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Conformational transition of polyelectrolyte as influenced by electrostatic complexation with protein Yiping Cao, Shugang Li, Yapeng Fang, Katsuyoshi Nishinari, and Glyn O. Phillips Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b01335 • Publication Date (Web): 28 Oct 2016 Downloaded from http://pubs.acs.org on November 3, 2016
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Biomacromolecules
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Conformational transition of polyelectrolyte as
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influenced by electrostatic complexation with protein
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Yiping Cao,† Shugang Li, † Yapeng Fang,*, †, ‡ Katsuyoshi Nishinari,†, ‡ and Glyn O. Phillips†
4
5 6
†
7
University of Technology, Wuhan 430068, China;
8
‡
9
Technology, Wuhan 430068, China.
10
Glyn O. Phillips Hydrocolloid Research Centre, School of Food and Biological Engineering, Hubei
Hubei Collaborative Innovation Centre for Industrial Fermentation, Hubei University of
*Corresponding author: Yapeng Fang, Email:
[email protected]; Tel: +86-(0)-27-59750470.
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12
13
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KEYWORDS: conformational transition, electrostatic complexation, theoretical modeling,
15
β-lactoglobulin, κ-carrageenan, gelling properties
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ABSTRACT: Conformation and conformational transitions are essential for the biological and
19
technological functions of natural polyelectrolytes, e.g., DNA. This study aims to clarify how the
20
conformational transition of natural polyelectrolyte is affected and tuned by electrostatic
21
complexation
22
protein/polyelectrolyte system, β-lactoglobulin (β-lg) and κ-carrageenan (κ-car), was used for the
23
investigation. The effect was found to be determined by the molecular state of β-lg/κ-car electrostatic
24
complexation and the molecular weight of protein. β-lg/κ-car complexation in soluble state had a
25
subtle effect on the coil-to-helix transition of κ-car, while that in insoluble state greatly suppressed it.
26
Based on the McGhee-Hippel theory, a quantitative model was successfully developed to describe
27
the effect of protein/polyelectrolyte electrostatic complexation on the conformational transition of
28
polyelectrolyte. The model can also provide additional information on the change of tertiary structure
29
of β-lg upon electrostatic complexation with κ-car. Moreover, it was found that β-lg or its
30
hydrolysates with a molecular weight larger than 2000 Da hindered the conformational transition of
31
κ-car, while those with a molecular weight lower than 1000 Da promoted it. The observations offer a
32
promising approach to controlling the conformational transition and related properties of
33
polyelectrolytes for technological applications.
34
INTRODUCTION
with
protein
as
encountered
in
many
biological
processes.
A
model
35
Natural polyelectrolytes exhibit diverse conformations in solutions, including random coil, single
36
helix, multiple helix, aggregate and sphere-like structure, etc.1-3 These conformations are involved in
37
many biological processes and in human diseases. For instance, DNA with mutated conformation
38
(e.g., hairpin, triplex, cruciform, and left-handed form) is associated with diseases such as myotonic
39
dystrophy, Huntington’s chorea, fragile X syndrome, and Friedreich’s ataxia.1 In cell biology, 2
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conformational equilibria of ubiquitin can determine its specific binding with protein or DNA, thus
41
regulating protein degradation, cell signaling/trafficking and DNA damage response.4 Conformation
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and conformational transitions of polyelectrolytes influence also their bioactivities. Triple helical
43
schizophyllan shows strong inhibition against tumor cells, whereas its single coil counterpart has
44
almost no bioactivity. This behavior is attributed to an increased interaction between the extended
45
chain conformation and the immune system.5 Sulphation or phosphatization is known to greatly
46
improve the inhibitivity of branched α-(1→4)-D-glucan against H-22 tumor cells. The effect is
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explained by a conformational transition from compact sphere to random coil upon the chemical
48
modification, which confers the polyelectrolyte more flexibility to bind with relevant receptors on
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the tumor cell surface.6 Conformational transitions of polyelectrolytes are applied in industry for
50
applications such as thickening, gelling, and stabilization.7
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Electrostatic complexation of polyelectrolyte with protein is also important in biological processes
52
including DNA replication, transcription and repair.8,
53
non-specifically to DNA via electrostatic interaction, and move subsequently along the DNA strand
54
until specific binding sites are identified.10 Protein/polyelectrolyte electrostatic complexation occurs
55
naturally in many living organisms, triggering a variety of biological functions, such as the adhesive
56
property of sandcastle worms provided by the complexation of highly polar proteins11 and the
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mechanical properties of mammalian cartilages provided by the ternary complexes of aggrecan,
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hyaluronan and cartilage link proteins12. Protein/polyelectrolyte complexation has found numerous
59
applications
60
separation/purification,13 enzyme stabilization/immobilization,11 drug delivery, and gene therapy14.
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In the food industry, protein/polyelectrolyte electrostatic complexation was used to design
in
the
biotechnological
and
9
Protein is considered first to bind
pharmaceutical
industries,
including
protein
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low-calorie and low-starch foods,15 encapsulate flavors and probiotics,16, 17 and stabilize emulsions
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and foams18.
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Coupling of protein/polyelectrolyte electrostatic complexation with conformational transition was
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frequently encountered in biological processes and technological applications. Our previous studies
66
have
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conformational transition via two interconnected mechanisms, namely, specific ionic binding and
68
chain stiffening.19 It is necessary now to clarify how the conformational transition of polyelectrolyte
69
is controlled by electrostatic complexation with protein. We demonstrate this behavior by reference
70
to a model protein/polyelectrolyte system, β-lactoglobulin (β-lg) and κ-carrageenan (κ-car).
shown
that
protein/polyelectrolyte
complexation
is
influenced
by
polyelectrolyte
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β-lg is the dominant globular protein found in whey protein.20 It has 162 amino acid residues, and
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a molecular mass of 18.4 kDa.20, 21 κ-car is a naturally anionic polysaccharide extracted from red
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seaweed species and is widely used in food, cosmetic, and pharmaceutical products as gelling agents,
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thickeners, stabilizers, and excipients.7 It is made up of alternating α-(1→3)-D-galactose-4-sulfate
75
and β-(1→4)-3,6-anhydro-D-galactose repeating units.7, 22 The temperature-induced conformational
76
transition of κ-car has been a subject of debate for many years. It is traditionally accepted that κ-car,
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in the presence of ions (e.g. K+), undergoes a conformational transition from random coil to double
78
helix and further to helical aggregates with lowering temperature.19,
79
elegantly showed that the κ-car chains first undergo a simple coil-to-helix transition, and then the
80
single helices interwine or supercoil into supermolecular strands with a hierarchical chirality
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amplification, using high-resolution atomic force microscopy.24-26 This represents a significant
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advancement of the debates on the conformational transition of carrageenans.
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EXPERIMENTAL SECTION
23
Recently, Schefer et al.
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Biomacromolecules
Materials
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β-lg was kindly supplied by Davisco, which according to the supplier has a minimum purity of >
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95%. β-lg has a weight average molar mass of Mw = 19.1 kDa, a polydispersity index of Mw/Mn =1.1,
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and a radius of gyration of Rg = 6.5 nm, as determined by gel permeation chromatography-multiple
88
angle laser light scattering (GPC-MALLS, Wyatt Technology Corporation, USA) at 25 oC in 0.1 M
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phosphate buffer (pH 7.0) using a Sepax SRT SEC-150 column (Sepax Technologies, USA). The
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isoelectric point (IEP) of the β-lg sample measured by Nano-ZS ZetaSizer (Malvern Instruments, UK)
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is 5.2 (Figure S1), which agrees with literature values.20, 21, 27
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κ-car was a gift from FMC biopolymer (Gelcarin GP-911NF). It was converted into the sodium
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salt using ion exchange resin (Amberlite IR-120, Sigma), and then lyophilized. The purified sample
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contains 6.32% Na, 0.067% K, 0.0027% Mg, and 0.0083% Ca as determined by atomic absorption
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spectrometry. The purification was to avoid the complicated effects of mixed counterions, allowing a
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precise molecular characterization of κ-car. The molecular parameters measured by GPC-MALLS at
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25 oC in 0.1 M NaI using a Shodex OHpak SB-805 separation column (GE Healthcare Co., USA) are:
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Mw = 467 kDa; Mw/Mn = 1.2; Rg = 85.0 nm, which represent the doubly-associated helical
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conformation of κ-car without further aggregation.19, 28 The zeta potential of κ-car, measured in the
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pH range of 3-8, is characteristic of strong polyelectrolytes with nearly a constant value of ca. -60
101
mV (Figure S1), which agrees with most literature reports.29
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Preparation of β-lg hydrolysates
103
Hydrolysates with different molecular weight were obtained by hydrolyzing β-lg to different
104
extents. 5.0 % β-lg was heated at 85 oC for 5 min, followed by hydrolysis using the enzyme papain
105
(3000 U, Biosharp) at the optimum conditions (45 oC, pH 4.5) for 4 h. The hydrolysate was separated
106
using ultrafiltration tube (MWCO 3000, Biosharp), and the filtrate was loaded into dialysis tube 5
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(MWCO 1000, Biosharp) for further separation. After dialysis against Millipore water at 4 oC for 96
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h, the hydrolysates inside and outside the dialysis tube were collected by freeze-drying and
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designated as S1 and S2, respectively. A total hydrolysate of β-lg (S3) was obtained by heat
110
treatment at 120 oC in 6.0 M HCl for 12 h, followed by removal of HCl using vacuum evaporation at
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60 oC. Although S1, S2 and S3 were mixtures of peptide fragments of different length and chemical
112
composition, as a whole, their average amino acid compositions were nearly the same as the mother
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protein β-lg without hydrolysis. This was confirmed by analysis using an automatic amino acid
114
analyzer (Hitachi, L-8900). Weight average molar mass measured by GPC-MALLS at 25 oC in 0.1
115
M phosphate buffer using a Sepax SRT SEC-150 column is: β-lg (19100 Da) > S1 (2053 Da) > S2
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( 921 Da). The weight average molar mass of S3 was calculated to be 127 Da, according to the amino
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acid composition of β-lg.20, 21
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Solution preparation
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Stock solutions of 0.06-1.80 wt% β-lg and 0.90 wt% κ-car were prepared by dissolving
120
appropriate amounts of the samples into 50 mM KCl. κ-car solutions were heated at 85 oC for 1 hour
121
under magnetic stirring. β-lg solutions were dissolved at ambient temperature overnight on a roller
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mixer. β-lg/κ-car mixtures at a fixed κ-car concentration (0.15%) and various β-lg/κ-car mixing
123
ratios (w/w, 0 < r IEP.31 It is because that both κ-car and β-lg are overall
188
negatively charged at this pH, with a zeta potential of -60 mV and -25 mV, respectively (Figure S1).
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The absence of electrostatic complexation at pH 5.6 is supported by the turbidity measurements in
190
Figure 1c where τ remains close to zero at all mixing ratios at 45 oC.
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In contrast, at pH 4.7 < IEP, the exothermic peak associated with the coil-to-helix transition of
192
κ-car was significantly reduced with increasing β-lg/κ-car mixing ratio (Figure 1d). The exothermic
193
peak tends to disappear completely at r > 8.00. This indicates that the conformational transition of
194
κ-car is greatly suppressed when mixed with β-lg. This can be attributed to the electrostatic
195
complexation between β-lg and κ-car, which impairs the helix formation of κ-car. Figures 1e plots
196
the enthalpy change (∆H) of the conformational transition of κ-car as a function of r at pH 4.7. ∆H
197
shows a two-step decrease with increasing r, and a turning point is located at r = 0.67. Beyond the
198
turning point (r > 0.67), the decrease in ∆H is apparently accelerated. It means that the
199
conformational transition of κ-car is more seriously impaired beyond the turning point.
200
Interestingly, the turning point in ∆H-r curve coincides with that obtained in τ-r curve at 45 oC
201
(Figure 1f). Turbidity is relatively low at r < 0.67, whereas it increases abruptly at r > 0.67. Similar
202
coincidence was found at other pHs, and the turning point is at r = 0.33, 1.45, 2.70 and 4.00, 10
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respectively, for pH = 4.4, 4.9, 5.0 and 5.1(see Figure S2). Turbidity was seen as a sensitive measure
204
of electrostatic complexation of protein/polyelectrolyte.11, 31, 32 The abrupt increase in τ at the turning
205
points is attributed to the transition from soluble to insoluble electrostatic complexes.31, 32 When β-lg
206
molecules crosslink different κ-car chains, large aggregates that are insoluble are formed, and thus
207
the turbidity increases.31, 32 Note that further decrease in pH below 4.4 caused extensive precipitation,
208
preventing the measurement of the conformational transition of κ-car. The evolution of electrostatic
209
complexes as a function of pH and mixing ratio has been detailedly studied in our previous works.29,
210
30
211
The molecular state of β-lg/κ-car complexation seems to control the conformational transition of
κ-car (Figure 2).
(a) cooling
(b) cooling
212 213
Figure 2. Schematic representation of the effect of β-lg/κ-car electrostatic complexation on the
214
conformational transition of κ-car upon cooling: (a) β-lg/κ-car complexation in soluble state; (b)
215
β-lg/κ-car complexation in insoluble state. κ-car and β-lg molecules are illustrated in black and red,
216
respectively. During the conformational transition, coiled κ-car chains transform into ordered helical
217
conformations (bold lines) which can be single helix, double helix, or even supermolecular strand.
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During initial stage of electrostatic complexation (Figure 2a), a small number of β-lg are bound to
219
κ-car, and the complexes remain soluble. β-lg molecules pose limited steric effect and κ-car
220
molecules have sufficient freedom to form helices through pairing. Therefore, the conformational
221
transition of κ-car is influenced to a limited extent by the electrostatic complexation with β-lg. As
222
electrostatic complexation further proceeds (Figure 2b), more and more β-lg are bound to κ-car, and
223
cross-linking between κ-car occurs. This causes the formation of insoluble β-lg/κ-car complexes.
224
κ-car molecules are sterically blocked by β-lg and have limited freedom to restructure to form
225
helices. The conformational transition of κ-car in this instance is greatly suppressed by electrostatic
226
complexation with β-lg. It should be pointed out that although the exact conformations of κ-car are
227
to some extent controversial (i.e., single helix, double helix, or supermolecular helical strand),24-26
228
the proposed mechanism, as schematically shown in Figure 2, applies to different situations.
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Theoretical modeling
230
As already discussed, the conformational transition of κ-car is greatly suppressed by electrostatic
231
complexation with β-lg, and the origin of this effect is κ-car being physically occupied with β-lg.
232
Assuming that the relative extent of conformational transition of κ-car is proportional to the number
233
of repeating units that are unoccupied with β-lg, a quantitative model can be developed to describe
234
the effect of protein/polyelectrolyte electrostatic complexation on the conformational transition of
235
polyelectrolyte (see Supporting Information), based on the DNA-protein binding theory proposed by
236
McGhee- Hippel:33
237
(1 − φ (r ) )φ (r ) + 1 − φ (r )
m
m −1
C p M wP N (1 − φ (r )) − Kmφ (r ) m r − CP =0 Pr m M w
(2)
238
where r is the protein/polyelectrolyte weight ratio; φ(r) is the relative extent of conformational
239
transition of polyelectrolyte; m represents the number of repeating units consecutively covered by 12
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one protein molecule and is the reciprocal of the binding stoichiometry n; K is the binding constant;
241
Cp is the molar concentration of polyelectrolyte; M wP and M wPr are the molecular weight of
242
polyelectrolyte and protein, respectively; N is the total number of repeating units of polyelectrolyte.
1.2
1.03
(a)
(b)
1.00
φ =0.93
0.9
0.97 0.6
φ
φ
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Biomacromolecules
0.94
φ =0.93
0.3 0.91 0
0.88 0
243
2
4
6
r =β -lg/κ -car
8
10
0
0.9
1.8
2.7
r =β -lg/κ -car
3.6
4.5
244
Figure 3. (a) Relative extent of conformational transition of κ-car (φ ) as a function of β-lg/κ-car
245
mixing ratio r at different pHs: pH=5.6 (■); pH=5.1 (●); pH=5.0 (◆); pH=4.9 (▲); pH=4.7 (×);
246
pH=4.4 (+); (b) Enlarged part of Figure a showing two-step decrease in φ. The solid lines represent
247
the curve fittings to Equation 2. The dot-dash lines at φ=0.93 mark the turning points of φ -r curves.
248
φ(r) could be experimentally measured as the ratio of the enthalpy change of conformational
249
transition of protein/polyelectrolyte mixture (∆H(r)) to that of pure polyelectrolyte (∆H(r=0)). Figure
250
3 plots φ as a function of β-lg/κ-car mixing ratio at different pHs. Except at pH 5.6, which is above
251
the IEP of β-lg, φ at pH = 5.1, 5.0, 4.9, 4.7 and 4.4 all exhibit a two-step decrease with r. The
252
decrease is more pronounced at lower pH. This is due to an increased electrostatic complexation of
253
β-lg/κ-car at lower pH, which exerts a stronger effect on suppressing the conformational transition of
254
κ-car. Intriguingly, the turning points of the two-step decrease at different pHs occur at the same
255
value of φ = 0.93 (Figure 3b). This probably indicates a common critical binding threshold beyond
256
which insoluble β-lg/κ-car complexes start to be formed and the conformational transition of κ-car is
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seriously inhibited. This threshold corresponds to 7% repeating units of κ-car being bound/occupied
258
with β-lg.
259
Taking N = 572, Cp= 6.42×10-6 mol/L, M wPr = 19100 Da and M wP = 233500 Da for the present
260
system, φ measured by DSC can be well fitted to Equation (2) (Solid lines in Figure 3a). The fittings
261
yielded the thermodynamic binding parameters m and K at different pHs, as listed in Table 1. The
262
parameters are in good agreement with those obtained from ITC measurements at 45 oC (see Figure
263
S3). The ITC results represent the binding of β-lg to κ-car in coil state and just prior to the
264
coil-to-helix transition, and therefore to large extent can approximate their dynamic binding during
265
the subsequent conformational transition. The agreement validates the theoretical model as
266
developed in Equation (2).
267
Table 1. Thermodynamic parameters of the binding of β-lg to κ-car obtained from DSC and ITC. Method
parameter
pH=4.4
pH=4.7
pH=4.9
pH=5.0
pH=5.1
m
25.2
17.1
13.9
13.2
12.2
K
1.7×108
2.3×106
3.1×103
3.5×102
5.1×101
m
25.5
16.6
14.2
12.8
12.3
K
1.9×108
2.1×106
3.0×103
3.9×102
6.2×101
DSC
ITC 268
m=1/n, n is the binding stoichiometry of β-lg to κ-car.
269
With lowering pH from 5.1 to 4.4, K increases from 5.1×101 to 1.7×108 M-1. The increase in K is
270
apparently attributed to an increase in electrostatic attraction. Note that the binding constants with
271
magnitude as high as 108 at lower pHs might represent strong out-of-equilibrium bindings, where
272
precipitation of β-lg/κ-car complexes occurred. m is also increased by lowering pH, but its change
273
around IEP (i.e., pH 5.1 and 5.0) is insignificant. Considering a repeating unit length of 1.03 nm for 14
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κ-car,34 m = 12.2 at pH 5.1 corresponds to a consecutive binding length of 12.6 nm, which is very
275
close to the diameter of β-lg (2Rg = 13 nm) as measured by GPC-MALLS. It implies that the weak
276
binding between β-lg/κ-car close to IEP is determined by the size of β-lg (most likely in dimeric
277
form).35 In contrast, m = 25.2 at pH 4.4, which corresponds to a consecutive binding length of 30.0
278
nm. This length is more than double the diameter of β-lg. It could be due to an increase in protein
279
surface charge, which increases its effective Debye length. Another possibility is that the strong
280
binding between β-lg/κ-car at lower pH induces a geometric elongation of β-lg.
281
DSC measurements (see Figure S4) show that at pH values (i.e., pH 5.1 and 5.6) close to IEP,
282
there is almost no difference in protein denaturation temperature between pure β-lg and β-lg/κ-car
283
mixture. However, at lower pHs (e.g., pH 4.4), the denaturation temperature of pure β-lg is
284
considerably higher than that in the β-lg/κ-car mixture. The reduction in denaturation temperature
285
indicates a less stable conformation or tertiary structure of β-lg upon electrostatic complexation with
286
κ-car. Furthermore, structural component analysis by attenuated total reflectance Fourier transform
287
infrared spectroscopy (ATR-FTIR) shows that the β-sheet and α-helix content of β-lg are both
288
decreased upon complexation with κ-car at lower pHs (see Figure S5). It was also found that protein
289
structures were altered upon complexation with polyelectrolyte in α-gliadin/gum arabic or
290
β-lg/acacia gum systems.32, 36 The structural changes revealed by DSC and ATR-FTIR may be
291
responsible for the geometric elongation of β-lg, as predicted by the theoretic model in Equation (2).
292
The situation is similar to the binding of proteins onto negatively charged surfaces (e.g., mica, silica)
293
at pHs < IEP or hydrophobic surfaces. The strong electrostatic interaction or hydrophobic interaction
294
upon adsorption onto the surfaces caused somewhat denaturation and spreading/elongation of the
295
proteins along the surfaces. 37-39
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On the other hand, β-lg was reported to exhibit a number of pH-induced, local and global
297
structural transitions between pH 1.0 and pH 13, including the dimer-to-monomer transition (pH
298
2.5-4.0), the Q-to-N transition (pH 4.5-6.0) and the Tanford transition (> pH 6.5), etc.35 In the pH
299
range investigated (pH 4.4-5.6), β-lg exists predominately as dimers, and undergoes a
300
conformational change from the compact native form (N) at pH 6.0 to the more extended acidic form
301
(Q) at pH 4.5. The hydrodynamic diameter of β-lg molecule was reported to increase from 5.16 nm
302
to 5.44 nm, accordingly.40 Such a pH-induced size expansion (only by 5%) could contribute to, but
303
was not fully responsible for the observed elongation of β-lg upon electrostatic complexation with
304
κ-car (Table 1). Moreover, Taulier and the coworker observed a slight change in β-lg tertiary
305
structure (i.e., increase in volume and compressibility) during the Q-to-N transition, which however
306
was not accompanied by any significant alteration in secondary structure.35 This slight change in
307
tertiary structure seems not to have conspicuous influence on the electrostatic complexation of β-lg
308
with κ-car, as the complexation behaviors at different pHs can be described by a common theoretical
309
model.
310
Effect of protein molecular weight
311
Since protein/polyelectrolyte electrostatic complexation suppresses the conformational transition
312
of polyelectrolyte via the physical hindrance of protein, it is desirable to investigate in details the
313
effect of protein molecular weight or size. For this purpose, β-lg was hydrolyzed into different
314
extents by enzymatic or acidic treatment, and three hydrolysates of different molecular weights were
315
fractionated using ultrafiltration and dialysis: S1 (2053 Da) > S2 (921 Da) > S3 (127 Da). The three
316
fractions have almost the same average amino acid compositions as the mother protein β-lg.
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(a)
1.2
Control lysine S3 S2 S1 β-lg
43
42
41
0.6
0.3
40
0 0
317
(b)
0.9
φ
To(oC) h
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biomacromolecules
2
4
r
6
8
10
0
2
4
r
6
8
10
318
Figure 4. Onset temperature (To) (a) and relative extent (φ ) (b) of the conformational transition of
319
κ-car, as affected by electrostatic complexation with β-lg or its hydrolysates. κ-car 0.15 %; pH = 4.7.
320
Figure 4 shows how differently the β-lg hydrolysates affect the conformational transition of κ-car
321
upon electrostatic complexation at pH 4.7. The effect of lysine was also compared, as it is one of the
322
most typical and abundant amino acids in β-lg. When the hydrolysate molecular weight is > 2000 Da
323
(e.g., β-lg and S1), the onset temperature (To) and relative extent (φ) of conformational transition of
324
κ-car are markedly reduced with increasing r, suggesting a suppressing effect of the hydrolysates.
325
When the hydrolysate molecular weight is around 1000 Da (e.g., S2), To and φ are nearly constant
326
with increasing r. It indicates that the electrostatic complexation in this range of hydrolysate
327
molecular weight has no effect on the conformational transition of κ-car. Significantly, when the
328
hydrolysate molecular weight is < 1000 Da (e.g., S3 and the amino acid lysine), To increases with
329
increasing r, while φ remains constant. The increase in To suggests that the hydrolysates stabilize the
330
helical conformation of κ-car, which can be formed at higher temperatures.
331
As discussed above, the suppressing effect of β-lg or its hydrolysates with a molecular weight
332
larger than 2000 Da is due to their steric effect, which physically hinders the conformational
333
transition of κ-car upon electrostatic complexation. K and m for S1 binding to κ-car at pH 4.7 were
334
calculated to be 4.1×102 M-1 and 6.8, respectively, based on the curve-fitting using Equation (2). The 17
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335
values are significantly smaller than those of 2.3×106 M-1 and 17.1 for β-lg binding to κ-car at pH 4.7,
336
indicating a decreased binding affinity with decreasing hydrolysate molecular weight. m = 6.8
337
corresponds to a consecutive binding length of 6.8 x 1.03 nm = 7.0 nm, which agrees well with the
338
size of S1 of 7.2 nm (containing 19 amino acids, each being approximately 0.38 nm in length).41
339
The steric effect disappears completely when the molecular weight of β-lg hydrolysates is
340
decreased to around 1000 Da. With further decreasing molecular weight below 1000 Da, the ionic
341
strength effect of the short hydrolysates becomes pronounced, due to the increasing number of amino
342
and carboxyl groups. This facilitates the coil-to-helix transition of κ-car and stabilizes the formed
343
helices during cooling. The promoting effect of β-lg hydrolysates with a molecular weight lower than
344
1000 Da is similar to that of adding electrolytes such as NaCl.22 The addition of NaCl was reported
345
to increase also the onset temperature of the conformational ordering of κ-car, while it did not
346
change noticeably the transition enthalpy at high ionic concentration. The promoting effect was
347
attributed to the screening of intra- or inter-molecular electrostatic repulsion and the alteration of
348
hydration properties of κ-car chains, which favors the formation of ordered helices.42
349
Application to tuning gelation and gel viscoelasticity
350
Since the conformational transition of κ-car underlies its gelation, we tried controlling the gelation
351
and gel viscoelasticity of κ-car using electrostatic complexation with β-lg or its hydrolysates. The
352
gelation and gel viscoelasticity of κ-car in mixtures with β-lg at pH 4.7 are shown in Figure 5.
353
During cooling, the storage modulus (G') of 0.15% κ-car exhibits an abrupt increase around 40.6 oC
354
(Figure 5a), indicating the occurrence of gelation.43 The onset temperature of gelation seems not to
355
be influenced by mixing with β-lg. However, the gel elasticity as characterized by G' is greatly
356
reduced with increasing r. The gelation is completely prevented at r = 10.0. Figure 5b displays the
357
frequency dependence of gel viscoelasticity of β-lg/κ-car mixture at 10 oC. For pure κ-car, both 18
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358
moduli are independent of frequency with G' > G'', indicative of a typical elastic gel.44 With
359
increasing r, G' and G'' become more and more frequency-dependent and cross over at high
360
frequencies. This is an indication of weak viscoelastic gels or viscous solutions.44 The results
361
demonstrate that by mixing with protein at different ratios electrostatic complexation can be used to
362
tune the gelling behavior of polyelectrolytes, to give viscoelastic properties ranging from elastic gels
363
to viscous solutions. The effect is in line with that on the conformational transition of κ-car, as
364
shown in Figure 3a.
1000 103
1000 103
(a)
r =0.00
10 10
r =2.00 r =6.67
G', G'' (Pa)
G' (Pa)
10 101
0.1 10-1 10
(b)
100 102
100 102
101 10
r =10.0
1010 0.1 10-1
15
20
25
T
365
30
35
40
45
0.01 10-2 10-2 0.01
0.1-1 10
(oC)
10 10
101 10
100 102
f (Hz)
366
Figure 5. (a) Gelation profiles of storage modulus G' versus temperature during cooling for κ-car at
367
different β-lg/κ-car mixing ratios r; (b) Frequency sweep of storage (G', closed symbol) and loss
368
moduli (G'', opened symbol) of β-lg/κ-car mixtures at 10 oC. κ-car 0.15 %; pH=4.7.
1000 103
1000 103
(a)
G', G'' (Pa)
101 10
1010
0.1 10-1 10
369 370
(b)
control
β -lg
100 102
100 102
G' (Pa)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biomacromolecules
S1
10 101
S2 S3
10 10
lysine
10-1 0.1
15
20
25
30
35
40
45
10-2 0.01 10-2 0.01
T (oC)
10-1 0.1
10 10
10 101
102 100
f (Hz)
Figure 6. (a) Gelation profiles of storage modulus G' versus temperature during cooling for κ-car in 19
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371
mixtures with β-lg and its hydrolysates; (b) Frequency sweep of storage (G', closed symbol) and loss
372
moduli (G'', opened symbol) of the mixtures at 10 oC. κ-car 0.15 %; r = 10.0; pH = 4.7.
373
Figure 6 demonstrates tuning the gelation and gel viscoelasticity of κ-car by electrostatic
374
complexation with β-lg hydrolysates of different molecular weights. At r =10.0 and pH = 4.7, the
375
presence of β-lg or S1 (> 2000 Da) significantly reduces the gelling ability and viscoelasticity of
376
κ-car (Figure 6a). The corresponding frequency sweep exhibits weak gel type or liquid behavior
377
(Figure 6b). The presence of S2 ( ～ 1000 Da) has almost no effect on the gelation and gel
378
viscoelasticity of κ-car. The gelation profile and frequency sweep of S2/κ-car overlap with those of
379
pure κ-car. In contrast, the low molecular weight hydrolysates, S3 and lysine (< 1000 Da), enhance
380
considerably the onset gelation temperature of κ-car, although the gel elasticity is barely affected
381
(Figure 6b). The results reveal different modulatory effects of protein and its hydrolysates on the
382
gelation and gel properties of polyelectrolyte. The effect of protein hydrolysates is in concert with
383
that on the conformational transition of κ-car, as shown in Figure 4.
384
CONCLUSIONS
385
The effect of protein/polyelectrolyte electrostatic complexation on the conformation transition of
386
polyelectrolyte was investigated both experimentally and theoretically by referring to β-lg/κ-car. The
387
effect was found to be related to the molecular state of electrostatic complexation: I) electrostatic
388
complexation in soluble state had subtle effect on the coil-to-helix transition of κ-car, which is due to
389
the relatively high freedom of κ-car at the initial stage of protein binding; II) electrostatic
390
complexation in insoluble state however greatly suppressed the conformational transition, which is
391
due to the high physical hindrance imposed by β-lg upon extensive protein binding. Based on the
392
McGhee-Hippel theory, a quantitative model was successfully developed to describe the effect of 20
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393
protein/polyelectrolyte electrostatic complexation on the conformation transition of polyelectrolyte.
394
Moreover, it was found that the effect was closely associated with the molecular weight of β-lg or its
395
hydrolysates. Larger hydrolysates (> 2000 Da) had an inhibitory effect on the conformational
396
transition of κ-car, whereas shorter hydrolysates (< 1000 Da) tended to promote it. The study
397
demonstrated the possibility of controlling the gelation and gel properties of polyelectrolytes via
398
electrostatic complexation with protein or its hydrolysates.
399
ASSOCIATED CONTENT
400
Supporting Information
401
Methods for measuring protein denaturation temperature (DSC) and secondary structure
402
(ATR-FTIR); Zeta potential of β-lg and κ-car (Figure S1); Enthalpy change and turbidity for
403
β-lg/κ-car mixtures as a function of mixing ratio at different pHs: 4.4, 4.9, 5.0 and 5.1 (Figure S2);
404
ITC (Figure S3); Denaturation temperature of β-lg (Figure S4); Secondary structure of β-lg (Figure
405
S5); Theoretical modeling. This material is available free of charge via the Internet at
406
http://pubs.acs.org.
407
AUTHOR INFORMATION
408
Corresponding Author
409
*Email:
[email protected]; Tel: +86-(0)-27-59750470.
410
Notes
411
The authors declare no competing financial interest.
412
ACKNOWLEDGEMENTS
21
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413
The research was supported by National Natural Science Foundation of China (31671811,
414
31322043, 31171751), projects from Hubei Provincial Department of Science and Technology
415
(2014BHE004, 2012FFA004) and Department of Education (T201307), program for New Century
416
Excellent Talents in University (NCET-12-0710), and project from Wuhan Science and Technology
417
Bureau (2015070504020218).
418
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419
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Table of Contents 1.2
Electrostatic complexation in soluble state
0.9
φ
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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Electrostatic complexation in insoluble state m −1 C MP (1 − φ (r ))φ (r ) + 1 − φ (r ) − Km φ (r ) m r p w − C N (1 − φ (r )) = 0
0.6
m
M wPr
P
m
0.3 0.0 0
476
2
4
6
r =β -lg/κ-car
8
10
24
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