Controlling the Ratio between Native-Like, Non-Native-Like, and

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Controlling the ratio between native-like, non-nativelike and aggregated #-lactoglobulin after heat treatment Roy J.B.M. Delahaije, Harry Gruppen, Evelien L. van Eijkvan Boxtel, Leonardo Cornacchia, and Peter A. Wierenga J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00816 • Publication Date (Web): 17 May 2016 Downloaded from http://pubs.acs.org on May 18, 2016

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Journal of Agricultural and Food Chemistry

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Controlling the ratio between native-like, non-

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native-like and aggregated β-lactoglobulin after

3

heat treatment

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Roy J. B. M. Delahaije1, Harry Gruppen1, Evelien L. van Eijk - van Boxtel2, Leonardo

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Cornacchia2 and Peter A. Wierenga1,*

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1

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WG, Wageningen, The Netherlands.

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2

Laboratory of Food Chemistry, Wageningen University, Bornse Weilanden 9, 6708

Nutricia Research, Uppsalalaan 12, 3584 CT, Utrecht, The Netherlands

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*Corresponding author:

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Phone: +31 317 483786

14

e-mail: [email protected]

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16

1

ACS Paragon Plus Environment


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Abstract

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The amount of heat-denatured whey protein is typically determined by pH 4.6-

19

precipitation. Using this method, a significant amount of non-denatured protein was

20

reported even after long heating times. Apparently, a fraction of the unfolded protein

21

refolds into the ‘native’ state rather than form aggregates. This fact is known and has

22

been

23

refolding/aggregation is however not fully understood. Therefore, this study

24

investigates the unfolding, refolding and aggregation process of β-lactoglobulin using

25

circular dichroism and size-exclusion chromatography to characterize different

26

folding variants and to quantify their content. The proteins remaining in solution at

27

pH 4.6 were confirmed to be native-like. The non-aggregated fraction contains

28

proteins with a native-like and two types of non-native-like conformations. The non-

29

aggregated fraction increased with decreasing temperature (60-90 °C) and

30

concentration (1-50 g/L) and increasing electrostatic repulsion (pH 7-8; 0-50 mM).

31

The native-like fraction in the non-aggregated fraction was independent of pH, ionic

32

strength and concentration, but increased with decreasing temperature.

explained

using

kinetic

models.

How

the

conditions

affect

the

33 34

Keywords

35

Unfolding, refolding, aggregation, temperature, concentration, pH, ionic strength,

36

structure, denaturation

2


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Introduction

38

Heat-induced denaturation of proteins has been extensively studied1-8. For whey

39

proteins, such as β-lactoglobulin, denaturation was described to lead to decreased

40

solubility at pH 4.62, 9-11 (i.e. close to the iso-electric point of the protein). The extent

41

of precipitation of whey proteins at pH 4.6 has in time evolved into a standard

42

protocol to quantify the proportion of ‘native’ and denatured proteins in heated

43

solutions12,

44

β-lactoglobulin (β-lg) decreased slowly during heating, even at relatively high

45

temperatures. For instance, 30 % native-like protein remained after 35 minutes

46

heating at 85 °C (5.8 g/L, H2O pH 6.6)14 and 40-50 % remained after 120 minutes at

47

80 °C (50 g/L, H2O pH 7.0)15. This cannot be attributed to limited unfolding, as

48

proteins unfold rapidly (i.e. within milliseconds to seconds) above their denaturation

49

temperature16-18. Recently, it has even been reported that β-lg (50 g/L, H2O pH 6.8

50

and 8.0) can be heated at 80 °C to complete unfolding and cooled down without

51

formation of aggregates19. This suggests that the unfolded proteins have (under these

52

conditions) a greater tendency to refold into their ‘native’ conformation than to form

53

aggregates. This raises the question whether the ‘native’ proteins determined after

54

heating are indeed in their native(-like) conformation, and whether they are correctly

55

quantified.

56

Upon heating, proteins are typically described to reversibly unfold, i.e. native(-like)

57

state (NL) ↔ unfolded state (U). In addition, the unfolded proteins can irreversibly

58

aggregate, i.e. U → aggregated state (A)15, 20. Moreover, it has been well established

59

that β-lg (partially) refolds into a non-native-like state (NNL). This was however

60

concluded based on indirect measurements such as a shift in the elution pattern in gel

61

electrophoresis and size-exclusion chromatography (SEC)21, 22. Another study showed

13

. Using this method, it was found that the amount of native-like

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an increase in exposed hydrophobicity, while no difference in tertiary structure was

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observed by intrinsic fluorescence23. Direct measurements of the secondary and

64

tertiary protein structure, using for example circular dichroism (CD), confirming the

65

structure of refolded β-lg are still lacking. The fact that β-lg may refold into a non-

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native-like conformation suggests that unfolded β-lg is not only in equilibrium with

67

its native(-like) state, but also with the non-native like state, i.e. NL ↔ U ↔ NNL. It

68

has to be noted that the unfolded state is often implicitly assumed to be in equilibrium

69

with the native(-like) state. This is most probably caused by the fact that it is

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experimentally difficult to distinguish these forms under these conditions.

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Upon unfolding, proteins expose their hydrophobic residues. This is typically

72

described to induce aggregation7, 24, 25, although the unfolded proteins may also refold

73

into their NL or NNL state. It is therefore of interest to identify how the balance

74

between the different states (i.e. NL, NNL and A) depends on the system conditions

75

(e.g. pH, concentration, T, time). Therefore, this study investigates the unfolding,

76

refolding and aggregation process in detail using CD and SEC to characterize and

77

quantify the content of native-like, non-native-like and aggregated protein in heat-

78

treated β-lactoglobulin solutions.

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Materials and methods

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Materials

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β-Lactoglobulin (β-lg; L-0130; Lot no. SLBC2933V) was purchased from Sigma-

83

Aldrich (St. Louis, MO, USA). The β-lg contained 92 ± 0.5 % protein based on

84

nitrogen content determined by Dumas26 (N x 6.33; based on the primary sequence,

85

http://www.uniprot.org; entry: P02754) using a Flash EA 1112 NC Analyzer (Thermo

86

Fischer Scientific Inc., Waltham, MA, USA). The protein contained ≥ 95 % β-

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lactoglobulin consisting of genetic variant A (67 %) and genetic variant B (33 %) as

88

confirmed by RP-HPLC (data not shown). All other chemicals were of analytical

89

grade and purchased from Sigma-Aldrich or Merck.

90 91

Fractionation of heated β-lactoglobulin

92

β-Lactoglobulin (20 g/L in 10 mM sodium phosphate buffer pH 7.0, as used

93

previously27) was heated at 80 °C for 900 s and cooled to 20 °C. Subsequently, the

94

sample (further referred to as H7) was fractionated using preparative scale size-

95

exclusion chromatography (SEC) and/or pH 4.6-precipitation.

96

Preparative scale SEC

97

The aggregated and non-aggregated proteins were separated on an Äkta Explorer

98

equipped with a Superdex 75 PG column (500 mL; GE Healthcare, Uppsala,

99

Sweden). The heat-treated β-lactoglobulin in 10 mM sodium phosphate buffer pH 7.0

100

was injected (10 mL) and eluted with 10 mM sodium phosphate buffer pH 7.0 at a

101

flow rate of 5 mL min-1. The elution was monitored at 280 nm. This resulted in an

102

aggregated and non-aggregated fractions. The non-aggregated fraction (further

103

abbreviated as HNA7) was collected, dialyzed against demineralized water to remove

104

salts and lyophilized.

105

pH 4.6-precipitation

106

To precipitate non-native-like proteins and aggregates12,

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β-lactoglobulin (H7) and the heated non-aggregated fraction (HNA7) was adjusted to

108

4.6 with 0.1 M HCl (further abbreviated as H4.6 and HNA4.6, respectively).

13

, the pH of the heated

109

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Differential Scanning Calorimetry (DSC)

111

The denaturation temperature of β-lactoglobulin was determined using a VP-DSC

112

MicroCalorimeter (MicroCal Inc., Northampton, MA, USA). β-Lactoglobulin was

113

dissolved in 10 mM sodium phosphate buffer pH 7.0 at a concentration of 20 g/L.

114

Subsequently, thermograms were recorded from 20 to 105 °C at a heating rate of 1 °C

115

min-1.

116 117

Secondary and tertiary structure

118

Far- and near-UV Circular Dichroism (CD)

119

Changes in the secondary and tertiary structures of β-lactoglobulin and the fractions

120

H7, HNA7, HNA4.6 and H4.6 were determined by far- and near-UV circular

121

dichroism, respectively. β-Lactoglobulin was dissolved in 10 mM sodium phosphate

122

buffer pH 7.0, 50 mM sodium phosphate buffer pH 7.0 or milliQ water adjusted to pH

123

8.0 with 0.1 M NaOH in a concentration of 0.2 or 2 g/L for far- and near-UV CD,

124

respectively. The fractions were diluted in 10 mM sodium phosphate buffer pH 7.0 to

125

a concentration of 0.2 or 2 g/L for far- and near-UV CD, respectively. Measurements

126

were performed using a J-715 spectropolarimeter (Jasco Corp., Tokyo, Japan) with a

127

sensitivity of 100 mdeg and a bandwidth of 2 nm. Prior to the measurements, the

128

spectropolarimeter was thermostated to the specific temperature. Far- and near-UV

129

CD measurements were performed in quartz cuvettes with an optical path length of 1

130

and 10 mm, respectively.

131

Spectrum measurement (constant temperature)

132

The CD spectra of β-lg and the fractions H7, HNA7, HNA4.6 and H4.6 were recorded

133

from 190-250 nm (i.e. far-UV CD) and 250-350 nm (i.e. near-UV CD) and averaged

134

from 10 runs. Measurements were performed at 20 °C and at 90 °C with a data point

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every 0.2 nm. The samples were thermostated at the specific temperature for 300 s

136

prior to the analysis. After subtracting the spectra of the protein-free sample from the

137

far-UV spectra of the sample, the relative content of secondary structure elements was

138

estimated using a non-linear least squares fitting procedure as described by de Jongh

139

et al.28.

140

Temperature profile (variable temperature)

141

The temperature trace of β-lg was recorded from 20 to 90 °C at a wavelength of 204

142

nm (far-UV CD) and 294 nm (near-UV CD), with a heating rate of 0.5 °C min-1 and

143

data collection every 0.2 °C.

144

Effect of heating time (constant temperature, variable time)

145

The ellipticity of β-lg at 80 °C was recorded for 300, 600, 1200 and 1800 s at a

146

wavelength of 204 and 294 nm for far- and near-UV CD, respectively. Data points

147

were collected every second. After the heat treatment, the solutions were cooled to 20

148

°C, and the spectrum was measured as described above.

149

Effect of heating temperature (variable temperature, constant time)

150

The ellipticity of β-lactoglobulin at 60, 70, 80 and 90 °C (i.e. temperatures around Td

151

29-31

152

UV CD, respectively. Data points were collected every second. After the heat

153

treatment, the solutions were cooled to 20 °C, and the spectrum was measured as

154

described above.

155

Calculation of the unfolded protein fraction

156

The unfolded protein fraction (native-like + non-native-like) was calculated from the

157

CD results by dividing the change in ellipticity at 204 or 294 nm by the maximum

158

change in ellipticity at these wavelengths (i.e. completely unfolded β-lg; 90 °C, 30

159

min).

) was recorded for 1800 s at a wavelength of 204 and 294 nm for far- and near-

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Intrinsic fluorescence

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In addition to near-UV CD, changes in the tertiary structure were determined from

162

changes in the exposure of tryptophan and tyrosine residues using intrinsic

163

fluorescence32. Native and heat-treated β-lg were dissolved at a concentration of 1 g/L

164

in 10 mM sodium phosphate buffer pH 7.0. These solutions were then excited at 282

165

nm (specific for phenylalanine, tyrosine and tryptophan), the emission spectrum was

166

collected

167

spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). The emission and

168

excitation slits were set to 5 nm, the scan speed was 600 nm min-1, the detector

169

voltage was 600 V and the measurements were performed at 25 °C.

from

300-420

nm

using

a

Varian

Cary

Eclipse

fluorescence

170 171

Size-exclusion chromatography (SEC)

172

Analytical scale

173

The amount of native-like, non-native like and aggregated protein was quantified with

174

SEC, as described previously27. Samples (20 µL) were injected on an Äkta Micro

175

equipped with a Superdex 75 10/300 GL column (GE Healthcare) and eluted with 10

176

mM sodium phosphate buffer pH 7.0 at a flow rate of 0.6 mL min-1. The elution was

177

monitored using UV absorbance at 214 and 280 nm. The column was calibrated with

178

globular proteins with a mass range of 13.7 - 67 kDa (α-lactalbumin, β-lactoglobulin,

179

ovalbumin and BSA). The fractions of native-like, non-native-like and aggregated

180

protein were calculated from the relative UV peak area. For the calculation of the

181

native-like, non-native-like and aggregated proteins the areas at pH 4.6 > 10 mL, at

182

pH 7.0 > 10 mL - pH 4.6 > 10 mL and at pH 7 < 10 mL were used, respectively.

183

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Results and discussion

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Structural characterization and stability

186

The far-UV CD spectrum of native β-lg showed extremes at 195, 212 and 218 nm

187

(figure 1A). From this, the secondary structure of β-lg (10 mM sodium phosphate

188

buffer pH 7.0, 20 °C) was estimated to consist of 56 % β-sheet, 26 % random coil, 9

189

% α-helix and 9 % β-turn structure. This is in agreement with previous literature

190

findings29, 33. The spectrum corresponding to the tertiary structure was dominated by

191

extremes at 287 and 294 nm (figure 1B), showing that the tryptophan (Trp) residues

192

are located in an apolar environment (i.e. located in the interior of the protein)34, 35.

193

Moreover, minor extremes were observed in the range ascribed to phenylalanine (Phe)

194

and tyrosine (Tyr)36, 37. This shows that these residues were also located in the interior

195

of the protein, as was previously observed in other studies16, 33, 38.

196

The denaturation temperature of β-lactoglobulin was found to be 70 °C using

197

differential scanning calorimetry (DSC; data not shown). This is in line with reported

198

denaturation

199

temperatures of the secondary and tertiary structure of β-lg, as determined by CD,

200

were 63.4 and 63.8 °C, respectively (figure 2). Moreover, it was observed that β-lg

201

was completely unfolded, i.e. loss of secondary as well as tertiary structure, at

202

temperatures superior to 85-90 °C. The lower denaturation temperatures determined

203

by CD compared to DSC has previously been reported33.

204

Unfolding of β-lg resulted in the disappearance of the extremes in the near-UV CD

205

spectrum corresponding to the tryptophan residues (figure 1B). This is attributed to

206

the exposure of the hydrophobic residues (Trp, Tyr and Phe) to a polar environment

207

(i.e. the solvent at the exterior of the protein), indicating a loss of tertiary structure. In

208

addition, unfolding was accompanied by a shift of the extremes in the far-UV CD

temperatures

of

70-75

°C29-31.

Surprisingly,

the

denaturation

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209

spectrum from 212 and 218 to 208 nm, as well as by a shift of the zero-crossing from

210

204 to 198 nm (figure 1A). This corresponds to a decrease in the content of β-sheet

211

structure (i.e. from 56 to 18 %) and a concomitant increase in α-helical and random

212

coil structure (i.e. from 9 to 28 % for α-helix and from 26 to 41 % for random coil

213

structure). A decrease in β-sheet content and an increase in α-helical content with

214

increasing temperature has previously been described for β-lg39, 40. This demonstrates

215

that, in some cases, the term unfolding is somewhat misleading since unfolded β-lg

216

still retains some secondary structure elements.

217 218

Kinetics and extent of unfolding

219

To obtain an overview of the kinetics and extent of β-lg unfolding, the effect of time

220

and temperature was studied. The effect of heating time on unfolding was determined

221

at 80 °C. The unfolded protein fraction reached a plateau, i.e. approximately 92 and

222

75 % for the secondary and tertiary structures respectively, as soon as the target

223

temperature was reached (~ 300 s) (figures 3A and C). This shows that the unfolding

224

and refolding process reached an equilibrium (i.e. N → U equals N ← U) on short

225

timescales. As a consequence, time did not influence the extent of protein unfolding

226

after the desired temperature was reached. Cooling to 20 °C resulted in a decrease in

227

the unfolded protein fraction from 92 to 58 % for the secondary and from 75 to 59 %

228

for the tertiary structure, respectively (figure 3A and C). This indicates that the

229

proteins, which were unfolded at 80 °C, partially refolded upon cooling.

230

The extent of protein unfolding at equilibrium increased with temperature (after 1800

231

s). An increase in temperature from 60 to 90 °C resulted in an increase of the

232

proportion unfolded protein from 43 to 92 % for the secondary and from 41 to 96 %

233

for the tertiary structure (figures 3B and D). A similar increase in unfolded protein

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fraction with increasing temperature was also observed based on intrinsic

235

fluorescence (data not shown). Above 80 °C, the unfolded protein fraction became

236

less dependent on temperature. This is caused by the fact that most proteins are in the

237

unfolded state at these temperatures (figure 2) (i.e. when the specified temperature

238

was reached). After cooling down, the unfolded proteins partially refolded, similar as

239

described for the time-dependent unfolding (figures 3D). However, CD does not

240

provide detailed insights in the structure of the refolded proteins. Based on the results,

241

it is, for example, still not clear whether the structure of the refolded proteins is

242

indeed native-like, and whether all proteins partially refold, or part of the protein

243

molecules completely refold into their native-like state. To answer these questions,

244

heated β-lactoglobulin was fractionated into separate fractions.

245

246

Unravelling the refolding and aggregation of β-lg

247

When native β-lg was heated for 900 s at 80 °C, the non-aggregated protein fraction

248

decreased with a concomitant increase in the aggregated protein fraction (figure 4A).

249

The secondary structure of heated β-lg (H7) was different from that of native β-lg

250

(figures 4B and C). After pH 4.6-precipitation, only a fraction of the non-aggregated

251

proteins remained in solution (figure 4A). These non-aggregated proteins (H4.6)

252

showed a secondary structure identical to that of native β-lg (figures 4B and C).

253

Hence, it was concluded that the non-aggregated proteins that remained soluble at pH

254

4.6 refolded into the native-like (NL) state. In addition, the non-aggregated proteins

255

that precipitated at pH 4.6 refolded into a non-native-like (NNL) state. This confirms

256

that, as assumed in literature, only the native-like proteins remain in solution at pH

257

4.6, whereas all NNL and aggregated proteins precipitate. Consequently, size-

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exclusion chromatography analysis prior to and after precipitation at pH 4.6 allows

259

quantification of the amount of and ratios between NL, NNL and aggregated β-lg.

260

261

Controlling protein unfolding and aggregation

262

Effect of time and temperature

263

The aggregated protein increased to approximately 50 % after 600 s at 80 °C (figures

264

5A and B). For longer heating times (> 600 s), the aggregated protein fraction levelled

265

off at 55 %. The time required to reach the maximum amount of aggregated protein is

266

in agreement with the time needed to reach the maximum amount of unfolded protein

267

after cooling (figures 3A and C). The NL protein fraction decreased to approximately

268

34 % after 1200 s at 80 °C with a concomitant increase in NNL protein fraction to

269

around 12 % (figure 5B). These results confirm that even after a long heating time at

270

80 °C a considerable amount of β-lg is in its NL conformation. This is in line with

271

previous studies14, 15. The ratio between NL and NNL β-lg shifted towards the NNL β-

272

lg with increasing heating times.

273

The NL protein fraction decreased with increasing temperature from around 76 % at

274

60 °C to around 22 % at 90 °C (figures 6A and B). At the same time, the NNL protein

275

fraction gradually increased from circa 1 % at 60 °C to 19 % at 90 °C (figure 6B).

276

This shows that the amount of non-aggregated (NL + NNL) protein decreased with

277

increasing temperature. Moreover, the ratio between NL and NNL β-lg shifted

278

towards NNL β-lg.

279

A comparison of the unfolded protein fraction (figures 3A-D) and the aggregated

280

protein fraction (figures 5B and 6B) shows that in these cases approximately 60 % of

281

the unfolded proteins aggregated (R2 = 0.93; figure 7). If all unfolded proteins would

282

irreversibly aggregate (N → U → A), the aggregated protein fraction (after cooling

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down) would have been equal to the unfolded protein fraction (at elevated

284

temperatures). It, therefore, clearly indicates that unfolded β-lg partially refolds, and

285

partially aggregates upon cooling. This is in agreement with the view of reversible

286

unfolding15, 20. Based on the clear correlation between the unfolded and aggregated

287

protein fraction (figure 7), it was concluded that the extent of aggregation can be

288

directed by controlling the level of unfolding.

289

Effect of protein concentration

290

The non-aggregated protein fraction (NL + NNL) decreased with increasing

291

concentration (figure 8A). This clearly shows that aggregation also depends on the

292

protein concentration. This is explained by the fact that the number of intermolecular

293

collisions increases with increasing protein concentration41, 42.

294

Effect of electrostatics

295

An increase in electrostatic repulsion between the protein molecules (e.g. from pH 7.0

296

to pH 8.0) was found to result in an increase of the native-like protein fraction after

297

heating (from around 34 to 52 % after 1800 s at 80 °C) (figures 5B and C and 8B).

298

Concomitantly, it reduced the aggregated protein fraction (e.g. from around 71 to 55

299

% from 50 mM to 10 mM sodium phosphate pH 7.0 after 1800 s at 80 °C) (figures 5B

300

and D and 8B). A similar effect of electrostatics was observed at different

301

temperatures (figures 6B-D). These observations show that the equilibrium between

302

refolding and aggregation shifts towards aggregation when the electrostatic repulsion

303

is reduced. This is in line with prior observations for α-amylase43 and is postulated to

304

be caused by a lower barrier for aggregation, as expected based on the DLVO theory

305

44

306

was not affected by changes in the electrostatic repulsion (i.e. changes in ionic

. Surprisingly, the ratio between the native-like and non-native-like conformation

13



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307

strength and pH) (figure 8B). This suggests that those changes only influenced the

308

barrier for aggregation.

309

From the above, it is concluded that the ratio between non-aggregated (NL + NNL)

310

and aggregated protein shifts towards non-aggregated protein with decreasing

311

temperature and concentration and increasing electrostatic repulsion. In addition, a

312

decrease in temperature results in a shift of the ratio between the NL and NNL state

313

towards the NL state. Concentration and electrostatic repulsion did not affect the ratio

314

between the NL and NNL state. These observations clearly indicate the importance of

315

for example protein concentration and salt in relation to refolding and aggregation. In

316

more complex systems such as whey concentrations and milk numerous other factors,

317

e.g. caseins45, may affect the refolding and aggregation behavior of βlactoglobulin.

318 319

Unravelling the folding variants of β-lg

320

The heat-treated β-lactoglobulin (900 s at 80 °C) was fractionated by size-exclusion

321

chromatography (SEC) (HNA7) or by SEC followed by pH 4.6-precipitation

322

(HNA4.6). The structure of the heated-treated, non-aggregated proteins obtained from

323

SEC (HNA7) was more similar to native β-lg compared to the heated β-lg (figure 9).

324

Nevertheless, the structure of HNA7 deviated from that of native β-lg. This difference

325

was caused by the fact that only part of the proteins completely refolded into the NL

326

state (i.e. HNA7 consists of NL + NNL). The secondary structure of the proteins that

327

remained soluble at pH 4.6 after the removal of aggregated proteins (HNA4.6) was in

328

close resemblance, but still different from that of native β-lg (figure 9). This is in

329

contradiction with prior observations that all aggregated and NNL proteins precipitate

330

at pH 4.6 (i.e. the secondary structure of H4.6 resembled that of N7, while the

331

secondary structure of HNA 4.6 was different; figure 4). Apparently, a fraction of the

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332

NNL β-lg (i.e. present as dimers based on SEC) does precipitate at pH 4.6 in the

333

presence of aggregates, but does not in the absence of aggregates. Consequently, two

334

types of NNL β-lg are distinguished; one which only precipitates in the presence of

335

aggregates (NNL-1), and one which precipitates in the presence and absence of

336

aggregates (NNL-2).

337

Based on the above, it is concluded that β-lg unfolds, partially refolds into the NL and

338

two types of NNL states and partially aggregates upon heating.

339

In conclusion, using circular dichroism it was confirmed that the soluble β-lg after

340

precipitation -at pH 4.6- of a heated protein solution indeed has an identical structure

341

as native β-lg. Before pH precipitation, the non-aggregated β-lg was present in a

342

native-like and two different non-native-like conformations. A method has been

343

introduced to quantify the fractions of NL, NNL, and aggregated β-lg in a heat-treated

344

solution. The ratio between non-aggregated (NL + NNL) and aggregated protein was

345

higher at low temperature, low protein concentration and high electrostatic repulsion

346

(i.e. decreasing ionic strength or shift of pH away from the iso-electric point). In

347

addition, the ratio between the NL and NNL state was independent of pH, ionic

348

strength and protein concentration, but shifted towards the NL state with decreasing

349

temperature.

350

351

Acknowledgements

352

L. Cornacchia acknowledges the support of NanoNextNL, a micro and

353

nanotechnology consortium of the Government of the Netherlands and 130 partners.

354

355

Note

356

The authors declare no competing financial interest.

15



Page 16 of 26

357

16


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30. Haug, I. J.; Skar, H. M.; Vegarud, G. E.; Langsrud, T.; Draget, K. I., Electrostatic effects on βlactoglobulin transitions during heat denaturation as studied by differential scanning calorimetry. Food Hydrocolloids 2009, 23, 2287-2293. 31. Relkin, P., Reversibility of heat-induced conformational changes and surface exposed hydrophobic clusters of β-lactoglobulin: their role in heat-induced sol-gel state transition. Int. J. Biol. Macromol. 1998, 22, 59-66. 32. Delahaije, R. J. B. M.; Wierenga, P. A.; Giuseppin, M. L. F.; Gruppen, H., Improved emulsion stability by succinylation of patatin is caused by partial unfolding rather than charge effects. J. Colloid Interface Sci. 2014, 430, 69-77. 33. Broersen, K.; Voragen, A. G. J.; Hamer, R. J.; de Jongh, H. H. J., Glycoforms of β-lactoglobulin with improved thermostability and preserved structural packing. Biotechnol. Bioeng. 2004, 86, 78-87. 34. Griffin, W. G.; Griffin, M. C. A.; Martin, S. R.; Price, J., Molecular basis of thermal aggregation of bovine β-lactoglobulin A. Faraday Trans. 1993, 89, 3395-3405. 35. Croguennec, T.; Mollé, D.; Mehra, R.; Bouhallab, S., Spectroscopic characterization of heat-induced nonnative β-lactoglobulin monomers. Protein Sci. 2004, 13, 1340-1346. 36. Kelly, S. M.; Jess, T. J.; Price, N. C., How to study proteins by circular dichroism. Biochim. Biophys. Acta, Proteins Proteomics 2005, 1751, 119-139. 37. Strickland, E. H.; Beychok, S., Aromatic contributions to circular dichroism spectra of protein. Crit. Rev. Biochem. Mol. Biol. 1974, 2, 113-175. 38. Manderson, G. A.; Creamer, L. K.; Hardman, M. J., Effect of heat treatment on the circular dichroism spectra of bovine β-lactoglobulin A, B, and C. J. Agric. Food Chem. 1999, 47, 4557-4567. 39. Prabakaran, S.; Damodaran, S., Thermal unfolding of β-lactoglobulin: characterization of initial unfolding events responsible for heat-induced aggregation. J. Agric. Food Chem. 1997, 45, 4303-4308. 40. Chamani, J.; Moosavi-Movahedi, A. A.; Rajabi, O.; Gharanfoli, M.; Momen-Heravi, M.; Hakimelahi, G. H.; Neamati-Baghsiah, A.; Varasteh, A. R., Cooperative α-helix formation of β-lactoglobulin induced by sodium n-alkyl sulfates. J. Colloid Interface Sci. 2006, 293, 52-60. 41. Majhi, P. R.; Ganta, R. R.; Vanam, R. P.; Seyrek, E.; Giger, K.; Dubin, P. L., Electrostatically driven protein aggregation: β-lactoglobulin at low ionic strength. Langmuir 2006, 22, 9150-9159. 42. Wolz, M.; Kulozik, U., Thermal denaturation kinetics of whey proteins at high protein concentrations. Int. Dairy J. 2015, 49, 95-101. 43. Olsen, S. N.; Andersen, K. B.; Randolph, T. W.; Carpenter, J. F.; Westh, P., Role of electrostatic repulsion on colloidal stability of Bacillus halmapalus α-amylase. Biochim. Biophys. Acta, Proteins Proteomics 2009, 1794, 1058-1065. 44. De Young, L. R.; Fink, A. L.; Dill, K. A., Aggregation of globular proteins. Acc. Chem. Res. 1993, 26, 614-620. 45. Yong, Y. H.; Foegeding, E. A., Effects of caseins on thermal stability of bovine β-lactoglobulin. J. Agric. Food Chem. 2008, 56, 10352-10358.

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18


Page 19 of 26


Figures and tables Proportion [%]

60

15

A

5

B

-2

30

-4

15

-6

0

native

0

θ [mdeg]

θ [mdeg]

10

0

45

heated

-5

-8 -10 -12 -14 -16

-10

Phe

-18

Tyr

Trp

-20

-15 190

200

210

220

230

240

250

250

260

260

270

280

Wavelength [nm]

290

300

310

320

330

340

350

Wavelength [nm]

Figure 1. Far-UV (A) and near-UV (B) CD spectra of native (solid) and heated (dotted; 90 °C, 30 min) β-lactoglobulin (10 mM sodium phosphate buffer pH 7.0, 20 °C). The panel in A shows the content of secondary structure elements of the native and heated β-lactoglobulin; α-helix (black), β-sheet (striped), random coil (grey) and β-turn (white). The dashed lines represent the wavelengths used to determine the unfolding kinetics (i.e. 204 and 294 nm).

0.8

1.2

0.02

1

0.01 0 20 30 40 50 60 70 80 90

0.6

Temperature [°C]

0.4 0.2 0 -0.2

B

0.03

Norm. θ

1

0.03

Normalized θ at 294 nm

A Norm. θ

Normalized θ at 204 nm

1.2

0.8

0.02 0.01 0 20 30 40 50 60 70 80 90

0.6

Temperature [°C]

0.4 0.2 0 -0.2

20

30

40

50

60

70

80

90

20

Temperature [°C]

30

40

50

60

70

Temperature [°C]

Figure 2. Temperature-dependence (20-90 °C) of the normalized ellipticity of far-UV (A) and near-UV (B) CD of β-lactoglobulin (10 mM sodium phosphate buffer pH 7.0, 0.5 °C min-1). The solid lines and the error bars represent the fit and the standard deviations, respectively. The panels represent the non-cumulative fits with the dashed lines representing the denaturation temperatures.

19


80

90


100

A

90

Unfolded protein fraction [%]


100

80 70 60 50 40 30 20 10 0

B

90 80 70 60 50 40 30 20 10 0

0

300

600

900

Time [s]

1200

1500

1800

20

100

30

40

30

40

50

60

70

80

90

50

60

70

80

90

Temperature [°C]

100

C

90



Page 20 of 26

80 70 60 50 40 30 20 10 0

D

90 80 70 60 50 40 30 20 10 0

0

300

600

900

Time [s]

1200

1500

1800

20

Temperature [°C]

Figure 3. Time- (A and C) and temperature-dependence (B and D) of the fraction of unfolded secondary (A and B) and tertiary (C and D) structure of heated β-lactoglobulin in 10 mM sodium phosphate buffer pH 7.0 measured at 80 °C (A and C, ), after 1800 s (B and D, ) and after cooling to 20 °C (A-D, ). The error bars indicate the standard deviations. The dashed and dotted lines in panels A and C indicate the time required to reach 80 °C and the equilibrium of the fraction of unfolded protein, respectively.

20


Page 21 of 26


A

Non-aggregated

A280 [mAU]

Aggregated

6

7

8

9

10

11

12

13

14

Elution volume [mL] B 6

θ [mdeg]

4 2 0 -2 -4 -6 190

200

210

220

230

240

250

260

Wavelength [nm] 70

C

Content [%]

60 50 40 30 20 10 0

N7

H7

H4.6

N/NL

NL+NNL+A

NL

Figure 4. Size-exclusion chromatograph (A), far-UV CD spectra (B) and content of secondary structure elements (α-helix (black), β-sheet (striped), random coil (grey) and β-turn (white)) (C) of native (dotted black, N7), heated, pH 7.0 (solid black, H7) and heated, pH 4.6 (dashed black, H4.6) β-lactoglobulin (0.2 g/L, 20 °C). The grey box in panel C indicates the heated samples and N, NL, NNL and A represent the native, native-like, non-native-like and aggregated state of the protein, respectively.

21



A

Aggregated

100

Non-aggregated

Page 22 of 26

B

90 80

Fraction [%]

A280 [mAU]

1800 s 1200 s 600 s 300 s

7

8

9

10

11

12

A

60 50

NL

40 30 20

NNL

10

0s 6

70

0

13

0

14

300

600

Elution volume [mL] 100

C

90

90

80

80

70

70

NL

60 50

A

40 30

NNL

20

Fraction [%]

Fraction [%]

100

1200

1500

D A

NL

30 20

0 900

1800

40

10 600

1500

50

0 300

1200

60

10 0

900

Time [s]

1800

NNL 0

Time [s]

300

600

900

1200

Time [s]

Figure 5. Time-dependence (T = 80 °C) of the unfolding and aggregation determined by size-exclusion chromatography at pH 7.0 (solid lines in A) and at pH 4.6 (dashed lines in A) and of the native-like (NL; ), non-native-like (NNL; ) and aggregated (A; ) fraction β-lactoglobulin in 10 mM sodium phosphate buffer pH 7.0 (B), in milliQ water pH 8.0 (C) and 50 mM sodium phosphate buffer pH 7.0 (D) (2 g/L, 20 °C). The solid lines in panel B-D are guides to the eye.

22


1500

1800

Page 23 of 26


Aggregated

A

100

Non-aggregated

B

90 80

Fraction [%]

A280 [mAU]

90 °C 80 °C 70 °C 60 °C

70

A

60 50 40

NL

30 20 10

20 °C 6

7

8

9

10

11

12

NNL

0

13

20

14

30

40

Elution volume [mL] 100

100

C

60

70

80

90

D

90

80

80

70

70

60

A

50 40

NL

30

Fraction [%]

Fraction [%]

90

50

Temperature [°C]

A

60 50 40 30 20

NL

10

10

NNL

0

0

20

NNL

20

30

40

50

60

70

80

90

20

30

Temperature [°C]

40

50

60

70

Temperature [°C]

Figure 6. Temperature-dependence (t = 1800 s) of unfolding and aggregation determined by size-exclusion chromatography at pH 7.0 (solid lines in A) and at pH 4.6 (dashed lines in A) and of the native-like (NL; ), non-native-like (NNL; ) and aggregated (A; ) fraction β-lactoglobulin in 10 mM sodium phosphate buffer pH 7.0 (B), in milliQ water pH 8.0 (C) and 50 mM sodium phosphate buffer pH 7.0 (D) (2 g/L, 20 °C). The solid lines in panel B-D are guides to the eye.

23


80

90


100

Page 24 of 26

N →U→A

Aggregated fraction [%]

90 80 70

N↔U→A

60 50 40 30 20 10 0 0

20

40

60

80

100

Unfolded fraction [%]

Figure 7. Aggregated protein fraction determined by SEC (figure 5B and 6B) as a function of the unfolded protein fraction determined from CD at 60-90 °C () (figure 3A-D). The dashed and solid lines represent completely irreversible (slope = 1.0; N→U→A) and partially reversible (slope = 0.6; R2 = 0.93; N↔U→A) unfolding, respectively.

100

A

90

90

80

80

70

70

Fraction [%]

Fraction [%]

100

60 50 40 30

B

60 50 40 30

20

20

10

10

0

0 1

2

20

Concentration [g

L-1]

50

0 mM pH 8

10 mM pH 7

50 mM pH 7

Figure 8. Effect of heating (80 °C, 1800 s) on the fraction of aggregated (black), native-like

(grey)

and

non-native-like

(white)

β-lactoglobulin

at

different

concentrations in 10 mM sodium phosphate buffer pH 7.0 (A) and in milliQ water pH 8.0, 10 mM sodium phosphate buffer pH 7.0 and 50 mM sodium phosphate buffer pH 7.0 at a concentration of 2 g/L (B) measured at 20 °C.

24


Page 25 of 26


70

A

B

6 60

Content [%]

θ [mdeg]

4 2 0 -2

50 40 30 20 10

-4

0

-6 190

200

210

220

230

240

250

260

Wavelength [nm]

N7 N/NL

H7 NL+NNL+A

HNA7 NL+NNL

Figure 9. Far-UV CD spectra (A) and content of secondary structure elements (αhelix (black), β-sheet (striped), random coil (grey) and β-turn (white)) (B) of native (dotted black, N7), heated, pH 7.0 (solid black, H7), heated, non-aggregated, pH 7.0 (solid grey, HNA7) and heated, non-aggregated, pH 4.6 (dashed grey, HNA4.6) β-lactoglobulin (0.2 g/L, 20 °C). The grey box in panel B indicates the heated samples and N, NL, NNL and A represent the native, native-like, non-native-like and aggregated state of the protein, respectively.

25


HNA4.6 NL+NNL-1


Page 26 of 26

For Table of Contents Only

26


Controlling the Ratio between Native-Like, Non-Native-Like, and

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