CHARGE-BASED SEPARATION OF PROTEINS USING

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CHARGE-BASED SEPARATION OF PROTEINS USING POLYELECTROLYTE COMPLEXES AS MODELS FOR MEMBRANE-LESS ORGANELLES Jéré J. van Lente, Mireille M. A. E. Claessens, and Saskia Lindhoud Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.9b00701 • Publication Date (Web): 16 Aug 2019 Downloaded from pubs.acs.org on August 17, 2019

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Biomacromolecules

1

CHARGE-BASED SEPARATION OF PROTEINS

2

USING POLYELECTROLYTE COMPLEXES AS

3

MODELS FOR MEMBRANE-LESS

4

ORGANELLES

5

Jéré J. van Lente 1,2, Mireille M.A.E. Claessens 1, Saskia Lindhoud* 1

6

1

7

Enschede

8

2

9

7522NB Enschede

10

*

Department of Nanobiophysics, University of Twente, Drienerlolaan 5, 7522NB

Membrane Science & Technology Cluster, University of Twente, Drienerlolaan 5,

Corresponding author

11 12

Abstract

1 ACS Paragon Plus Environment

Biomacromolecules 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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Membrane-less organelles are liquid compartments within cells with different solvent

14

properties than the surrounding environment. This difference in solvent properties is

15

thought to result in function related selective partitioning of proteins. Proteins have also

16

been shown to accumulate in polyelectrolyte complexes but whether the uptake in these

17

complexes is selective has not been ascertained yet. Here we show the selective

18

partitioning of two structurally similar but oppositely charged proteins into polyelectrolyte

19

complexes. We demonstrate that these proteins can be separated from a mixture by

20

altering the polyelectrolyte complex composition and released from the complexed by

21

lowering the pH. Combined, we demonstrate that polyelectrolyte complexes can separate

22

proteins from a mixture based on protein charge. Besides providing deeper insight into

23

the selective partitioning in membrane-less organelles, potential applications for selective

24

biomolecule partitioning in polyelectrolyte complexes include drug delivery or extraction

25

processes.

26

Membrane-less organelle(s), polyelectrolyte complex(es), protein(s), protein separation,

27

selectivity


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Biomacromolecules

28 29

Introduction

30

In cells, many (bio)chemical reactions and processes necessary for functioning require

31

environmental conditions that deviate from those in the cytosol. These processes are

32

often performed in specialized compartments called organelles. In some organelles, like

33

the nucleus and the mitochondria, compartmentalization is achieved by membrane

34

encapsulation. Alternatively, cells create micro-environments by inducing liquid-liquid

35

phase separation resulting in the formation of membrane-less organelles (MLOs). For

36

several MLOs the presence of RNA and specific intrinsically disordered RNA-binding

37

proteins (RBPs) with intrinsically disordered regions has been reported to drive phase

38

separation and the formation of MLOs

39

polyelectrolytes; polymeric macromolecules consisting of charged monomeric subunits.

40

RNA is a strong polyanion while RBPs are typically weak polycations 7. For these MLOs,

41

phase separation is driven by complex coacervation 8–10.

1–6.

Both RNA and RBPs are natural



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Although the exact function is not known for all MLOs, specific biological functions

43

typically require the controlled accumulation and release of (bio)molecules

44

Additionally, MLOs need to partition specific compounds with a high degree of specificity

45

as the cytosol contains a large variety of different compounds, many of which share

46

structural and physicochemical similarities. MLO misfunction may lead to undesired

47

biological consequences

48

several neurodegenerative diseases has been reported to drive liquid-liquid phase

49

separation by coacervation. These tau droplets can serve as an intermediate towards the

50

formation of amyloid deposits of tau found in neurodegenerative diseases 16.

51

Since the ability to specifically and dynamically accumulate and release compounds is an

52

emergent property of MLOs it may also be possible to induce this behavior in alternative

53

systems that phase separate via polyelectrolyte complexation. Oppositely charged

54

polyelectrolytes can phase-separate into polyelectrolyte complexes (PECs) in aqueous

55

solution. The properties of PECs consisting of synthetic polyelectrolytes resemble those

56

of MLOs. Several studies have reported that proteins can accumulate in PECs

15.

3,11–14.

For example, the hyperphosphorylation of tau observed in

17–23.


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Biomacromolecules

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However, it is unclear if more complex behavior like the selective accumulation of

58

compounds also emerges in PECs.

59

In this study we investigated the ability of PECs composed of the weak polyelectrolytes

60

poly(acrylic acid) (PAA) and poly(allylamine hydrochloride) (PAH) to dynamically

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discriminate between two oppositely charged protein species, lysozyme and succinylated

62

lysozyme. Previous research has focused on two-component systems containing a

63

protein and an (oppositely charged) polyelectrolyte 24–29. Such systems have been shown

64

to be able to separate proteins by selective interaction with a polyelectrolyte29–32. In these

65

works, a specific protein in a mixture has a higher affinity to the added polyelectrolyte,

66

allowing the specific protein to complexate with the polyelectrolyte into a coacervate,

67

leaving the other proteins in solution. In our system the polyelectrolyte complex is formed

68

by two oppositely charged polyelectrolytes which both interact with the protein, resulting

69

in a three-component system. This allows us to change the ratio between the

70

polyelectrolytes and thus gives us an additional parameter by which we can tune

71

partitioning of proteins into the PECs.



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Lysozyme is a common antimicrobial enzyme that has been reported to partition in a PEC

73

system

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charge at physiological pH with very similar structure 33 to native lysozyme.

75

PAH and PAA are commonly used polyelectrolytes with known phase behaviour. PECs

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of these polyelectrolytes have been observed previously to enrich proteins

77

model systems are less complex compared to MLOs and may help provide a better

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physicochemical understanding of how complex coacervation contributes to intracellular

79

organization. In this study we find that the partitioning of both lysozyme and succinylated

80

lysozyme strongly depends on the PEC composition with maximal protein partitioning into

81

PECs observed at distinct but different charge ratios. At the charge ratio where maximal

82

partitioning is observed the partitioning coefficient remains constant for a range of protein

83

concentrations indicating that the PECs behave as a solvent for the protein. Sharp

84

transitions were observed between complete and no protein partitioning, both as functions

85

of the PEC composition and of the solution pH. We demonstrate that the sharp transitions

86

and difference in PEC composition at which maximal partitioning is observed can be

17.

Succinylated lysozyme is chemically modified to hold an equal but opposite

21,34.

PEC


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Biomacromolecules

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exploited to separate structurally similar proteins of opposite charge from a mixture. We

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suggest that the mechanism responsible for the composition- and pH-dependent

89

partitioning behaviour may be exploited by MLOs.

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Experimental (Materials and Methods)

91

Materials

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Commercially available materials used were poly(acrylic acid) (PAA) (Polysciences, Cat#

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06567, MW = ±6,000), poly(allylamine hydrochloride) (PAH) (Sigma-Aldrich, 283215, MW

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= ±17,500 Da) and lysozyme (Sigma-Aldrich, L6876). Succinylated lysozyme was made

95

as previously described

96

1.00317.1000) or NaOH (Merck, 1.06462.1000). Protein concentrations were determined

97

using UV-Vis at 281.5 nm on a Shimadzu UV-2401PC spectrophotometer, using a

98

molecular extinction coefficient of 2.635 L g-1 cm-1 for both proteins 35.

99

Charge Concentration and ratio

33.

Stock solutions were adjusted to pH 7 - 7.4 with HCl (Merck,



Page 8 of 44

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To determine the charge ratio, both polyelectrolytes were assumed to be fully charged at

101

pH 7. Under this assumption, the charge of any amount of polyelectrolyte is a function of

102

the molecular weight of the composite monomers. Lysozyme and succinylated lysozyme

103

respectively have charges of +7 and -7 at pH 7-7.4

104

defined as:

105

𝐹

―

=

[𝑛 ― ] [𝑛 ― ] + [𝑛 + ]

17,33,36,37.

The charge ratio F- was

#(1)

106

107

were [n-] and [n+] are the negative (PAA) and positive (PAH) charge concentrations

108

respectively

109

charge ratios. The number of charges per polyelectrolyte molecule is a function of

110

monomer weight and remained constant. To change F- the concentration of PAA was

111

kept constant while the concentration of PAH was varied. Variation in the order of addition

112

of the polyelectrolytes did not give different results. Lysozyme partitioning into PECs was

113

evaluated for a range of polyelectrolyte and protein concentrations (supplementary figure

17,28,38.

Different ratios of polyelectrolyte are mixed to result in different F-


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1). From these experiments we decided to continue experimentation with concentrations

115

of 1 g/L PAA and 1 g/L protein.

116

The optimal charge ratio Fopt- was defined as the F- corresponding to the lowest

117

concentration of protein in the supernatant.

118

Protein supernatant measurements

119

Compounds are mixed as follows: first mixtures of like-charged molecule were prepared,

120

then these mixtures were combined, thoroughly vortexed and left to equilibrate for 2 days.

121

Protein concentration was set at 0.8-1 g/L unless otherwise specified. Samples were then

122

centrifuged at 12500 g for 30 minutes. Protein concentration in the supernatant was then

123

determined by measuring the absorbance spectra of appropriately diluted supernatant on

124

a Shimadzu UV-2401PC spectrophotometer as previously described. If supernatant

125

samples showed an absorbance of over 0.01 AU at 400 nm, this was taken as indicative

126

of the presence of dissolved complexes and the sample was discarded as the presence

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of dissolved complexes interferes with the protein concentration determination. Protein

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concentration in the sample supernatant was compared to a control containing only 9 ACS Paragon Plus Environment


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protein, similar to other studies 19. The presence of PAH or PAA had a negligible influence

130

on the protein concentration measurements (supplementary figure 2).

131

For experiments investigating supernatant protein as a function of pH a pH-sensitive

132

electrode (Mettler Toledo, InLab Flex-Micro) was used. Diluted (10 mM) HCl and NaOH

133

were used to adjust the pH to the desired values.

134

Determination of partition coefficient and partition free energy

135

To determine the partition coefficient and free energies, the supernatant protein

136

concentration was measured as described previously. Additionally, the complex mass

137

was calculated by measuring empty sample tubes and sample tubes with the dilute

138

supernatant phase removed. As an approximation, the PEC density was taken as equal

139

to that of water. From this data, the protein concentration in the complex was calculated

140

and the partition coefficient and partition free energies for the systems when equilibrated

141

were calculated via:

142

𝐾𝑝𝑎𝑟𝑡𝑖𝑡𝑖𝑜𝑛 =

[𝑝𝑟𝑜𝑡𝑒𝑖𝑛]𝑐𝑜𝑚𝑝𝑙𝑒𝑥 [𝑝𝑟𝑜𝑡𝑒𝑖𝑛]𝑠𝑢𝑝𝑒𝑟𝑛𝑎𝑡𝑎𝑛𝑡

#(2)


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Biomacromolecules

𝛥𝐺𝑝𝑎𝑟𝑡𝑖𝑡𝑖𝑜𝑛 = ― 𝑅𝑇𝑙𝑛(𝐾𝑝𝑎𝑟𝑡𝑖𝑡𝑖𝑜𝑛)#(3)

143

144

Protein release from PEC

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To evaluate whether protein partitioning was reversible, the ability of the PECs to release

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proteins was investigated using a pH change. First, proteins were partitioned at their

147

optimal charge ratio Fopt- = 0.65 for lysozyme or Fopt- = 0.55 for succinylated lysozyme.

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The supernatant protein concentration was then measured as previously described and

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1 µl of 1 M HCl was added (resulting in a measured pH of approximately 4) to lower the

150

pH. After 2 more days to equilibrate, supernatant protein concentration was measured

151

again. Supernatant protein concentrations were compared to control samples not

152

containing polyelectrolytes.

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Protein analysis on polyacrylamide gel

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Polyacrylamide gel electrophoresis was used to qualitatively distinguish between

155

lysozyme and succinylated lysozyme. For the different steps (A-D) of the protocol shown

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in figure 3A, supernatant samples were frozen at -80 °C until evaluation. A polyacrylamide

157

gel solution consisting of 65% 0.3M tris(hydroxymethyl)aminomethane (Tris) (Merck, 11 ACS Paragon Plus Environment


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1.08382.0500), adjusted to pH 8.5, 10%-acrylamide (Merck, 1.00639.1000), 0.1%

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ammonium persulfate (Bio-Rad, 1610700), and 0.1% Tetramethylethylenediamine

160

(Sigma-Aldrich, T7024) in milliQ water was prepared. A comb was inserted approximately

161

halfway the gel to create sample slots. The solution was left to polymerize for 45 minutes

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under a layer of isopropanol (Merck, 1.09634.1000). Afterwards the isopropanol was

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decanted and leftovers were removed by rinsing the gel with demineralized water.

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Undiluted supernatant was thawed and mixed 1:1 with sample-buffer consisting of 0.12M

165

tris(hydroxymethyl)aminomethane (Tris) (Merck, 1.08382.0500), 20% glycerol (Merck,

166

356350) and 0.02% bromophenol blue (Bio-Rad, 161-0404). Of the sample/sample-

167

buffer mixture 30 µl was transferred to the individual sample slots on the gel. The

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electrophoresis was done at 90 V for 3 hours in running buffer consisting of 26 mM Tris

169

and 192 mM glycine (Sigma, G8898) in milliQ water.

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After electrophoresis, the gel was fixed for 1 hour in a 30% methanol (ATLAS & ASSINK

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CHEMIE, 0360.01.210.5), 10% acetic acid (Merck, 1.00063.1000) solution and then

172

washed with milliQ water for 30 minutes and 1 hour. The gel was left to stain in Imperial


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Biomacromolecules

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Protein Stain (Thermo Scientific, Prod# 24615) overnight before destaining with milliQ

174

water twice for 1 hour. The gel was imaged with a ProteinSimple Fluorchem M and

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ImageJ was used to evenly remove background intensity from the images.

176

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Results

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Protein partitioning depends on PEC composition

179

Intracellular MLOs are able to partition proteins from the cytosol

180

complexes have been reported to do the same

181

lysozyme enrichment in PDMAEMA/PAA PECs is a function of the composition of the

182

PEC F- (equation 1), with maximal partitioning into the PEC at F- = ~0.63 17. To investigate

183

whether enrichment in PECs depends on the protein properties such as the charge of the

184

protein, we investigated the accumulation of lysozyme and chemically modified

185

succinylated lysozyme as functions of F-. Both proteins are structurally nearly identical

186

but carry a net opposite charge at neutral pH

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proteins in PAH/PAA PECs, F- was varied and the amount of protein in the supernatant

33.

17–21.

12.

Polyelectrolyte

We previously reported that

To investigate the enrichment of both



Page 14 of 44

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was measured. In figure 1A, we show images of the PEC-containing samples after

189

centrifugation within sample tubes. The polyelectrolytes have formed a visco-elastic

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dense white solid-like precipitate. In a total volume of 250 µl, the PEC volume makes up

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around 5 µl (2%) with the remaining volume consisting of the dilute supernatant aqueous

192

phase.

193

194

Figure 1. Partitioning of lysozyme (○) and succinylated lysozyme (●) in PAH/PAA PECs.

195

Individual measurements are shown as dots, the lines are drawn to guide the eye. A)

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Images of samples after centrifugation. The numbers in the images correspond to the F-


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Biomacromolecules

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values at which the samples were prepared, indicated by the white numbers. B) Protein

198

in the supernatant as a function of F- at a protein concentration of 0.8 – 1 g/L. Protein

199

concentration in the supernatant is expressed as a percentage of the control system

200

without polyelectrolytes. C) Partition coefficient of the proteins into the PECs at their Fopt-

201

as a function of added protein.

202

203

Figure 1B shows distinct partitioning profiles for lysozyme and succinylated lysozyme

204

between the PEC and dilute supernatant phase. Both proteins show a minimum in the

205

supernatant protein concentration as a function of F-. At this minimum the protein has

206

maximally accumulated in the PEC. For both proteins we also observe an F- region where

207

no partitioning takes place and nearly all protein is found in the supernatant.

208

Interestingly, the partitioning of lysozyme and succinylated lysozyme follows a mirrored

209

pattern. We defined the optimal partitioning charge ratio Fopt- as the charge ratio with

210

maximum protein partitioning into the PEC. Fopt- was determined to be Fopt- = F- = 0.65 for

211

lysozyme and Fopt- = F- = 0.55 for succinylated lysozyme. Note that the optimal partitioning 15 ACS Paragon Plus Environment


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ratio Fopt- for neither lysozyme nor succinylated lysozyme is found at the (calculated) equal

213

net charge of F- = 0.50. The deviation of Fopt- from F- = 0.50 is not explained by the

214

additional charges brought in by the proteins which, when included, would shift the Fopt-

215

of lysozyme to 0.63 and not affect the Fopt- of succinylated lysozyme. If the partitioning

216

was a solely charge-driven process, we would expect maximum protein partitioning at F-

217

= 0.50. That the Fopt- of both proteins deviates from 0.50 indicates that, regardless of the

218

charge of the protein, both polyanions and polycations are required for proteins to

219

accumulate in PECs. This may indicate that the selective partitioning of proteins into

220

PECs is an emergent property of PECs. The necessity for an excess of positive- or

221

negative charges compared to positive charges (i.e. Fopt- not equal to 0.5) has been

222

observed previously for protein-polyelectrolyte systems

223

mechanism has been established. Charge patchiness of the protein and charge

224

regulation phenomena have been suggested as possible reasons 22.

225

If the protein enrichment in PECs was solely governed by charge-charge interactions one

226

would expect the partitioning of lysozyme in PECs to increase with higher values of F-.

28,38,

although no clear


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Biomacromolecules

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However, we observe that the supernatant lysozyme increases at F- values higher than

228

Fopt-. The total PEC mass decreases at high F-, as PAA has less PAH available to form

229

PECs. At high F-, it is likely that smaller soluble PAA-lysozyme complexes form instead.

230

At low F-, the same happens for soluble succinylated lysozyme-PAH complexes.

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In a previous study we have enriched lysozyme in a PDMAEMA/PAA complex coacervate

232

system and observed a 90-95% decrease of the protein in the supernatant phase and

233

concomitant accumulation in the PEC phase.

234

here we report a decrease of 99.8% of lysozyme in the supernatant at a comparable F-

235

(0.65 vs 0.63). Interestingly, Zhao et al. used a similar PAH/PAA system to partition

236

bovine serum albumin (BSA) but only saw a decrease of 50% of the supernatant protein

237

concentration 21. Our experimental findings and the literature combined suggests that the

238

partitioning behaviour of proteins in polyelectrolyte complexes is likely dependent on the

239

structural and physicochemical properties of the polyelectrolytes and the partitioned

240

protein. Future research in which multiple polyelectrolyte and protein systems with

17.

For the PAH/PAA system investigated



Page 18 of 44

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distinctly different properties are evaluated is necessary to elucidate the exact nature of

242

the responsible interactions and mechanisms.

243

Protein partitioning coefficient are protein concentration dependent

244

The PAH/PAA PECs studied here form a separate aqueous phase in which proteins can

245

be localized. The partitioning between the dilute phase and the PEC phase can be

246

quantified by the partitioning coefficient Kpartition (equation 2) which we show as a function

247

of the protein concentration (cprotein) in figure 1C. In this figure two regimes of Kpartition as

248

a function of cprotein are visible. For low cprotein up to 2 - 3 g/L, Kpartition >1,000 were found.

249

At higher cprotein, the Kpartition decreases, presumably because the PEC becomes saturated

250

with proteins. In this regard PAH/PAA PECs behave as normal solvents, despite being in

251

a solid-like phase.

252

The Kpartition values for (succinylated) lysozyme in PEC/Water systems are within range of

253

reported Kpartition values for small molecules such as heptane in octanol/water systems 39–

254

41.

255

18,19,22

Comparable or lower Kpartition are reported for proteins in polypeptide coacervates or in other, synthetic polyelectrolyte systems

23.

Interestingly, BSA completely 18

ACS Paragon Plus Environment

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Biomacromolecules

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partitioned into polypeptide coacervates

257

PAH/PAA PECs

258

polypeptide coacervate system, lysozyme was found to have a noticeable higher

259

maximum Kpartition (~1000) compared to other proteins 22, though this Kpartition was still lower

260

than that for lysozyme in the PAH/PAA PECs. It is important to note that different

261

quantities of PECs and protein concentrations can give an inaccurate partition coefficient

262

if the experimental conditions are not below that of the saturation of the PEC.

263

Like polyelectrolytes, the intrinsically disordered regions of some proteins have been

264

shown to undergo liquid-liquid phase separation

265

MLOs from such proteins and investigated the partitioning of fluorescent proteins into the

266

protein-rich phase. In these phases partition coefficients up to 27 were found, depending

267

on the type of fluorescent protein and any additional protein modification

268

differences in partitioning of proteins between the dilute and coacervate phases of

269

different polyelectrolytes suggest that the exact partitioning properties of systems depend

270

on the polyelectrolyte and protein species.

21.

19

whereas only half of BSA was partitioned in

In one study where multiple proteins were evaluated in the a

42,43.

Schuster et al. prepared model

20.

The



Page 20 of 44

271

The protein partitioning between the PAH/PAA PECs and the dilute supernatant is a

272

passive equilibration process; no active energy-consuming biological mechanism is

273

required to enrich the proteins in the PECs. As such, the accumulation of protein in the

274

PECs is associated with a gain in free energy. At their Fopt-, we report a partition free

275

energy of -20.2 ± 0.3 kJ/mol (mean ± standard deviation, n = 4) for lysozyme and -19.5 ±

276

0.5 kJ/mol (n = 5) for succinylated lysozyme at a protein concentration of 0.8 – 1 g/L

277

(equation 3). In comparison; for a system of phase-separated complexes consisting of

278

disordered regions of proteins, a partition free energy of -8 kJ/mol for single-stranded

279

DNA and 2 kJ/mol for double-stranded DNA was reported 44.

280

Protein partitioning is pH-dependent

281


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Biomacromolecules

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Figure 2. The effect of pH on the partitioning of lysozyme (○) and succinylated lysozyme

283

(●) into PECs. A) PECs are prepared at F- = 0.65 or 0.55 for lysozyme or succinylated

284

lysozyme respectively while the pH of the system is varied. Individual measurements are

285

represented by dots. Lines are drawn to guide the eye. B) Release of proteins from the

286

PECs by lowering the pH from ±7.7 to 4. N = 3.

287

288

In figure 1B we modulated the partitioning of lysozyme and succinylated lysozyme in the

289

PECs by changing the composition in terms of F-. An alternative method to effectively

290

alter F- is by changing the pH of the solution. At low pH values, polyanionic PAA will

291

become less negatively charged while the charge of the polycationic PAH remains

292

unaffected. At high pH values, PAA charge remains unaffected while PAH becomes less

293

positively charged. As a consequence, a pH decrease increases the total negative charge

294

in the complex and is equivalent to lowering the F- via compositional changes and vice

295

versa. Additionally, lysozyme remains positively charged at pH < 10 (pI = 11.35) while

296

succinylated lysozyme undergoes a net charge shift in the evaluated pH range (pI = 4.5)



Page 22 of 44

297

from negative to positive 37. Lysozyme and succinylated lysozyme remain stable at room

298

temperature for pH values as low as 3 and 3.5 respectively

299

suggest that proteins recovered from PECs remain functional 46.

300

To evaluate the effect of pH on the partitioning of the proteins, PAH/PAA PECs were

301

prepared at F- = 0.65 and 0.55 for lysozyme and succinylated lysozyme respectively, at

302

a pH between 4 and 12. In figure 2A we show that the shape of the partitioning curve of

303

the proteins as a function of the pH is similar to the F- dependence shown in figure 1B:

304

for both proteins a region in which none to very little partitioning and a region of maximum

305

partitioning into the PECs is observed. In the presence of lysozyme, at pH > 10, the

306

presence of soluble complexes resulted in light scattering which obscured the

307

measurements and the protein concentration could therefore not be accurately

308

determined.

309

The pH-dependent partitioning of proteins in PECs and the sharp transitions in partitioning

310

as a function of pH and composition offers an interesting strategy to recover proteins from

311

the PECs. This approach was previously shown to work for BSA in polypeptide complexes

33,45.

Earlier studies also


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Biomacromolecules

312

19.

To investigate whether changes in pH also shift the equilibrium distribution and result

313

in the release of lysozyme and succinylated lysozyme from PECs, the systems were first

314

equilibrated at Fopt-. Subsequently, the pH was lowered from ±7.7 to 4, where according

315

to figure 2A the proteins are found in the dilute supernatant phase. Indeed, we show in

316

figure 2B that lowering of the pH recovers all lysozyme and nearly all succinylated

317

lysozyme from the PAH/PAA PECs.

318

PECs are also known to be sensitive to ionic strength. An increase in salt concentration

319

is known to disrupt polyelectrolyte complexes and recover partitioned protein47. Protein

320

release using changes in ionic strength was however found to be a less efficient as

321

lowering the pH (supplementary figure 3). Additionally, the disruption of the complex via

322

salt addition leads to soluble complexes which interfered with the spectroscopic

323

determination of the protein concentration.

324

Protein separation using PECs



Page 24 of 44

325

326

Figure 3: Separation of lysozyme and succinylated lysozyme from a protein mixture

327

containing 1 g/L of both lysozyme and succinylated lysozyme. A) Schematic

328

representation of the experimental procedure. The protein species were qualitatively and

329

quantitatively measured at the points indicated as A1,2 – D1,2. B) Qualitative analysis of


Page 25 of 44 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

330

the protein species present in the supernatant using SDS-PAGE electrophoresis. C)

331

Quantitative UV-vis analysis to determine total supernatant protein concentrations.

332

333

The ability to selectively partition proteins based on F- composition (figure 1B) and release

334

proteins by adjusting the pH (figure 2B) opens up the possibility separate lysozyme or

335

succinylated lysozyme from a mixture of the two in PECs. Figure 1B shows that at the F-

336

for which maximal partitioning into PECs is observed for one protein, the other protein

337

remains in the dilute supernatant phase. We therefore hypothesized that if we start with

338

a mixture of lysozyme and succinylated lysozyme and add polyelectrolytes at Fopt- for one

339

of the proteins, it will selectively partition that protein, while the other protein remains in

340

the supernatant.

341

Following this strategy, we separated a 1:1 mixture of lysozyme and succinylated

342

lysozyme using PAH/PAA PECs via the procedure illustrated in figure 3A. After each step

343

the total protein concentration and composition of the dilute phase was quantitatively and

344

qualitatively investigated by UV-vis (figure 3C) and gel electrophoreses (figure 3B) 25 ACS Paragon Plus Environment


Page 26 of 44

345

respectively.

346

measurement, only one of the proteins was dominantly present in the supernatant, and

347

thus only one of the proteins was present in the PEC. Quantification by UV-vis

348

spectroscopy (figure 3C) shows that the total relative concentration of supernatant protein

349

is either approximately half of the total protein concentration or nearly zero. Taken

350

together, the results show that PAH/PAA PECs can be used to selectively separate either

351

lysozyme or succinylated lysozyme from a mixture of the two proteins.

352

Discussion

353

Previously, single polyelectrolytes have been used to selectively form complexes with

354

proteins from mixtures, resulting in the polyelectrolyte-protein complex forming a separate

355

phase

356

polyelectrolytes PAH and PAA can also separate proteins based on charge. The protein

357

partitioned by the PAH/PAA PEC was found to be dependent on the PEC composition F-,

358

which is a tunable factor. Depending on F-, PAH/PAA PECs can act as selective solvents

359

with high partitioning coefficients for either lysozyme or succinylated lysozyme. From

29–32.

The gel electrophoresis experiments (figure 3B) verified that for each

We have demonstrated that PECs consisting of oppositely charged


Page 27 of 44 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

360

figure 1B and figure 2A, we observe that PAH/PAA PECs have very steep transitions

361

between no partitioning and full partitioning of proteins with very high partition coefficients

362

as a function of PEC composition and solution pH. The exact region of the transitions

363

depended on the charge of the protein and we hypothesize that this region is also

364

dependent on other physicochemical properties of the protein and the constituent

365

polyelectrolytes. We suggest that MLOs in biological systems may have similar steep

366

transitions that can be manipulated by the cell via composition changes or variations in

367

pH. Interestingly, we observed for lysozyme and succinylated lysozyme that maximum

368

partitioning did not occur at F- = 0.5.

369

Cells might be able to alter their MLO composition by manipulating the RNA or RBP

370

concentrations by production, recruitment from other cellular components, or degradation

371

mechanisms. An early model suggests that cells could make such adjustments 48. Protein

372

modifications via phosphorylation, sumoylation and methylation are also known to

373

influence phase separation, providing an additional mechanism for the cells to control

374

MLO solvent properties

44,48–50.

In line with this it has recently been shown that cells are



Page 28 of 44

375

able to regulate the dissolution and formation of specific MLOs during and after mitosis

376

by regulating the presence of certain kinase enzymes

377

primary structure of RBPs may have drastic effects on complex coacervation and solvent

378

properties as they affect the RBP’s charge and isoelectric point. Minor protein

379

modifications may thus result in a steep transition between maximum and no partitioning

380

of proteins. One study where artificial membraneless compartments consisting of

381

customized RNA and synthetic polycations were made showed that enzymes can indeed

382

be partitioned and retain a level of activity in at least partially synthetic complexes 52.

383

The cytosolic pH is generally very tightly regulated to a slightly alkaline (7 – 7.4) value 53.

384

However, figure 2A shows that for PECs, only very slight variations in pH are required to

385

make proteins switch from full to no partitioning in PAH/PAA PECs. Similar steep

386

transitions might be found in MLOs, allowing changes in intracellular pH to influence

387

protein partitioning behaviour. Variation in intracellular pH has been reported to vary

388

depending on the cell’s phase in the cell cycle and exact intracellular location

389

notably, a consistent drop in cytosolic pH from physicological conditions to 5.5 has been

51.

Additionally, changes in the

53,54.

Most


Page 29 of 44 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

390

observed for proliferating yeast

55.

Variations in both more alkaline and acidic directions

391

occur at different phases during mitosis

392

observed to disappear during mitosis and reappear afterwards, while the centrosome and

393

spindle assemblies are MLOs that plays key roles in cell division

394

gradients are present within migrating cells when different functionalities are required

395

within the cell depending on the distance from the migrating leading edge 59.

396

Beyond gaining insight into the discrimination of coacervate phases between proteins

397

based on charge and into mechanisms by which MLOs can regulate protein partitioning

398

in the cell, we suggest possible applications. For these applications it is important to

399

realize that PECs behave as solvents. Understanding the factors that influence the

400

partitioning behaviour of these tunable aqueous solvents may open new directions for the

401

extraction and concentration of molecules from wastewater streams. Partitioning for

402

various small molecules from solution has been reported 60–62. The same principle is worth

403

investigating for other compounds.

56,57.

Interestingly, several MLOs have been

51,58.

Additionally, pH



Page 30 of 44

404

Another field where PECs might be promising is controlled drug delivery 27,63,64, especially

405

with the possibility of a triggered release system

406

suggested that PECs can show reduced cytotoxicity compared to free drug

407

have tunable drug release rate based on environmental pH 67.

408

Conclusion

409

Membrane-less organelles have the ability to partition intracellular proteins and act as an

410

additional organizing mechanism for the regulation of intracellular processes

411

ability to selectively partition the desired protein(s) while excluding other cytosolic

412

compounds is essential for MLO functioning. Polyelectrolyte complexes have been shown

413

previously to be able to enrich a variety of proteins from solution into PECs 17–21, but the

414

ability to selectively partition proteins starting from a mixture using tunable PECs

415

consisting of oppositely charged polyelectrolytes had not yet been shown. In this study

416

we showed that a high degree of selectivity is possible based on protein net charge, even

417

when the proteins are otherwise structurally very similar.

65.

Early-stage experimentation has 66

and can

3,11–14.

The


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Biomacromolecules

418

Finally, beyond insight into MLOs, intracellular regulation, and potential new avenues to

419

explore diseases, more direct applications of the ability of PECs to selectively and tunably

420

partition proteins, biomolecules or other organic compounds can be found in waste- or

421

surface water treatment and in drug delivery systems.

422

423

ASSOCIATED CONTENT

424

Supporting Information

425

The Supporting Information is available free of charge on the ACS Publications website

426

Figure S1: Influence of PAA and PAH on absorbance spectra

427

Figure S2: Effect of total polyelectrolyte- and protein concentration

428

Figure S3: Release of Proteins from PECs via salt addition

429

430

Corresponding Author



Page 32 of 44

431

Dr. Saskia Lindhoud

432

[email protected]

433

Author Contributions

434

JvL conducted experiments and processed results. MC and SL were responsible for

435

supervision. SL directed the study. All authors contributed to experimental design,

436

manuscript writing and editing.

437

Acknowledgements

438

The authors acknowledge funding from the Netherlands Organization for Scientific

439

Research (NWO) VENI (# 722.013.013) and VIDI (# 723.015.003) grants.

440 441

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Biomacromolecules

74x44mm (300 x 300 DPI)



Figure 1. Partitioning of lysozyme (○) and succinylated lysozyme (●) in PAH/PAA PECs. Individual measurements are shown as dots, the lines are drawn to guide the eye. A) Images of samples after centrifugation. The numbers in the images correspond to the F- values at which the samples were prepared, indicated by the white numbers. B) Protein in the supernatant as a function of F- at a protein concentration of 0.8 – 1 g/L. Protein concentration in the supernatant is expressed as a percentage of the control system without polyelectrolytes. C) Partition coefficient of the proteins into the PECs at their Fopt- as a function of added protein.


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Biomacromolecules

Figure 2. The effect of pH on the partitioning of lysozyme (○) and succinylated lysozyme (●) into PECs. A) PECs are prepared at F- = 0.65 or 0.55 for lysozyme or succinylated lysozyme respectively while the pH of the system is varied. Individual measurements are represented by dots. Lines are drawn to guide the eye. B) Release of proteins from the PECs by lowering the pH from ±7.7 to 4. N = 3.



Figure 3: Separation of lysozyme and succinylated lysozyme from a protein mixture containing 1 g/L of both lysozyme and succinylated lysozyme. A) Schematic representation of the experimental procedure. The protein species were qualitatively and quantitatively measured at the points indicated as A1,2 – D1,2. B) Qualitative analysis of the protein species present in the supernatant using SDS-PAGE electrophoresis. C) Quantitative UV-vis analysis to determine total supernatant protein concentrations.


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CHARGE-BASED SEPARATION OF PROTEINS USING

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