Phase Separation Behavior of Supercharged Proteins and

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Phase separation behavior of supercharged proteins and polyelectrolytes Chad S. Cummings, and Allie C. Obermeyer Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00990 • Publication Date (Web): 06 Dec 2017 Downloaded from http://pubs.acs.org on December 13, 2017

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Biochemistry

Phase separation behavior of supercharged proteins and polyelectrolytes Chad S. Cummings, Allie C. Obermeyer* Department of Chemical Engineering, Columbia University, New York, NY 10027, United States

*Corresponding author: Allie C. Obermeyer; email: [email protected]; phone: 212-8531315

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ABSTRACT

Membraneless organelles, like membrane-bound organelles, are essential to cell homeostasis and provide discrete cellular sub-compartments. Unlike classical organelles, membraneless organelles possess no physical barrier, but rather arise by phase separation of the organelle components from the surrounding cytoplasm or nucleoplasm. Complex coacervation, the liquidliquid phase separation of oppositely charged polyelectrolytes, is one of several phenomena that is hypothesized to drive the formation and regulation of some membraneless organelles. Studies to examine the molecular properties of globular proteins that drive complex coacervation are limited as many proteins do not complex with oppositely charged macromolecules at neutral pH and moderate ionic strength. Protein supercharging overcomes this problem and drives complexation with oppositely charged macromolecules. In this work, several distinct cationic supercharged green fluorescent protein (GFP) variants were designed to examine the phase behavior with oppositely charged polyanionic macromolecules. Cationic GFP variants phase separated with oppositely charged macromolecules at various mixing ratios, salt concentrations, and pH values. Efficient protein incorporation in the macromolecule rich phase occurred over a range of protein and polymer mass fractions, but protein encapsulation efficiency was highest at the midpoint of the phase separation regime. More positively charged proteins phase separated over broader pH and salt ranges than proteins with lower charge density. Interestingly, each GFP variant phase separated at higher salt concentrations with anionic synthetic macromolecules compared to anionic biological macromolecules. Optical microscopy revealed that most variants, depending on solution conditions, formed liquid-liquid phase separations, except for GFP/DNA pairs which formed solid aggregates at all tested conditions.


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Biochemistry

INTRODUCTION

Similar to classical membrane-bound organelles, membraneless organelles help maintain cell homeostasis by compartmentalizing important reactions and partitioning cellular components. However, membraneless organelles, such as P granules, nucleoli, and stress granules, possess no boundary layer and behave like liquid droplets that rapidly assemble and disassemble in response to changes in the cellular environment.1 Recent discoveries have indicated that proteinpolyelectrolyte complexes, in the form of complex coacervates, are important to the formation and function of membraneless organelles2–4 and could contribute to disease pathology.5,6 Complex coacervation, a type of liquid-liquid phase separation of oppositely charged macromolecules, is one of several phenomena, including simple coacervation and cytoplasmic clustering, that are critical to the formation and function of these non-membrane-bound organelles.7,8 These same protein-polyelectrolyte coacervates can also be incorporated into materials used for numerous applications including therapeutics,9,10 sensors,11,12 and diagnostics.13 When complexed with another macromolecule, protein stability to extreme pH,14 temperature,15 or solvent conditions16 is improved compared to non-complexed protein. Despite the biological relevance and potential applications, the design parameters for complex coacervation of globular proteins are not well established. Protein-polyelectrolyte phase separation is initiated by the electrostatic attraction of oppositely charged molecules followed by thermodynamically favorable entropic gains associated with surface bound counter-ion release.17 Following complexation, protein-polyelectrolyte mixtures can phase separate creating a macromolecule rich and a macromolecule dilute phase. The nature of the phase separation, liquid-liquid, liquid-solid, or a combination of both, depends on the concentration of water, polyelectrolyte, and salt.18–20 Liquid-liquid phase separations can 3 ACS Paragon Plus Environment

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coalesce over time and exhibit rapid exchange of their macromolecular components. In some situations, it is possible for liquid protein-polyelectrolyte phase separations to transform into more solid-like precipitates over time.6,21 The protein and polymer molecular properties that govern whether a liquid-liquid or liquid-solid phase separation will form are not yet fully understood. While globular protein complexation with oppositely charged polyelectrolytes provides many advantages, many native proteins often do not phase separate at neutral pH and moderate ionic strength. One way to drive complexation and phase separation over a wider range of pH and ionic strengths is through protein supercharging,22 which increases the overall charge density on the protein surface. Protein supercharging modifies the surface properties of globular proteins while maintaining the folded structure, and can be accomplished through chemical23,24 or biological protein modification techniques.25,26 Common amino acid targets for chemical supercharging include glutamic acid, aspartic acid, and lysine due to their charge and chemical reactivity. Beneficially, many of these amino acids are commonly on the surface of the protein, which enables a large effect on net surface charge following modification. For biological methods of supercharging, these same amino acids are also frequently targeted for mutation as well as other polar, surface accessible residues (e.g. Asn, Gln, Thr). After supercharging, proteins are more likely to form complexes with oppositely charged macromolecules and less likely to self-aggregate.27–30 We are interested in understanding how the molecular properties of proteins affect complex coacervation and phase separation. Specifically, we are interested in understanding how protein surface charge effects the nature of phase separation. In order to determine the basic molecular parameters for globular protein phase separation with polyanions, the phase separation behavior


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Biochemistry

of supercharged superfolder green fluorescent protein (sfGFP) with both synthetic and biological macromolecules was investigated. Green fluorescent protein was chosen as a well characterized and diagnostically relevant model protein.25,31–33 Four anionic macromolecules, poly(acrylic acid) (PAA), poly(styrene sulfonate) (PSS), deoxyribonucleic acid (DNA), and ribonucleic acid (RNA), were chosen as representative synthetic and biological polyanions. These polymers were selected to incorporate both biological and synthetic polyanions as well as both strong (PSS, DNA, RNA) and weak (PAA) ionizable groups. We selected biological and synthetic polyanions as it is important to consider how secondary structure in biological macromolecules might alter the phase behavior when compared to unstructured synthetic macromolecules.

The phase

separation behavior of the GFP/polyanion mixtures was examined using an initial turbidimetry screen over a broad range of protein/polyanion mixing ratios. The nature of the phase separated state, either liquid or solid, was determined using optical and fluorescence microscopy. The effect of pH and salt concentration on protein-polymer phase separation was determined for each of the twenty protein-polyanion mixtures, and GFP incorporation in the dense phase was determined. These experiments provide insight in to how protein surface charge and polyanion structure affect phase separation behavior. Phase separation with biological polyanions occurred at a wider range of mixing ratios, while phase separation with synthetic polyanions occurred at wider salt and pH ranges. At the midpoint of the two phase region, phase separation of most GFP/polyanion pairs was solid-like, but the state of the mixtures was predictably modified by varying mixing ratios or solution ionic strength. By integrating the phase behavior of each GFP/polyanion pair, it was possible to elucidate molecular characteristics that were predictive of the phase separation behavior of this model system. MATERIALS AND METHODS


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Materials and general methods Low dispersity poly(styrene sulfonate) (PSS) (Mn = 26,500, Ð=1.03) and poly(acrylic acid) (PAA) (Mn=26,500 g/mol, Ð=1.20) were purchased from Polymer Source, Inc. (Montreal, Quebec, Canada). Double stranded deoxyribonucleic acid (DNA) sodium salt from Oncerhynchus keta (D1626) and type VI ribonucleic acid (RNA) (total RNA – single and double stranded) from Torula yeast (R6625) were purchased from Sigma Aldrich. NiCo21 (DE3) competent cells were purchased from New England Biolabs (Ipswich, MA, USA). All other chemicals were purchased from Fisher Scientific (Pittsburgh, PA, USA). All chemicals were used as received. Absorbance and fluorescence measurements were taken on either an Infinite M200 Pro plate reader (Tecan Group Ltd., Mannedorf, Switzerland) or a Cary 60 ultraviolet/visible spectrophotometer (Agilent Technologies, Santa Clara, CA). Optical microscopy images were collected using an EVOS FL Auto 2 imaging system equipped with a GFP (470/22 nm) LED light cube and a 20X 0.4NA Plan Fluor objective (Invitrogen, Carslbad, CA, USA). GFP cloning Amino acid mutations for each of the GFP variants were based on amino acid mutations previously reported for supercharged GFP.25 Specific mutations for each variant were selected by viewing the crystal structure of GFP (PDB: 2B3P) and selecting uniformly distributed residues for each variant (superfolder GFP amino acid sequence and amino acid mutations for each GFP variant included in the Supporting Information, Table S1). Mutagenesis and sub-cloning into an expression vector (pET) was completed by GenScript (Piscataway, NJ, USA). Prior to protein expression, DNA plasmids were transformed into NiCo21 (DE3) and sequence verified. Protein expression and purification


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GFP was overexpressed in NiCo21 (DE3) cells in LB media. Prior to induction, cells were grown (37 ºC, 200 rpm) to an OD600 between 0.8-1.0. Expression of GFP was induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and cells were grown for 16-20 h between 16-37 ºC (37 ºC: sfGFP, GFP (+2), GFP (+8), GFP (+14); 28 ºC: GFP (+20); or 16 ºC: GFP (+26)). Cells were harvested by centrifugation (4000 rpm, 10 min), re-suspended in cell lysis buffer containing 50 mM sodium phosphate (pH 8.0) and 1 M sodium chloride (NaCl), and stored at -20 ºC until cell disruption. Immediately prior to cell lysis by sonication, RNase A (225 µg per L culture) and DNase I (200 µg per L culture) were added to re-suspended cells. Cells were lysed in an ice bath using probe tip sonication for 10 min (2 s pulse on, 4 s pulse off). Following sonication, the soluble fraction was separated from insoluble components by centrifugation (8000 rpm, 45 min). GFP was purified from other soluble components using NiNTA agarose chromatography under native conditions (wash buffer: 50 mM sodium phosphate, 300 mM NaCl, 35 mM imidazole (pH 8.0), elution buffer: 50 mM sodium phosphate, 300 mM NaCl, 250 mM imidazole (pH 8.0)). Purified GFP was concentrated by centrifugal ultrafiltration (molecular weight cut off (MWCO) 10 kDa) and buffer exchanged into 10 mM Tris (pH 7.4) by dialysis (MWCO 3.5 kDa). GFP was stored in solution at 4 ºC until use. Sample preparation The concentration of GFP was determined by measuring the absorbance at 488 nm (ε = 83,000 L mol-1 cm-1).34 GFP solutions were stored at 1 mg mL-1 in 10 mM Tris (pH 7.4) at 4 ºC in the dark. Polymers were stored as solids at room temperature (PAA, PSS), 4 ºC (DNA), or -20 ºC (RNA) and dissolved at 1 mg mL-1 in 10 mM Tris (pH 7.4) just prior to use. Because dissolution of PSS, PAA, and RNA resulted in a large decrease in solution pH, the pH was adjusted to 7.4


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using NaOH prior to experimentation. For DNA, solution pH was constant at 7.4 even after polyanion dissolution, so no adjustment was necessary. Turbidimetry assay GFP and polyanion solutions were dissolved in 10 mM Tris (pH 7.4) at 1 mg mL-1. GFP and polyanion solutions were mixed together at protein mass fractions ranging from 0 to 1 in increments of 0.04, corresponding to different charge fractions for each GFP/polyanion mixture. Absorbance (λ=600 nm), as a marker of phase separation in GFP/polyanion mixtures, was measured in triplicate after 15 seconds of shaking in tissue culture treated polystyrene 96 well half area plates (Corning) using a plate reader. Charge fraction in mixtures was calculated by converting protein mass fraction to charge fraction using the predicted GFP charge as calculated by PROPKA. Percent transmittance (%T) was calculated from absorbance (A) using the formula A = -log(%T). Additionally, time between sample preparation and analysis was constant for each GFP/polyanion pair (t < 10 min). Encapsulation assay Encapsulation of GFP in the dense phase of GFP/polyanion mixtures was measured at mixing ratios determined from initial turbidity analysis. GFP and polyanions were dissolved at 1 mg mL1

in 10 mM Tris (pH 7.4) and mixed at a minimum of five protein/polymer ratios centered on the

midpoint of the two phase region. Phases were separated by centrifugation of the GFP/polyanion solutions for 10 min at 12,500 rpm. Supernatant was removed, and the pellet was re-dissolved in an equal volume of 10 mM Tris (pH 7.4) with 1 M NaCl. A high concentration of salt was required to dissolve the phase separated pellet into a homogenous mixture. The dense phase of PSS and GFP(+20) or GFP(+26) did not dissolve at 1 M NaCl. For these samples, the dense phase was diluted an additional 10-fold in order to obtain a homogeneous solution prior to


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Biochemistry

fluorescence and absorbance measurements. GFP concentration was measured in the dilute and dense phases by measuring both fluorescence (λex =488 nm; λem=510 nm) and absorbance (λ=488 nm). Fluorescence and absorbance measurements were converted to concentrations (mg mL-1) using a calibration curve made for each GFP variant in 10 mM Tris (pH 7.4) with 1 M NaCl. Salt (NaCl) titration Dependence of the GFP/polyanion phase separation on the salt concentration was determined by titrating NaCl into phase separated mixtures. Both GFP and polyanions were dissolved at 1 mg mL-1 in 10 mM Tris (pH 7.4). GFP/polyanion mixtures were prepared at the protein mass fraction corresponding to the midpoint of phase separation in bulk turbidity experiments (specific ratios, Table S2). Percent transmittance (%T) of the phase separated mixtures (25 ºC, 850 rpm in Peliter cuvette holder) was measured after each addition of 10 mM Tris (pH 7.4) with 6 M NaCl. Measurements were taken until %T stabilized for 10 additions. Effect of pH on phase separation Dependence of the GFP/polyanion phase separation on pH was determined by titration with sodium hydroxide (NaOH). Both GFP and polyanions were dissolved at 1 mg mL-1 in 10 mM Tris (pH 7.4). GFP/polyanion mixtures were combined at the protein mass fraction corresponding to the midpoint of phase separation in bulk turbidity experiments (Table S2). Percent transmittance (%T) of the mixtures was measured after each addition of 1 M (points far away from critical pH) or 0.1 M (points close to critical pH) NaOH. The pH of the solution was concurrently measured by a microelectrode placed in the cuvette. Samples were kept at 25 ºC using a Peltier cuvette holder and stirred at 850 rpm. Microscopy


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GFP and polyanions were dissolved at 0.2 mg mL-1 or 1 mg mL-1 (GFP(+2)) in 10 mM Tris (pH 7.4), and mixed together in a glass bottomed 384 well plate (Nunc) and incubated for 16 h at 25 ºC. GFP/polyanion mixtures were mixed at the protein mass fraction corresponding to the midpoint of phase separation in bulk turbidity experiments (specific ratios found in Table S2). A few additional mixing ratios, centered around this midpoint, were also investigated. Images were acquired at 20X magnification in both GFP (λex =470/22 nm, λem=510/42 nm) and phase contrast channels. Dynamic light scattering (DLS) Z-average particle size of GFP/polyanion complexes was measured using dynamic light scattering for GFP(+8), GFP(+14), GFP(+20), and GFP(+26) with RNA and for GFP(+14) with all polyanions. Samples were prepared at 1 mg mL-1 total macromolecule concentration and mixed at varying negative charge fractions (from 0.0-1.0). For each sample, DLS measurements were conducted at 25 °C, repeated in triplicate, and involved collection of 10 independent measurements of 10 seconds each. Error bars indicate standard deviation of the three measurements.

RESULTS AND DISCUSSION Protein supercharging can be used to study and understand the molecular properties of globular proteins required for in vitro and in vivo complex coacervation.35–37 In order to determine the effect of protein surface charge on phase separation with oppositely charged macromolecules, we designed and expressed five engineered cationic variants of superfolder green fluorescent protein (sfGFP). With unmodified sfGFP32 as a template (expected net charge, -4), 3-5 amino acid point mutations were selected for each successive cationic GFP variant. This resulted in a panel of five


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Biochemistry

cationic GFP variants with expected net charge of +2, +8, +14, +20, and +26 (Figure 1a) as predicted by the computational tool PROPKA, which predicts the pKa values of ionizable groups in proteins based on the folded structure.38,39 Nomenclature used for the supercharged GFP variants, GFP(+2), GFP(+8), GFP(+14), GFP(+20), and GFP(+26), corresponds to the calculated net charge for each of the variants.

Figure 1. (a) Expected net charge of green fluorescent protein (GFP) variants as calculated using PROPKA. (b) Electrostatic surface potential representations of the solvent accessible surface area for GFP variants used in this study as calculated using the linearized Poisson-Boltzmann equation with Adaptive Poisson-Boltzmann Solver (ABPS) (±10 kBT/e, blue = positive and red = negative). Each specific mutation for the supercharged variants used in this study was selected from a library of 29 total mutations incorporated in a previously designed supercharged sfGFP,25 which


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has an expected net charge of +35, according to PROPKA calculations. Since it is possible for charge patches on proteins to affect phase separation behavior,40 the crystal structure of sfGFP (PDB: 2B3P) was used as an aid to select point mutations in order to maintain an isotropic charge distribution for each variant (Figure 1b). Each amino acid modification replaced either an anionic or neutral amino acid with a cationic amino acid (lysine or arginine). In many cases, a negative residue was removed, resulting in a net charge difference of +2, while other mutations replaced a neutral amino acid, resulting in a net +1 difference per mutation. In order to maintain an equal (+6) charge difference between each supercharged GFP variant, each successive variant contained 3-5 additional mutations from the previous variant depending on the specific amino acids that were mutated. The GFP variants were expressed in E. coli41 and purified using affinity chromatography. All supercharged GFP variants expressed at a moderate level (>10 mg/L and up to 80 mg/L culture), but expression levels decreased as the expected GFP net charge increased. SDS-PAGE analysis revealed the proteins were of high purity and confirmed that the molecular weight of the variants was similar to native sfGFP (Figure S1). Proteins with increasing positive charge density showed slightly lower electrophoretic mobility due to the increase in charge. After purification, GFP concentration was calculated by measuring the absorbance at 488 nm (ε = 83,000 L mol-1 cm-1). Additionally, it was confirmed that the chromophore in each supercharged GFP variant was predominantly in the anionic phenolic state that absorbs at 488 nm (Figure S2).33 Deoxyribonucleic acid (DNA), ribonucleic acid (RNA), poly(acrylic acid) (PAA), and poly(styrene sulfonate) (PSS) were selected as model polyanions to examine phase separation behavior of positively supercharged GFP variants with oppositely charged macromolecules. PAA and PSS both had narrow polydispersity (Ð = 1.2 and 1.03 respectively), and nominal


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Biochemistry

molecular weights (Mn = 26.5 kDa) similar to sfGFP (28 kDa). Double stranded DNA (1.3 x 106 Da, ~2000 bp)42 isolated from Oncerhynchus keta and total RNA (double and single stranded) from Torula yeast were obtained from commercial suppliers. Turbidity at 600 nm was used to evaluate the bulk phase separation properties of each GFP/polyanion pair. All five supercharged GFP variants were mixed separately with all four anionic polymers at a total macromolecule concentration of 1 mg mL-1 in 10 mM Tris (pH 7.4), resulting in a total of 20 GFP/polyanion pairs. Phase separation of each of the mixtures was measured over a broad range of protein mass fractions, and mass fraction was converted to negative charge fraction using the formula: = fraction, M

-

is total negative charge (

∗

charge (

∗

!"#$ "%& !#' ).

, where f

)

-

is the negative charge

and M+ is total positive

Each of the positively charged GFP variants phase

separated with oppositely charged polyanions at a wide range of negative charge fractions (Figure 2). It was expected that the phase separation would be centered on a charge fraction of 0.5 to achieve charge neutrality. For each of the GFP/polyanion pairs, the midpoint was shifted to higher negative charge fractions (more polyanion). This shift indicates formation of the phase separated state favored excess polyanion or complexation with polymer resulted in protein charge regulation.22 As a control, turbidity of native sfGFP/polyanion pairs was also measured. Not surprisingly, since native sfGFP is negatively charged (expected net charge -4), sfGFP/polyanion pairs resulted in no detectable phase separation over all GFP mass fractions. However, when sfGFP was mixed with polycations (poly(allylamine) and poly(quaternized 4vinylpyridine) it did phase separate and demonstrated a shift to higher positive charge fractions (more polycation, Figure S3). The biological polyanions, DNA and RNA, demonstrated a 13 ACS Paragon Plus Environment

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broader range of phase separation than the synthetic polyanions, PAA and PSS. This effect could be due to the narrow dispersity of the synthetic polymers as the DNA and RNA used here have a broad range of molecular weights. As the protein surface charge increased, the midpoint of turbidity shifted to lower negative charge fractions (more protein), irrespective of the polyanion (Table S2). Since GFP(+2) has an equal number of positive and negative residues, the phase separation of GFP(+2) with all polyanions was unexpected as proteins with similar charge density generally do not phase separate with oppositely charged macromolecules.22 Therefore, we hypothesized that a charge patch on the surface of GFP drives complexation and phase separation with polyanion macromolecules. For PAA and PSS, the midpoint of GFP(+2) phase separation occurred approximately at the same negative charge fraction as the more highly charged variants, but, had higher turbidity. The turbidity of GFP(+2) was expected to be lowest of all the variants due to weak complexation with oppositely charged polyanions because of the relatively low protein surface charge. However, this unpredicted behavior could arise from the experimental pH being most similar to the isoelectric point of GFP(+2) or an artifact due to the non-quantitative relationship between turbidity and fraction phase separated.


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Biochemistry

Figure 2. Bulk phase separation properties of GFP and polyanion mixtures. Turbidity profiles of cationic GFP charged variants with (a) poly(acrylic acid), (b) poly(styrene sulfonate), (c) DNA, and (d) RNA. Complexes were mixed at varying protein and polymer ratios (total polyelectrolyte concentration 1 mg mL-1) in 10 mM Tris (pH 7.4). Top x-axis corresponds to sfGFP/polyanion pairs (black open circle), while the bottom x-axis corresponds to all cationic GFP variants. The hydrodynamic diameter of GFP/polyanion mixtures correlated well with turbidity profiles (Figure S4). At mixing ratios where phase separation occurred, GFP/polyanion mixtures formed micron size aggregates as measured by dynamic light scattering. At charge fractions outside the observed increased turbidity region, GFP(+14) formed ~50-200 nm complexes with both PAA and PSS (Figure S4b), indicating soluble complexes formed even in the absence of increased solution turbidity. The narrow size of the non-phase separated region precluded this analysis for RNA and DNA complexes with GFP. 15 ACS Paragon Plus Environment

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While it was clear that each of the GFP/polyanion mixtures phase separated at a broad range of protein mass and charge fractions, it was not apparent whether the nature of the phase separation was liquid-liquid, liquid-solid, or a mixture of the two. In order to determine the state of the phase separation, GFP/polyanion pairs were mixed at ratios corresponding to the midpoint of turbidity from the initial turbidity screens (specific ratios, Table S2). Microscopy experiments were conducted at a macromolecule concentration five times lower than initial bulk turbidity screens, due to the brightness of GFP. Conveniently, the phase separation behavior (solid-liquid or liquid-liquid) exhibited at this lower concentration was equivalent to that at 1 mg mL-1 as determined using phase contrast microscopy (data not shown). Evaluation of the phase behavior by microscopy indicated that almost all the GFP/polyanion pairs underwent a liquid-solid phase separation, or precipitation, at the midpoint of the phase separated region (Figure 3). Solid precipitates can be visualized as opaque non-uniform aggregates when observed using optical microscopy, and solid phase separation can result in complex formation that is not reversible.19 Conversely, liquid-liquid phase separation results in the formation of optically clear, spherical droplets that are dynamic in nature. While most GFP/polyanion pairs phase separated as solids, each pair still had high levels of fluorescence in the phase separated state indicating the structure of GFP was not greatly affected during phase separation. Interestingly, several of the RNA/GFP pairs (GFP(+2), (+14), and (+20)) formed a liquid coacervate upon phase separation at the optimal charge fraction. With the observation that these GFP/RNA pairs had lower GFP mass fractions, we hypothesized that the GFP/synthetic polyanion pairs may also phase separate as liquids at lower protein mass fractions. Indeed, at lower mass fractions of GFP, both GFP/PSS and GFP/PAA pairs formed liquid phase separated coacervate droplets, while at higher protein mass fractions, phase separation was still as a solid (Figure S5). This trend indicates that mass


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Biochemistry

fraction of protein may be more important than charge neutrality when predicting liquid-liquid versus liquid-solid phase separation. DNA phase separated as a solid with each GFP variant at all tested mixing ratios, which is attributed to the double stranded nature of the genomic DNA used in this study.43,44

Figure 3. Nature of phase separation for GFP/polyanion mixtures. Microscopy images of GFP/polyanion mixtures showing the formation of a liquid coacervate (labeled L) or precipitate (unlabeled). Samples were mixed at 0.2 mg mL-1 total macromolecule concentration (except for GFP(+2)/polyanion pairs: 1 mg mL-1) and incubated at 25 ºC for 16 hours prior to imaging. Scale bar 25 µM. Even though the absolute turbidity values and phase morphology were different for each GFP/polyanion pair, encapsulation of GFP in the macromolecule rich phase was greater than 90


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percent for most of the GFP/polyanions pairs at the optimal protein/polymer mixing ratio determined by turbidity (Figures 4 and S6). To determine the amount of GFP incorporation in the polyelectrolyte rich phase, GFP/polyanion pairs were mixed at 1 mg mL-1 total macromolecule concentration at several ratios centered on the midpoint of phase separation for each pair, and centrifugation was used to separate the two phases. Following separation, it was clear that, for most GFP/polyanion pairs, there existed a macromolecule dense phase with a high concentration of GFP and a macromolecule dilute phase with a low concentration of GFP. The concentration of GFP in the dense phase was estimated to be at least 10-20 mg/mL for most GFP/polyanion pairs (Figure S7). The only exception was for GFP(+2)/polyanion pairs, which showed modest to moderate levels (30-80%) of GFP incorporation in the dense phase. Encapsulation of GFP in the dense phase for each of the GFP/polyanion pairs was highest at the optimal mixing ratio and as either additional protein or polymer was added the encapsulation efficiency decreased. Similar to the turbidity profiles, the biological macromolecules displayed a broader range of high GFP encapsulation compared to synthetic polyanions. This broad range of encapsulation is hypothesized to arise from the higher molecular weight of the biological polyions as well as the increased dispersity of these poly(nucleotides). Away from peak encapsulation, the degree of GFP incorporation in the dense phase fell more drastically at lower negative charge fractions (more GFP) compared to higher negative charge fractions (less GFP). At negative charge fractions higher than the optimal charge fraction, polyanion was in excess in the mixture, which likely permitted all GFP to be encapsulated with excess polymer in the macromolecular dilute phase. Conversely, at negative charge fractions lower than the midpoint of encapsulation, GFP was in excess compared to polyanion, which resulted in the dilute phase containing a larger fraction of GFP.


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Figure 4. Encapsulation efficiency of cationic GFP variants in the coacervate or precipitate phase with (a) poly(acrylic acid), (b) poly(styrene sulfonate), (c) DNA, and (d) RNA. Protein and polymer were mixed in 10 mM Tris (pH 7.4) at ratios centered on the midpoint of phase separation (f

–

midpoint)

as measured by bulk turbidity experiments. The fraction of GFP phase

separated is plotted as a function of the charge fraction relative to the midpoint. Titration of NaCl into the model protein/polyelectrolyte mixtures was used to determine the critical salt concentration for the phase separation of each GFP/polyanion pair. GFP and polyanion were mixed at a total macromolecule concentration of 1 mg mL-1 in 10 mM Tris (pH 7.4) with 0 M NaCl. Transmittance (%T) was measured after stepwise additions of 6 M NaCl in 10 mM Tris (pH 7.4) until the GFP/polyanion pairs fully dissolved (> 90% T). Increasing the net charge on GFP variants resulted in phase separation with the polyanions at increasing salt


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concentrations (Figures 5 and S8). A control experiment examining the addition of 10 mM Tris (pH 7.4) with no salt resulted in no difference in turbidity values up to 20 vol% addition of salt free titrant (Figure S9). Interestingly, dissolution of each GFP/polyanion pair with the synthetic polymers, PAA and PSS, occurred at higher salt concentrations than with the biological macromolecules. The effective salt concentration range of phase separation for each GFP/polyanion pair was hypothesized to be dependent on the net charge of GFP when comparing across GFP variants and on the pKa of the polymer ionizable groups when comparing across polyanions. While it was clear net charge of the protein was important for predicting the effect of salt on phase separation, the pKa of the polyanion charged group did not appear to have an effect on the critical salt concentration for phase separation. For DNA, RNA, and PSS their pKa value is ~1,45,46 while PAA is between 4-5.47 Thus, we hypothesized PAA would dissolve at the lowest salt concentration compared to other polyanions, but in actuality DNA and RNA dissolution occurred at the lowest salt concentrations. This fact indicates that additional factors, including macromolecule length, dispersity, charge density, or hydrophobicity, beyond polyanion pKa could be important for GFP/polyanion complex salt stability.48 Since both DNA and RNA are biological macromolecules and possess varying degree of secondary structure, the structural organization of the polyanion may also contribute to the differences in GFP/polyanion complex salt stability more so than the polyanion pKa. Additionally, the high molecular weight and large dispersity of these biological macroions could play a role in their dissolution at relatively low ionic strengths. Some GFP/polyanion pairs, most notably GFP/PAA, exhibited a plateau in solution turbidity at intermediate salt concentrations before completely dissolving. We hypothesized this plateau could correspond to a transition from solid-liquid phase separation to liquid-liquid phase


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separation. To test this hypothesis, GFP(+14)/PAA and GFP(+20)/PAA pairs were imaged at varying salt concentrations centered on the plateau. Phase separation behavior was examined for GFP/PAA pairs when pre-mixed at a constant NaCl concentration (Figure S10) and when initially mixed with 0 mM NaCl followed by titration of salt (Figure S11). GFP/PAA pairs initially mixed with no salt and then subjected to NaCl titration did exhibit phase separation behavior similar to what was observed during larger scale mixing, indicating that the observed plateau most likely corresponds to a solid to liquid phase transition. This was also confirmed for an additional polyanion/protein pair (RNA/GFP(+26), Figure S12). For both tested mixing conditions, GFP/PAA pairs underwent liquid-liquid phase separation rather than solid-liquid at higher NaCl concentration. However, the transition to a liquid-liquid phase separated state occurred at lower salt concentrations for premixed GFP/PAA pairs compared to GFP/PAA pairs titrated with NaCl. The difference in the salt concentration required to form liquid-liquid phase separations for each mixing regime indicates phase separation behavior of GFP/polyanion pairs is dependent on mixing conditions, a result consistent with previous studies.19,49


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Figure 5. Effect of salt concentration on GFP/polyanion phase separation. GFP and (a) poly(acrylic acid), (b), poly(styrene sulfonate), (c) DNA, and (d) RNA (total polyelectrolyte concentration 1 mg mL-1) were mixed in 10 mM Tris (pH 7.4) at the ratio corresponding to the midpoint of phase separation from bulk turbidity experiments. Percent transmittance (%T) was measured after small additions of 10 mM Tris (pH 7.4) with 6 M NaCl.


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Similar to the behavior seen for each of the GFP/polyanion pairs at different salt concentrations, increasing the net GFP charge resulted in phase separation over a broader pH range (Figure 6). Since there should be no electrostatic driving force for complexation at pH values above each supercharged GFP’s isoelectric point (pI), it was predicted that the GFP/polyanion complexes would form a single miscible phase at the pI of each variant. Surprisingly, for all of the GFP variants except GFP(+2), dissolution of the complex occurred at a pH greater than the calculated pI (Figure S13), indicating that phase separation occurred between two polyelectrolytes with nominally the same net charge. The phase separation of two like charged polyelectrolytes has been observed previously,50,51 but it is also possible that complexation of the polyanion results in charge regulation22,52 of the protein. Polymer complexation may increase the apparent pKa values of ionizable groups on the protein, leading to dissolution at pH values above the expected protein pI. For GFP(+2)/polyanion pairs, all mixtures showed complete dissolution at pH values lower than the pI of GFP(+2), providing support for the weaker association between GFP(+2)/polyanion pairs.


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Figure 6. Effect of pH on GFP/polyanion phase separation. Turbidimetric titration of phase separated mixtures of cationic GFP with (a) poly(acrylic acid), (b) poly(styrene sulfonate), (c) DNA, and (d) RNA. Samples were prepared in 10 mM Tris buffer and percent transmittance and pH were measured after small additions of NaOH. GFP/polyanion phase separations were mixed at the midpoint of phase separation determined by bulk turbidity. In order to develop protein complex coacervation as an effective strategy for applications in drug delivery and enzyme catalysis, protein/polymer complexes must, depending on the 24 ACS Paragon Plus Environment

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application, phase separate at a specific pH, temperature, and salt concentration. Since many properties of proteins and polyelectrolytes affect the phase separation behavior, it is essential to understand how molecular properties impact phase separation at different solution conditions. The phase behavior of the panel of proteins tested here enabled the identification of important molecular parameters that predict phase behavior. Several protein molecular properties were screened for the ability to predict phase separation of the mixtures with respect to solution pH and salt concentration. While not equaling the exact pH of GFP/polyanion complex dissolution, protein isoelectric point (pI, as calculated using PROPKA) was predictive of the critical pH value, defined as the point where turbidity reached 50% - halfway between complete dissolution (0%) and maximum complexation (100%), for each of the four polyanions (Figure 7a). Rather than pI, the positive-to-negative amino acid residue ratio was capable of predicting of the critical salt concentration ([NaCl] at 50% turbidity) for each variant (Figure 7b). Although, in the case of predicting the critical salt concentration, PSS demonstrated significantly different dependence on this ratio compared to the three other polyanions, which we hypothesized arose from the low dispersity, synthetic nature, and low pKa of ionizable groups of PSS. Each of these factors tend to argue for uniform complexation from one complex to another which could explain the surprisingly high salt stability. Other protein and polymer molecular properties including total positive residues, positive-negative residue difference, surface charge density, expected protein charge, and polymer pKa were less reliable as predictive factors for either the critical pH or critical salt concentration (Tables S3 and S4, Figures S14 and S15).


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Figure 7. Predictive indicators of phase separation conditions. (a) Correlation between predicted protein isoelectric point (pI) and critical pH for phase separation and (b) correlation between ratio of protein positive-to-negative residues and critical salt concentration for phase separation. CONCLUSION In this work, amino acid point mutations were used to create five distinct positively supercharged sfGFP variants. Each of the supercharged GFP variants phase separated when mixed with the polyanions DNA, RNA, PAA, and PSS. Phase separation encompassed a broader solution charge fraction range for the biological polyanions, DNA and RNA, compared to the synthetic polyanions, PSS and PAA. Each GFP variant phase separated over a wide range of pH and salt concentrations, with increasing net charge on the GFP variants increasing the range over which phase separation was observed. Opposite of the behavior observed with respect to charge fraction, DNA and RNA phase separated over narrower pH and salt concentration ranges compared to PAA and PSS. From this work, we gained valuable insight into the molecular properties of proteins and polyanions that affect globular protein phase separation. Most GFP/polyanion pairs phase separated as solid precipitates at the midpoint of the phase separated region, but most of the pairs phase separated as liquid-liquid at lower mass fractions of proteins. The exception was for mixtures with DNA, which formed solid precipitates at all tested


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conditions. The ability of relative macromolecule concentration in this model system to drive liquid-liquid or liquid-solid phase transitions may provide insight into the aberrant solidification process observed in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS).21,53 Additionally, at physiologically relevant salt concentrations the solid precipitates transitioned to a liquid coacervate, behavior that is reminiscent of membraneless RNA/protein bodies. Using this panel of model proteins and polymers, predictive parameters were established for the critical pH and salt concentration for globular protein phase separation with polyanions. The predicted isoelectric point of the protein strongly predicted the critical pH values of GFP/polyanion pairs, while the ratios of positive-to-negative amino acids were correlated with the critical salt concentrations. Each of these relationships can be used to design future protein-polymer phase separation mixtures for applications ranging from protein and nucleic acid delivery to enzyme compartmentalization and stabilization.

ASSOCIATED CONTENT Supporting Information. Additional experimental information can be found in the supporting information including: specific amino acid mutations, SDS-PAGE gel, absorbance scans of GFP variants, specific turbidity midpoint ratios of all variants, full salt dissolution curves for GFP/PSS, optical images of GFP/polyanion pairs at varying protein mass fractions and salt concentrations, phase separated concentration calculations, linear fits of predictive (or nonpredictive molecular properties), representative size measurements of GFP/polyanion pairs, turbidity profile of sfGFP with polycation, non-normalized encapsulation profiles, and relative pH dissolution curves. AUTHOR INFORMATION 27 ACS Paragon Plus Environment

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Corresponding Author *Allie Obermeyer, [email protected], 500 W. 120th St. New York, NY 10027 Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT Funding Information The work was supported by the Fu School of Engineering and Applied Sciences at Columbia University. REFERENCES (1) Guo, L., and Shorter, J. (2015) It’s Raining Liquids: RNA Tunes Viscoelasticity and Dynamics of Membraneless Organelles. Mol. Cell 60, 189–192. (2) Sleeman, J. E., and Trinkle-Mulcahy, L. (2014) Nuclear bodies: new insights into assembly/dynamics and disease relevance. Curr. Opin. Cell Biol. 28, 76–83. (3) Strom, A. R., Emelyanov, A. V., Mir, M., Fyodorov, D. V., Darzacq, X., and Karpen, G. H. (2017) Phase separation drives heterochromatin domain formation. Nature 547, 241–245. (4) Larson, A. G., Elnatan, D., Keenen, M. M., Trnka, M. J., Johnston, J. B., Burlingame, A. L., Agard, D. A., Redding, S., and Narlikar, G. J. (2017) Liquid droplet formation by HP1α suggests a role for phase separation in heterochromatin. Nature 547, 236–240. (5) Ambadipudi, S., Biernat, J., Riedel, D., Mandelkow, E., and Zweckstetter, M. (2017) Liquid– liquid phase separation of the microtubule-binding repeats of the Alzheimer-related protein Tau. Nat. Commun. 8, 275. (6) Jain, A., and Vale, R. D. (2017) RNA phase transitions in repeat expansion disorders. Nature 546, 243–247. (7) Wei, M.-T., Elbaum-Garfinkle, S., Holehouse, A. S., Chen, C. C.-H., Feric, M., Arnold, C. B., Priestley, R. D., Pappu, R. V., and Brangwynne, C. P. (2017) Phase behaviour of disordered proteins underlying low density and high permeability of liquid organelles. Nat. Chem. 9, 1118– 1125. (8) Feric, M., Vaidya, N., Harmon, T. S., Mitrea, D. M., Zhu, L., Richardson, T. M., Kriwacki, R. W., Pappu, R. V., and Brangwynne, C. P. (2016) Coexisting Liquid Phases Underlie Nucleolar Subcompartments. Cell 165, 1686–1697. (9) Blocher, W. C., and Perry, S. L. (2017) Complex coacervate-based materials for biomedicine. Wiley Interdiscip. Rev.: Nanomed. Nanobiotechnol. 9, doi: 10.1002/wnan.1442. 28 ACS Paragon Plus Environment

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Phase Separation Behavior of Supercharged Proteins and

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