure of membrane proteins in optically turbid media [58]. An alternative approach is to correct for absorption induced flattening using the mathematical approach recently proposed by Gerdova et al. [59]. A limitation of the approach taken by Gerdova et al. is that the absorption spectrum of the particle/scattering body is not determined independently, whereas in the SRCD approach the spectrum of the particle is independently determined. 3.3. Near UV CD analysis of protein conformation Aromatic amino acids and disulphide bonds produce CD signals in the near UV region between 260 and 320 nm [50,60]. The CD absorption by these groups is very sensitive to environmental changes and hence is an additional measure of changes to the tertiary structure of a protein. In a rather definitive review Strickland [60] outlines that aromatic amino acids tend to have characteristic CD absorptions in the near UV region (Fig. 2D); i) tryptophan — main absorption around 290 nm with fine structure between 290 and 305 nm, ii) tyrosine — main absorption around 275 to 282 nm whose fine structure can be obscured by the tryptophan absorptions, iii) phenylalanine — produces sharp CD fine structure between 270 and 255 nm, prominent phenylalanine CD absorptions occur as same sign pairs of similar intensity separated by 6 nm, and iv) cysteine residue disulphide bonds — usually give near-UV absorptions that begin at long wavelengths gradually increasing in intensity to give one or two broad peaks above 240 nm [50,60]. Unlike Far-UV CD the theory behind near-UV CD has not advanced to the point to enable quantitative analysis of protein tertiary structure [60]. Nonetheless, near-UV CD measurements have given valuable qualitative information on the conformational changes that proteins undergo [61,62•,63] under defined physical stresses (heat or extreme pHs) [64,65•]. 3.4. Tryptophan emission fluorescence spectroscopy Tryptophan emission fluorescence spectroscopy is widely used to study protein conformational changes in solution [66]. In a fluorimeter, light excites intrinsic fluorophores such as aromatic amino acids to a high energy state and then the emission of the absorbed light from the sample is recorded. The emission energy of tryptophan is highly sensitive to the hydrophobicity of its local environment (Fig. 2B). At an excitation wavelength of around 295 nm, a tryptophan residue buried in the hydrophobic core of a protein or incorporated in a hydrophobic solvent emits at wavelengths with a maximum around 335 nm (Fig. 2B) [67–70]. In comparison, the emission of tryptophan exposed to an aqueous solvent, for example due to denaturation of a globular protein, is red-shifted towards a higher wavelengths with a maximum around 355 nm (Fig. 2B) [61,67,68,71]. The emission spectra therefore provide useful information about the local environment of tryptophan, which can reflect protein tertiary structural change.

In normal fluorescence spectroscopy, light excites the sample at an incident angle of 90°. When a considerable amount of light is absorbed or scattered (e.g. by an emulsion) insufficient emitted fluorescence reaches the detector resulting in poor signals. In order to study protein conformational change in emulsions, front-face tryptophan fluorescence spectroscopy has been used by a number of groups [67,72,73 •,74,75 •,76,77 •,78–81]. With the incidence angle set to between 30 and 60° (but not 45°) in a front-face set-up, light penetrates the first few 10's of micrometres and a portion is reflected to the detector at 90° and a good signal can be obtained [66]. 4. Conformational changes of various proteins upon adsorption to emulsion oil/water interfaces 4.1. Conformation of β-lactoglobulin at oil/water interfaces β-Lg (Fig. 3A) is the major whey protein found in bovine milk, and belongs to the lipocalin class of proteins [82]. It is a globular protein with a molecular weight (Mw) of 18.4 kDa that naturally exists as a dimer at neutral pH and as tetramers and octamers at acidic or basic pHs [83 •]. The β-Lg structure consists of nine anti-parallel β-sheets which wrap around to form a hydrophobic β-barrel with one long α-helix and four short 310 helices on the outer surface (Fig. 3A) [27 ••,84,85]. Circular dichroism studies have found that β-Lg in solution contains 40–50% β-sheet and 10–15% α-helix structure [84,86,87]. β-Lg contains five cysteine residues per monomer; four form two intramolecular disulphide bonds (Cys66–Cys160 and Cys106–Cys119) and one free cysteine (Cys121). The existence of a reactive cysteine residue means that β-Lg readily undergoes disulphide exchange reactions to produce gels when exposed to heat, pressure and basic pHs [83•]. The conformation of β-Lg adsorbed to oil/water interfaces has been examined using FTIR [27 ••,28 ••,29 ••,88], CD [18 ••,28••,89••,90•] and fluorescence emission spectroscopy [89 ••]. Fang and Dalgleish were one of the first groups to use FTIR to study the conformational changes of β-Lg upon adsorption to the oil/water interface of soybean oil emulsions [27 ••]. Adsorption to emulsion oil/water interfaces caused a decrease in intramolecular β-sheet, no change in α-helix, an increase in unordered structure and an increase in intermolecular β-sheet. Emulsions prepared with 1% β-Lg underwent quicker and more extensive conformational re-arrangement than 2% β-Lg. There was some effect of pH, emulsions prepared at pH 6 underwent faster and more extensive denaturation than those prepared at pH 7 [27••]. Furthermore, in emulsions prepared at pH 6, β-Lg had much larger amounts of unordered structure and (possibly) intermolecular β-sheet structure whereas in emulsions at pH 7, β-Lg had higher α-helical content [29••]. Unadsorbed protein in the serum phase exhibited a spectrum almost identical to native β-Lg in solution indicating that adsorption rather than homogenisation caused the conformational change, which agrees with the results of Lefevre and Subirade [29••]. Husband et al. [28••] made a considerable advance in the analysis of protein structure in emulsions by developing a refractive index matched emulsion (RIME) method that permitted the use of CD spectroscopy on emulsions. Using FTIR and RIME-CD it was observed that upon adsorption to the interface of n-tetradecane emulsions the main structural elements of β-Lg were retained. FTIR measurements indicated that upon adsorption there was an increase in intermolecular β-turns whereas the intramolecular β-sheet content appeared similar. RIME-CD measurements indicated an increase in the positive CD absorption at 190 nm, a move in the zero crossing to lower wavelengths, and an increase in intensity of the negative absorptions at 208 and 220 nm. SELCON analysis by the authors indicated that these changes in the CD were indicative of an increase in α-helical content. The authors raised concerns over this due to the discrepancy between the FTIR and CD measurements. The observation of a significant increase in α-helical content of β-Lg upon adsorption to emulsion oil/water interfaces by Husband

J. Zhai et al. / Current Opinion in Colloid & Interface Science 18 (2013) 257–271

A) β-lactoglobulin

B) α-lactoalbumin

261

C) Lysozyme

D) Bovine serum albumin E) Myoglobuin

Fig. 3. The structures of common food proteins depicted in quasi-3D with the ribbon diagram of their secondary structures. Blue and red elements in the space fill structure depict positive and negative surface charges respectively. Protein structures were drawn using protein crystal databank files (3BLG, 1HFX, 132L, 3V03 and 1MBN) and with the aid of Pymol software v1.5.0.3 with APBS tools.

et al. was confirmed by Zhai et al. [18 ••,89 ••] and Day et al. [90 •]. Using SRCD spectroscopy Zhai et al. examined the conformation of β-Lg in tricaprylin and n-hexadecane emulsions at ambient and at elevated temperatures [18 ••,89 ••]. Far UV SRCD measurements indicated that β-Lg adsorption to oil/water interfaces caused a large increase in α-helix and a concurrent decrease in β-sheet secondary structure (Fig. 4(1)A). The α-helical content of β-Lg increased from 16% in solution, to 24% when adsorbed at the tricaprylin/water interface, and 50% at the n-hexadecane/water interface. At the same time, the percentage of β-sheet structure decreased from 33% in solution to 28% and 15% for β-Lg adsorbed at the tricaprylin/water and n-hexadecane/water interfaces, respectively [89••]. Near-UV SRCD measurements indicated that there was a considerable disruption of the tertiary structure of β-Lg, bands associated with tryptophan (285 and 293 nm) and tyrosine (277 nm) disappeared upon adsorption to both oil/water interfaces (Fig. 4(2)A). These changes in near-UV SRCD spectra were accompanied by shifts in the fluorescence emission maxima of tryptophan from 335 nm in solution to 330.5 and 328.5 nm at the tricaprylin/water and n-hexadecane/water interfaces respectively [89 ••]. Similar shifts in β-Lg tryptophan fluorescence emission maxima were also observed by Sakuno et al. at triacylglycerol (TAG)/water and diacylglycerol (DAG)/water interfaces [88].

A strong increase in β-Lg intermolecular β-sheet content was also found in the work of Lefevre and Subirade who studied the conformation of β-Lg at the interface of n-hexadecane/water emulsions [29••]. Lefevre and Subirade commented that the increase in β-Lg intermolecular β-sheet upon adsorption to emulsion interfaces was analogous to its structure of heat induced gels in solution. The authors highlighted that this increase in intermolecular β-sheets was indicative of protein aggregation at the interface, and proposed that this was one possible explanation for the high viscoelastic nature of β-Lg interfaces. These results were countered by the recent work of Sakuno et al. who saw relatively minor changes in intermolecular β-sheet formation upon the adsorption of β-Lg to the interface of TAG and DAG emulsions [88]. Sakuno et al. also observed a considerable decrease in intramolecular β-sheet content, a large increase in the α-helical content and an increase in unordered structure. The changes observed were greater at the DAG/water interface than at the TAG/water interface. The presence of a free sulfhydryl group on Cys121 contributes to the changes in the structure of β-Lg at emulsion interfaces. A number of studies demonstrated that the free cysteine at Cys121 participated in intra-[22,91 •,92] and inter-[8••,9 •,91•] droplet sulfhydryl-disulphide exchange. Dickinson and Matsumura [22] were the first to demonstrate that intra-droplet sulfhydryl-disulphide exchange did not occur due to

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MRE (103 deg cm2 dmol-1 res-1)

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Fig. 4. Far-UV (1) and Near-UV SRCD (2) spectra of A) β-Lactoglobulin B) α-Lactalbumin C) lysozyme and D) myoglobin when they are i) in solution (black), and adsorbed at ii) tricaprylin/water emulsion interfaces and iii) n-hexadecane/water emulsion interfaces. Detailed explanation of sample preparation and data collection can be obtained from these references. A (Zhai et al. [89••]), B (Zhai et al. [97•]) and C and D (Day et al. [90•]) were reproduced with permission.

emulsification, but rather during emulsion ageing at room temperature. Damodaran and Anand [91•] suggested that at extended times interdroplet β-Lg polymerisation can also occur under ambient conditions. Kim et al. [8••,9•] demonstrated that heating of β-Lg emulsions also caused inter-droplet sulfhydryl-disulphide exchange. Interestingly SRCD measurements by Zhai et al. found that β-Lg underwent limited conformational change upon heating to 76 °C and that the conformational changes were much less than β-Lg in solution [89 ••]. In addition, the conformational change during heating of β-Lg adsorbed at the n-hexadecane/water interface was less than at the tricaprylin/water interface. These contrasting observations might be understood from Fang and Dalgleish who proposed that adsorption may cause movement of the α-helix away from the rest of the molecule and increase the accessibility of the free sulfhydryl group at Cys121 [27 ••]. 4.2. Conformation of α-lactalbumin at oil/water interfaces α-Lactalbumin (α-La) is a small globular protein (Mw 14.2 kDa) that has one large domain of three α-helices and one sub-domain of three small antiparallel β-sheets, which are connected by a calcium binding loop (Fig. 3B) [83 •,93]. The lack of a free cysteine (α-La has four disulphides), and the existance of a Ca 2 + binding pocket play important roles in the stability and conformation of α-La. An important feature of α-La is its ability to adopt a molten globule state under various conditions such as at low pHs, high temperatures, at moderate concentration of denaturant, or in its Apo (non-calcium bound) form [64,94••,95]. The molten globule state of a protein is characterised by the protein having disordered tertiary structure but native-like secondary structure. The molten globule state of α-La has important

implications for protein folding at interfaces as Bañuelos and Muga [96] found that α-La binds to lipid bilayers in a molten globule-like conformation. Dickinson and Matsumura [94••] in the '90s speculated that proteins might adopt a molten globule state upon adsorption to emulsion oil/water interfaces. Fang and Dalgleish used FTIR to study the conformational changes of apo-α-La upon adsorption to soybean oil/water interfaces [46 •]. When α-La was adsorbed at the interface of the emulsion, Fang and Dalgleish observed an increase in β-sheet structure, an increase in turn structure, a decrease in 310 helical content and a sharpening of FTIR bands associated with α-helical structure. Unlike their observations with β-Lg [27 ••], the changes to secondary structure of α-La showed only marginal evolution with emulsion ageing and/or emulsion pH variation. Emulsions prepared with lower α-La contents (1 wt% as opposed to 2 wt%) showed greater loss of the 310 helix content but the two bands associated with the α-helix were more distinct and perhaps had greater intensity in emulsions prepared at pH 6. Similar to emulsions stabilised by β-Lg, there was no change in the conformation of the protein in the serum phase of the emulsion. Zhai et al. [97 •] also examined the conformation of α-La at both n-hexadecane and tricaprylin oil/water interfaces using SRCD. The Far-UV CD and SRCD of α-La indicate that α-La in solution has 29.9% α-helix and 18.0% β-sheet [65 •, 98]. Adsorption of α-La to emulsion oil/water interfaces showed a systematic increase in α-La helical content rising to 45.8% at the tricaprylin/water interface and 58.5% at the n-hexadecane/water interface (Fig. 4(1)B) [97•]. In parallel, the β-sheet content decreased to 8.3% at the tricaprylin/water interface and 9.9% at the n-hexadecane/water interface. Zhai et al. also probed the changes in α-La tertiary structure using front-face fluorescence emission

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spectroscopy and near-UV SRCD [97•]. Adsorption of α-La to emulsion oil/water interfaces resulted in a 6.5 nm red-shift of the emission wavelength maximum to 335 nm at the tricaprylin/water interface and a 12 nm red-shift to 340.5 nm at the n-hexadecane/water interface. These results demonstrated the movement of one or more tryptophan residues to a more hydrophilic environment when α-La was adsorbed at oil/water interfaces, suggesting a perturbation in the native tertiary structure. When adsorbed at emulsion oil/water interfaces the deep broad minimum between 260 and 290 nm in the near-UV SRCD spectrum (arising from tyrosine residues and disulfide bonds) and the small peak at 295 nm (tryptophan residues) disappeared (Fig. 4(2)B), suggesting loss of the globular structure of α-La [61,65•,95,98]. Unlike β-Lg, α-La does not have a free cysteine which would suggest it does not undergo polymerisation at emulsion interfaces. However, Damodaran and Anand found that there was a small degree of dimerisation of α-La that occurred during the storage of emulsions stabilised only by α-La [91•]. It was proposed that dimerisation was caused by free radical catalysed protein–protein cross-linking rather than disulphide exchange [91 •]. There is very little information on the effect of heat on disulphide exchange of α-La adsorbed at emulsion interfaces. However, Zhai et al. studied the conformational changes that α-La underwent at elevated temperatures and found that similar to β-Lg, the conformation of α-La in the adsorbed state was also more resistant to conformational change than in the solution state [97 •]. This was particularly true for α-La adsorbed at the n-hexadecane/water interfaces where α-La barely underwent any change above 34 °C. However, the conformation of α-La adsorbed at the tricaprylin/water interface underwent a significant conformational change [97 •].

4.3. Conformation of β-casein at oil/water interfaces The caseins (β, αs1, αs2 and Κ) are the major protein component of bovine milk comprising approximately 80% [82]. The most studied is β-casein (209 residues, Mw 24.04 kDa, β-Cas) known for its hydrophobic C-terminus and highly phosphorylated N-terminus. In solution, FTIR and CD measurements indicate that β-Cas adopts an open conformation with many short sequences (n ≥ 4) of polyproline II type structure [99]. Overall structural analysis revealed that β-Cas has approximately 12– 20% α-helix and around 20–35% β-sheet, with the rest constituting turns and disordered structure [62 •,99–102]. There have been numerous studies of the structure and conformation of β-Cas at air/water interfaces, but comparatively fewer at oil/water interfaces [62 •,102,103]. Caessens et al. [62 •] found using CD that adsorption to a Teflon surface induced ordering of β-Cas secondary structure; the α-helical content increased from 13% to 20% and the random coil content decreased from 72% to 65%. By examining the conformational changes of various β-Cas peptides, Caessens et al. identified that the N-terminal amphipathic peptide [1–105/107] showed more adsorption-induced structure increase than the hydrophobic C-terminus [106/108/114–209] [62•]. Wong et al. [102] found a similar trend when they examined β-Cas adsorption to emulsion oil/water interfaces using SRCD. The extent of structure induction correlated slightly with the polarity of the oil phase, changing from 5% α-helix (35% β-sheet and 60% unordered structure) to 20% α-helix (23% β-sheet and 57% unordered structure) at the tricaprylin/ water interface and to 23% α-helix (23% β-sheet and 54% unordered structure) at the n-hexadecane/water interface [102]. What is interesting is that both studies appear to observe an increase in α-helical structure at the expense of β-sheet structure (or more likely polyproline II structure as it is hard to discriminate between the two with CD). Front face tryptophan fluorescence emission spectroscopy measurements by Wong et al. showed (as expected) a blue shift in the emission wavelength maximum from 349 nm in solution to 339.5 nm at the tricaprylin/water interface and 337 nm at the n-hexadecane/water interface [102].

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4.4. Conformation of bovine serum albumin at oil/water interfaces Bovine serum album (BSA, Fig. 3D) is a large globular protein (Mw 66.3 kDa) with a highly ordered internal structure that is stabilised by 17 disulphide bonds, and has one free thiol (Cys34) [104,105]. The 17 disulphide linkages in BSA are positioned as eight sequential Cys–Cys pairs in a repeating series of loop-link-loop structures between the α-helical sub-domains [105,106]. The far-UV CD spectrum of BSA in solution (neutral pH) reflects a strongly α-helical structure, with relative proportions of 73% α-helix, 2% β-sheet, 8% turn and 17% unordered structure [90 •] which agrees well with the X-ray crystallographic data [106,107]. The initial evidence for changes in BSA structure upon adsorption to oil/water interfaces was provided by a number of studies using front-face fluorescence emission spectroscopy [67,73•,75•,77•]. Castelain & Genot [67] found that the wavelength of the emission wavelength maximum of BSA adsorbed at the dodecane/water emulsion interface was blue-shifted by as much as 15 nm (from 335 nm to 320 nm) as compared to that of BSA in solution, suggesting that the environment of the tryptophan residues became more hydrophobic. The blue-shift was slightly larger in the case of dodecane emulsions than for sunflower oil and miglyol emulsions [73•]. Blue-shifts in the emission wavelength maximum of human serum albumin (HBA) and/or BSA adsorbed to water-in-oil emulsion interfaces have also been reported by Zelisko et al. [108] and Jorgensen et al. [109], indicating structural changes around the tryptophan residues upon its adsorption to the interface. Using Raman spectroscopy, Meng et al. [110] found a reduction in the normalised intensity of the tryptophan band near 750 cm−1, and a reduced intensity ratio of the tyrosine doublet at 850–830 cm−1 in the Raman spectra when BSA was adsorbed at a mineral oil/water interface compared to BSA in solution. They interpreted these changes as an indication of the exposure of these hydrophobic residues and changes in the tertiary structure of BSA at the interface. However, Meng et al. concluded that there was no significant change in BSA's secondary structure because the Raman spectrum (in the amide I region, 1654 cm−1) of BSA at the interface was similar to the spectrum of BSA in solution. Jorgensen et al. [109] investigated the conformation of BSA adsorbed at the water-in-oil emulsion interface using FTIR and was able to show that the secondary structure of the proteins changed to some extent, 12% for BSA and 9% for HSA, due to the difference in the area overlap and a slight shift in the position of the α-helix band. Similarly, changes in the amide I band of the BSA adsorbed to a hydrophobic dipalmitoylphosphatidylcholine (DPPC) liposome was observed by McClellan & Franses [111]. The changes measured by FTIR corresponded to an 8% reduction in α-helix content and a 10% increase in β-sheet structure of BSA adsorbed layer, indicating some unfolding of BSA upon adsorption. Husband et al. examined the structure of BSA adsorbed at n-tetradecane/water interfaces using FTIR and RIME-CD [28 ••]. The FTIR spectra of the amide I band showed a decrease at 1653 cm−1 as well as an increase at 1633 cm−1 suggesting a decrease in α-helix and an increase in β-sheet structure in BSA after adsorption. CD measurements confirmed a 9% reduction in α-helix and a 14% increase β-structure content of BSA at the oil/water interface. A reduction in α-helix accompanied by an increase in β-sheet and unordered structures of BSA at emulsion oil/water interfaces was also seen in a recent study by Day et al. using SRCD [90•]. Interestingly Day et al. saw that the reduction in BSA α-helical content scaled with the oil phase polarity, reducing by 12% at the tricaprylin/water interface, and 21% at the less polar n-hexadecane/water interface compared with BSA in solution [90•]. At the same time, the β-structures (sheet and turns) increased [90•]. Near-UV CD was used in the studies of Husband et al. [28••] and Day et al. [90•] to monitor changes in the environment of the aromatic groups of BSA adsorbed at emulsion interfaces. Husband et al. saw small changes in the near-UV CD spectra of BSA upon adsorption; these changes were mainly within the tyrosine and tryptophan bands (280–290 nm). Consistent with front-face fluorescence measurements

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which showed that the tryptophan emission wavelength maximum shifted towards lower wavelengths (more hydrophobic environment) with less polar oils [73•], Day et al. also demonstrated that the changes in BSA tertiary structure upon adsorption to the n-hexadecane/water interface were greater than at the tricaprylin/water interface [90•]. 4.5. Conformation of lysozyme at oil/water interfaces Lysozyme is a small globular protein comprising 129 amino acid residues with a Mw of 14.4 kDa. It has 4 short α-helices between the residues 4–15, 24–36, 88–99 and 108–115, which makes up one domain encompassing the N- and C-terminal segments of the protein connected via two disulphide bonds (Cys6–Cys127 and Cys30–Cys115) (Fig. 3C) [112,113]. The second domain of the protein is made up with a triple stranded antiparallel β-sheet (41–60) and a long loop (61–78) [112,114]. There are also two disulphide bonds (Cys64–Cys80 and Cys76–Cys94) within this domain. CD spectroscopy of lysozyme in solution (pH 7) indicates that it consists of 36% α-helix, 15% β-sheet, 15% turn and 34% unordered structure [90 •,115]. The lack of a free cysteine means that the four Cys–Cys in lysozyme, particularly the two within the α-helix domain do not participate in disulphide-exchange reactions and hence the conformation of lysozyme has high pH and thermal stability [113,116]. There have been a few studies examining the conformation of lysozyme adsorbed at emulsion oil/water interfaces by FTIR [117], FT-Raman Spectroscopy [118], differential scanning microcalorimetry (micro DSC) [23 •] and SRCD [90 •]. Corredig and Dalgleish [23 •] using micro-DSC found a substantial reduction in the denaturation enthalpy of lysozyme upon adsorption to emulsion interfaces. Corredig & Dalgleish also found that once adsorbed, the structure of lysozyme at emulsion oil/water interfaces was more stable to heat-induced unfolding than lysozyme in solution. This observation was later confirmed by Day et al. who examined the heat induced changes to lysozyme's conformation at emulsion oil/water interfaces using SRCD [90•]. Using a combination of FTIR, DSC and fluorescence spectroscopy, Jorgensen et al. [119•] found small changes in the secondary and tertiary structure of lysozyme when it was adsorbed to solid lipid nanoparticles. The changes in FTIR spectra were mainly related to broadening of the absorption bands, in particular of the α-helix band at 1655 cm−1. Broadening of the amide I region around the α-helix peak was also observed by Adam et al. [117] who studied lysozyme adsorbed to different lipid layers. Jorgensen et al. also revealed a minor blue shift in fluorescence emission maximum for lysozyme adsorbed at the oil/water interface. The conformation of lysozyme adsorbed at n-hexadecane/water and tricaprylin/water interfaces of emulsions was recently examined by Day et al. [90 •]. Far-UV SRCD measurements (Fig. 4(1)C) indicated that the changes in lysozyme conformation upon adsorption to emulsion interfaces (4–7% reduction in α-helical and 3–7% increase in β-structure content (sheet and turns)) were small, relative to changes found for β-Lg, α-La and BSA. Adsorption of lysozyme to the emulsion interfaces also resulted in moderate changes to its near-UV SRCD spectra and a considerable reduction in the intensity of the peaks in the region of 280–300 nm (Fig. 4(2)C). The changes in the near-UV spectra of lysozyme adsorbed to oil/water emulsion interfaces suggest that the environment around the tryptophan and tyrosine residues was altered upon its adsorption. However, the presence of peaks in the tryptophan region of the near-UV indicated that some of the tertiary structure was retained when lysozyme was adsorbed to emulsion interfaces. These studies indicated that lysozyme underwent some (re) unfolding upon adsorption with a small loss of α-helix structure, but most of the secondary and tertiary structures were retained. This limited ability of lysozyme to undergo conformational change at emulsion interfaces is in contrast to many other proteins, and could be due to its high internal cohesion [112,113]. By comparing the particle size of the emulsions prepared in the absence or presence of reducing and/or denaturing agents (e.g. dithiothreitol, (DTT), urea

and DTT + urea), Poon et al. showed that the emulsifying activity of lysozyme was markedly enhanced when the disulphide bonds were reduced by DTT. In contrast only a small improvement in emulsifying activity of lysozyme occurred when the 76–94 disulphide bond was removed in a genetically modified lysozyme [120]. These findings indicate that the position of disulphide bonds is likely to play a key role in maintaining the 3D structure of lysozyme upon adsorption to emulsion oil/water interfaces. It appears that the two disulphide linkages that bring the α-helical structures at N- and C-terminus together play an important role in stabilising the lysozyme structure and prevent rapid exposure of the internal hydrophobic regions of the protein to the oil surface. Thus the emulsification properties of native lysozyme is not as active as when the disulphide bonds are broken [121•]. 4.6. Conformation of other proteins at oil/water interfaces Myoglobin is a single-chain globular protein (M w 17.6 kDa) containing no cysteine residues [122]. The structure of myoglobin is predominantly stabilised by the interactions between the functional heme group and by hydrophobic interactions and hydrogen bonds [123]. Myoglobin was the first proteins to have its crystal structure determined by X-ray crystallography more than half century ago [124]. It consists of 8 helices, many of which are amphipathic, and a hydrophobic core (Fig. 3E). The solution structure of myoglobin consists of 75% α-helical structure with the rest being largely turns (10%) and unordered structure (13%) [90•,125]. Day et al. used SRCD to study myoglobin and showed that it underwent considerable changes in its structure upon adsorption to emulsion oil/water interfaces [90 •]. There was a clear reduction in the intensities of the positive peak at 193 nm and the negative peaks at 208 nm and 222 nm in the far-UV SRCD spectra when myoglobin was absorbed at the oil/water emulsion interfaces (Fig. 4(1)D). The changes in the intensities of myoglobin far-UV spectra were associated with a loss of 19–28% helical structure and corresponding increases in both β-sheet and unordered structure. The extent of the reduction in α-helical structure was also correlated with the polarity of the oil phase. Near-UV SRCD showed that the peaks corresponding to tryptophan and tyrosine (294, 289, 282 and 273 nm) diminished when myoglobin was adsorbed at emulsion oil/water interfaces (Fig. 4(2)D), suggesting a substantial loss of myoglobin tertiary structure upon adsorption to the interface [90•]. The structural changes of soy proteins upon adsorption to emulsion interfaces were recently studied by Keerati-u-rai et al. [126•] and Miriani et al. [127] using fluorescence spectroscopy. The emission wavelength maximum of the emulsions showed a red shift of 7–12 nm with respect to those of native proteins (β-conglycinin or glycinin) indicating an increased exposure of the tryptophan residues to the aqueous phase after adsorption. These authors postulated that the tryptophan residues located in the extended region of the β-conglycinin subunits protrude into the aqueous phase, whereas the tryptophan-free core hydrophobic region of β-conglycinin is anchored at the interface [127]. Fluorescence spectroscopy coupled with SRCD measurements were also used by Wong et al. to investigate the conformation of deamidated gliadins adsorbed at emulsion oil/water interfaces [102]. The tryptophan fluorescence emission wavelength maximum underwent a blue shift when deamidated gliadins were adsorbed at emulsion interfaces, the magnitude correlating with oil phase polarity (− 6 nm at the tricaprylin/water interface, − 9 nm at the n-hexadecane/water interface). The blue shift of the emission wavelength maximum suggested that the tryptophan residues located at the C-terminus (where the helical structure resides) of deamidated gliadins were in a more hydrophobic environment when the protein was adsorbed at emulsion interfaces [102]. However SRCD measurements indicated that the secondary structure of deamidated gliadins was largely unchanged after adsorption. Gliadins have up to 4 pairs of disulphide bonds, all located at the C-terminal hydrophobic domain, this could be

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one of the factors that restrict protein conformational re-arrangement upon adsorption to emulsion interfaces. The conformations of both soy proteins and deamidated wheat proteins appear to be particularly resistant to heat-induced re-arrangements once they are adsorbed at oil/water interfaces [127,128]. 5. Trends in protein conformation at emulsion interfaces A number of key topics/trends have been mentioned over the past few decades of research into protein folding at emulsion interfaces including; an interfacial molten globule state, whether proteins have a preference to adopt specific conformations at interfaces, the thermal stability of the interfacial conformation and the impact of the oil phase on protein folding at emulsion interfaces. The recent increase in experimental information about protein folding at emulsion interfaces perhaps allows re-examination of these topics. One of the clearest trends is the propensity for proteins to undergo greater conformational re-arrangement at non-polar n-alkane/water interfaces than at the more polar triglyceride/water interfaces. This idea grew out of observations that proteins had different susceptibilities to digestion by Leaver and Dalgleish [25••]. Spectroscopic measurements (mainly using SRCD) have confirmed the greater conformational change at n-alkane/water interfaces with several proteins, β-Lg [89••], α-La [97•], myoglobin [90•], BSA [90•], lysozyme [90•] and to a small extent β-casein [102]. Furthermore, recent observations on the interfacial rheology and digestibility of β-Lg at the two different interfaces also highlighted the differences in protein conformation [129]. The main thermodynamic driving force for protein adsorption at an oil/water interface is the solvation of non-polar side chains in the hydrophobic oil phase. The extent of conformational re-arrangement is likely to be influenced by the gain in free energy associated with the transfer of hydrophobic residues to the oil phase, hence the influence of oil phase polarity. A second related trend is that proteins undergo less heat induced rearrangement at interfaces than in solution, and that this rearrangement is lower at non-polar n-alkane/water interfaces than at polar triglyceride/ water interfaces. This is seen in two recent studies which compared denaturation of β-Lg and α-La in solution, at n-hexadecane/water and at tricaprylin/water interfaces (see Sections 5.1 and 5.2) [89••,97•]. A recent study also showed a slight resistance to heat induced unfolding when lysozyme and BSA were adsorbed at n-hexadecane/water interfaces (see Sections 5.4 and 5.5) [90•]. The stability of the protein conformation at the interface might be influenced by how the interface changes the energy balance between the native and denatured states. In solution protein denaturation is governed by the balance between the energy penalty associated with ordering of water molecules around exposed hydrophobic residues and the gain in configurational entropy associated with an open protein structure. When a protein is adsorbed at an interface, the energy penalty associated with moving hydrophobic residues from the oil phase to the water is larger than that in solution, hence the stabilisation effect of the oil on protein unfolding. The strength of this stabilising effect would depend on; i) how good the oil is as a solvent for each amino acid residue (hence the influence of oil phase polarity) and ii) the number of residues solvated in the oil phase. Naturally this stabilising effect would only influence local protein structure, and hence parts of the protein (those facing or protruding into the water phase) could be unaffected, particularly for large proteins. One slightly controversial trend is whether proteins prefer a specific conformation upon adsorption to emulsion interfaces. Zhai et al. [97,128 •,128] recently proposed that proteins prefer an α-helix rich conformation at interfaces after SRCD measurements showed that a β-sheet rich (β-Lg) and an α-helix rich protein (α-La) both adopted conformations with very high α-helical contents upon adsorption, which was supported by earlier FTIR measurements in other studies [28 ••]. This hypothesis was further explored in a very recent study on lysozyme, BSA and myoglobin which all showed a reduction in their α-helical content upon adsorption but the major structural motif

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was still α-helix [90 •]. Studies on the emulsification properties of peptides derived from domains of BSA or myoglobin showed that the α-helical structure of these peptides is either retained or increased following adsorption to oil/water interfaces [130,131], indicating that the α-helix conformation plays a vital role for the peptide to be adsorbed at oil/water interfaces. Lefevre and Subirade [29 ••] proposed that intermolecular β-sheets play an important role in the structure of proteins at oil/water interfaces. FTIR measurements by Lefevre and Subirade indicated the formation of intermolecular β-sheets, which they suggest is an indication of surface aggregation of β-Lg [29 ••]. It was thought that the conformation of β-Lg at the interface did not completely satisfy the hydrogen bonding and hydrophobic interaction requirements of the protein and hence favoured 2D aggregation of β-Lg. The presence of intermolecular β-sheet was thought to indicate that aggregation of β-Lg at oil/water interfaces follows much the same pattern as in solution. The concepts of Zhai et al. and Lefevre and Subirade are not necessarily mutually exclusive as they could be describing what is happening to different parts of the protein. One question that some have asked is whether the proposed hypotheses for structural preference upon adsorption are influenced by a perceived bias to β-sheet structures in FTIR and to α-helix structures in CD. This is a valid question as it is true that the much higher signal intensity of α-helix structure in CD influenced early SELCON analysis based on poly-proline motifs. However, the analysis of CD spectra has advanced considerably with the advent of DichroWeb© which uses the SRCD spectra of known proteins to analyse secondary structure contents [56,132]. Likewise for FTIR measurements, correct assignment of individual absorptions to specific structural moieties can be difficult. In particular it can be difficult to correctly assign absorptions arising from “turns”. But perhaps the biggest challenge with FTIR is that techniques for quantitative analysis of secondary structures are in their infancy, are very complex and the results can be influenced by a number of experimental variables (baseline noise, atmospheric moisture and protein concentration at interfaces). In trying to understand if/why there is a preferred conformation of a protein upon adsorption to emulsion interfaces it is best to start with protein folding in solution. Two concepts are particularly relevant, the Levinthal paradox and the fact that the conformation a protein adopts is unrelated to its overall thermodynamic stability [133]. To explain the speed of protein folding Levinthal suggested that a protein's conformation is under kinetic rather than thermodynamic control, i.e. that the native conformation corresponds to an easily accessible local free energy minimum. When proteins adsorb to emulsion interfaces they need to adopt an amphiphilic conformation in a restricted semi-2D space (i.e. the low penetration of proteins into the oil phase). Restriction of the physical space in which to form structural motifs forces the protein to adopt the most compact amphiphilic conformation — which of the two amphiphilic moieties possible, α-helix and β-sheet, the most simple to form is an α-helix. Hence, it is not surprising that upon adsorption proteins have a tendency to adopt a conformation which has large amounts of α-helix. This trend is reflected in the conformation of transmembrane proteins, the vast majority of which have a very high α-helical content in the transmembrane domains [134]. A final concept that has been used to describe the structure of proteins at interfaces is the concept of an interfacial molten globule state. Dickinson and Matsumura [94••] proposed that proteins adopt a “molten globule” state at oil/water interfaces after the experimental evidence suggested that proteins lost their tertiary structure, but retained much of their native secondary structure which is the hallmark of a classical molten globule state. In the case of all the proteins presented in this review, adsorption to oil/water interfaces leads to loss of all near-UV absorption bands (except lysozyme, however the intensity was reduced considerably), which is indicative of disruption of the tertiary structure (Fig. 4(2)). Moreover, it is clear that these proteins do not exhibit near

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native secondary structure either (Fig. 4(1)). Hence, upon adsorption to emulsion interfaces proteins do not form a molten globule intermediate as such. However, they do adopt a conformation which is rich in secondary structure and almost devoid of tertiary structure. A question that arises is whether the structures observed at fluid interfaces to date are the “native” interfacial structure or simply a result of the conditions each emulsion was formed under. This question can be answered in part from studies that compared interfaces created from native α-La and α-La already in the molten globule state [94••,98]. Matsumura et al. [98] found that the interface created from α-La molten globule state had a lower interfacial rheology than the native α-La. This result suggests that in each case α-La had a different conformation at the interface, and hence is influenced by the initial state of the protein. This might suggest that like proteins in solution, the conformation of proteins adsorbed at interfaces is under kinetic control, i.e. that there may not be one single preferred conformation that is representative of the “native” interfacial conformation but rather many that reflect the history the protein has undergone. 6. Implications of protein conformational changes at emulsion interfaces A key question is whether the denaturation of proteins at oil/water interfaces has any implications for the performance/properties of emulsions, and whether there is benefit from deliberately trying to induce protein denaturation. These questions are best understood via a brief examination of three key properties of emulsions which are related to the re-arrangement of proteins at interfaces, including emulsification capability, emulsion stability and protein digestibility. 6.1. Emulsification capability The denaturation of a protein involves unfolding of its structure into a more open form, and hence one property that might logically be affected is the area the protein occupies at the interface (i.e. emulsification capability). Known more commonly as the emulsion activity index (EAI), emulsification capability is a measure of the area of interface stabilised per gramme of protein [135]. Pre-denaturation of proteins using chemical denaturants such as urea or DTT has been shown to improve the emulsification capability of proteins. In proteins that contain cysteine residues, reduction of disulphide bond(s) for example in lysozyme, soy protein isolate etc., can lead to an increased emulsification capability [121 •,136,137]. Disulphide bond reduction leads to an increase in protein conformational mobility, which in turn allows the protein to rearrange and orient hydrophobic residues to the oil phase. This effect is dependent on the location of disulphide bond(s) because the gain in emulsification capability is greater for lysozyme than for BSA, despite BSA having far more disulphide bonds [121 •]. However, in other proteins such as β-Lg and BSA disulphide bond reduction results in a reduced emulsification capability [121 •,138]. This is because β-Lg and BSA already have significant conformational freedom at interfaces, and reduction of their disulphide bonds disrupts the organisation of hydrophobic regions of the protein that interact with the oil phase. Similarly, disruption of the intramolecular non-covalent forces within the protein structure via exposure to urea (or guanidine HCl) generally leads to an increased emulsification capability (except for BSA) [121•,138]. For globular proteins the increase in emulsification capability is caused by the opening of the structure, whilst for more unstructured open proteins (i.e. caseins) the self-association of these proteins is disrupted [121•]. Interestingly, the emulsification capability of β-Lg and BSA was improved when they were pre-denatured in the presence of both urea and DTT [121•,138]. One explanation is that combined urea and DTT impart maximum conformational freedom to the proteins and relieves any conformational constraint that results from disulphide bond reduction. However, chemical denaturants such as urea and DTT

are far from food grade, and several studies have focussed on using thermal or high hydrostatic pressure treatments to change protein structures in an attempt to improve emulsification capability [139,140]. Studies on proteins in solution have found that denaturation induced by these treatments can increase the area occupied by a protein molecule at the interface; however it generally leads to protein aggregation, loss of protein solubility and/or reduced surface activity; all of which reduce emulsification capability [139,140]. Together these studies highlight that, if appropriate conditions are used, deliberate denaturation of proteins in solution can increase the area they occupy at an interface (emulsification capability), which could also have important implications for the stability of these emulsions due to different orientation of amino acid side chains.

6.2. Emulsion stability Many proteins used to stabilise food emulsions are globular, which generally form a compact interface with little extension into the bulk aqueous phase (i.e. minimal steric repulsion). The major colloidal interactions of globular protein stabilised emulsions are thus a balance between Van der Waals and hydrophobic attractive forces and electrostatic repulsive forces. Van der Waals forces result from the net emulsion entity, but hydrophobic and electrostatic forces arise from the protein surface layer and are hence affected by the orientation of amino acids at the interface (i.e. protein conformation). In the absence of salt, emulsion droplets stabilised by globular proteins (i.e. β-Lg or α-La) do not flocculate, even when heated above the (solution) thermal denaturation temperature. However, most food emulsions contain moderate amounts of monovalent and divalent salts. At moderate ionic strengths (150 mM NaCl), β-Lg-stabilised emulsions are prone to flocculation due to electrostatic screening. Upon heating, inter-droplet hydrophobic and sulfhydryl–disulphide interactions occur between the β-Lg interfacial layers accentuating droplet aggregation [5,7–9]. The effect of protein conformation on emulsion stability has been highlighted in a number of recent studies [8••,9•,89••,97•]. Kim et al. [8••] found that the flocculation of β-Lg emulsions depended on the order of the salt addition and heating sequence (Fig. 5A). When β-Lg stabilised n-hexadecane/water emulsions were heated in the absence of salt, the emulsions that were heated above 70 °C underwent less flocculation than those heated below 70 °C, when salt was added at room temperature after the heat treatment. Kim et al. equated the differences in emulsion stability to lower hydrophobic attraction as a result of conformational re-arrangement during heating [8••]. Kim et al. also showed that pre-heating β-Lg emulsions in the absence of salt to 95 °C made them more stable to subsequent heating in the presence of salt, suggesting that heating moved the proteins' conformation (principally the di-sulphide chemistry) into a more stable one. In other studies, Zhai et al. showed that oil/water emulsions created using n-hexadecane were more stable to heating in the presence of 120 mM NaCl than emulsions created with triglyceride oils [89 ••,97•] (Fig. 5B). While the authors commented that the lower refractive index of n-hexadecane oils would slightly reduce the VDW attractive force, they also observed differences in droplet surface charge (ξ-potential) and found greater conformational re-arrangement of these two proteins at n-hexadecane/water interfaces than tricaprylin/ water interfaces. The changes in droplet charge and protein conformation were thought to be the main factors causing the difference in emulsion stability. The emulsion stability work of Kim et al. and Zhai et al. highlighted that specific conformations of proteins at emulsion interfaces are more stable to heat + salt induced flocculation than other conformations. Whilst we are only just beginning to understand the details of these specific conformations, these studies also highlight that deliberate denaturation of proteins may lead to much greater resistance of emulsions to heating in the presence of salt, an important challenge in a number of emulsion based food products.

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6.3. Protein digestibility The rate and extent of protein digestion are influenced by the protein structure because they affect enzyme access to cleavage sites [141–143]. Changes in the protein conformation upon its adsorption to emulsion oil/water interfaces are therefore likely to change the hydrolysis behaviour of proteins. In fact, the early evidence for protein re-arrangement at interfaces came from studies that revealed that β-Cas and β-Lg had different digestion behaviours at emulsion interfaces (and polystyrene particle surfaces) than in solution [24,25••,26]. The recent renewed interest in understanding food digestion has seen a focus on understanding the digestion of proteins adsorbed at emulsion interfaces under physiological conditions [143,144]. One of the first such studies by Macierzanka et al. [145••] showed that β-Lg adsorbed at olive oil/water interfaces underwent rapid proteolysis (30%) during the initial stages of gastric digestion. The protein was subsequently completely broken down under the in vitro duodenal conditions (Fig. 6). The finding that the gastric digestion of β-Lg absorbed at emulsion interfaces was faster than the protein in solution was subsequently confirmed by Sarkar et al. [146,147] for β-Lg at soy oil/water emulsion interface and by Nik et al. [148,149] for whey protein isolate stabilised oil-in-water emulsions. It is well known that β-Lg in solution is resistant to proteolysis by pepsin [142,150–152]. The high structural integrity of β-Lg, with aromatic amino acids buried within its hydrophobic core, means that cleavage sites are not easily accessible by pepsin. This resistance to enzymatic proteolysis has been proposed to contribute to the allergenicity of β-Lg in humans [151]. The changes in the conformation of β-Lg as a result of un(re)-folding at the emulsion interface expose the hydrophobic regions containing most of the aromatic amino acid residues which are the most active peptic cleavage sites [153], and therefore significantly decreases the resistance of β-Lg to proteolysis by pepsin. Leaver and Douglas [24,25••] found that the digestion of β-Cas by trypsin at emulsion interfaces was faster than in solution. Similarly Macierzanka et al. found that digestion of β-Cas at the emulsion interface under in vitro gastric conditions was twice as fast as digestion in solution and also led to different peptide products [145••]. The digestion behaviour of β-Cas also appears to be different at different oil/water interfaces. When β-Cas was adsorbed at tetradecane/water interfaces certain trypsin-sensitive bonds were not readily hydrolysed, however they

A

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were hydrolysed when β-Cas was at soya oil/interface [25••]. Studies by Sakuno also showed that the susceptibility of β-Lg to proteolysis was different at TAG and DAG oil/water interfaces [88]. A similar observation was made recently by Maldonado-Valderrama et al. [129] who showed that pepsin digestion of β-Lg adsorbed at olive oil/water interfaces was higher than at tetradecane/water interfaces. The authors proposed that the lower polarity of tetradecane allowed an increased orientation of the hydrophobic residues towards this phase, enhancing the interaction with the oil and reducing access of pepsin [129]. The difference in the digestion behaviour of β-Cas and β-Lg suggests that the polarity of the oil phase influences the conformation of these proteins at oil/water interfaces. These studies have consistently shown that adsorption of proteins to oil/water interfaces alters their digestion as a result of adsorption induced conformational re-arrangement which in turn alters the susceptibility of a protein to proteolysis. In addition, the peptides produced during the hydrolysis of proteins adsorbed at emulsion interfaces are different from those in solution. Recent studies have highlighted that this can have important implications not only for protein digestion but potentially also for fat digestion [149]. Furthermore, adsorption-induced changes to the digestion of protein allergens (e.g. wheat, nut, dairy, egg, mustard and sunflower proteins) could have important implications in the testing and formulation of foods for allergy sufferers [154]. Understanding the molecular conformation of proteins at interfaces is only one aspect of the hierarchical structure of proteins at interfaces, and spectroscopic measures of protein folding at interfaces are well complimented by monolayer techniques such as surface tension measurements [155,156], neutron and X-ray reflectivity [19], and other techniques [30 •]. By far the most understanding has come from interfacial/surface tension measurements where fundamental models of the adsorption and unfolding of proteins at interfaces have been described [155,156]. Of particular note is the work of Miller et al. who have conducted a number of detailed studies [155–157]. 7. Concluding remarks In the past several decades our understanding of protein conformation at interfaces has evolved from indirect evidence of conformational change (i.e. calorimetry, digestion kinetics etc.) to direct measurement of the specifics of conformational change. The conformations of several

B

40 i) heating + 0mM NaCl

Mean particle size (µm)

Particle Diameter (µm)

35

Temperature (oC)

ii) TAG: heating + 120mM NaCl iii) TAG: 120mM NaCl + heating

30

iv) Hexa: heating + 120mM NaCl

25

v) Hexa: 120mM NaCl + heating

20 15 10 5 0 20

30

40

50

60

70

80

90

Temperature (oC) Fig. 5. A) Impact of heating (20 min at each temperature) and order of salt addition on the aggregation of 5 wt.% n-hexadecane oil in water emulsions stabilised by 0.5 wt.% β-Lg (pH 7). B) Impact of oil phase polarity on the aggregation of β-Lg (0.3 wt.%) stabilised tricaprylin (10 wt.%) oil in water emulsions [i, ii & iii] or n-hexadecane (10 wt.%) oil in water emulsions [iv and v]. The emulsions were; i) heated in the absence of salt, ii) and iv) heated in the absence of salt, and then exposed to 120 mM NaCl, or iii) and v) heated in the presence of 120 mM NaCl. Note: particle size of the emulsions was measured 24 h after heat treatment, which lasted for 20 min. Reproduced with permission from Kim et al. [8••].

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Fig. 6. SDS-PAGE analysis of gastric and duodenal digestions of: (A) β-Lg in solution and B) in emulsion. Reproduced from Macierzanka et al. [145••].

proteins adsorbed at emulsion oil/water interfaces have now been characterised using several techniques. Synchrotron radiation circular dichroism has facilitated quantitative analysis of protein structure in emulsions, but needs to be paralleled with considerable advancement of FTIR so that quantitative structural analysis of proteins adsorbed to fluid interfaces becomes more accurate. Whilst there is still a growing understanding of the implications of these measurements, several trends in protein conformation have been identified. It is now known that proteins can undergo considerable conformational re-arrangement upon adsorption to emulsion interfaces. Over time more appropriate definitions for these structures (e.g. molten globule and interfacial conformation) and processes (e.g. denaturation, vs. unfolding, vs. conformational re-arrangement) will be found. It also appears that upon adsorption to emulsion oil/water interfaces, most proteins lose their tertiary structure but retain large amounts of (non)native secondary structure. Two secondary structure motifs appear prominent in the structure of proteins adsorbed to protein interfaces, intermolecular β-sheets and α-helices. Further measurements on more diverse proteins, and especially under different emulsion preparation conditions, are needed to probe the generality of these initial observations. The conformation of proteins at interfaces considerably affects the properties of the emulsion or the interface. However, a direct relationship between the conformations measured at the molecular scale and the emulsion properties is yet to be established, although some might say a subjective one exists. Overall the recent advances in the measurement of protein conformation at emulsion oil/water interfaces have generated a number of questions/hypotheses that will need to be tested with further detailed study. References and recommended reading •,•• [1] Dickinson E. Caseins in emulsions: interfacial properties and interactions. Int Dairy J 1999;9:305–12. [2] Foegeding EA, Davis JP, Doucet D, McGuffey MK. Advances in modifying and understanding whey protein functionality. Trends Food Sci Technol 2002;13:151–9. [3] Hunt JA, Dalgleish DG. Adsorption behavior of whey protein isolate and caseinate in soy oil-in-water emulsions. Food Hydrocolloids 1994;8:175–87. • ••

Of special interest. Of outstanding interest.

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