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1 DOI: /NCHEM.1110 Peptide nucleotide microdroplets as a step towards a membrane-free protocell model Shogo Koga 1,2, David S. Williams 1, Adam W. Perriman 1 & Stephen Mann. 1 1 Centre for Organized Matter Chemistry, School of Chemistry, University of Bristol, Bristol BS8 1TS, UK. s.mann@bris.ac.uk 2 Mitsubishi Chemical Group Science and Technology Research Centre Inc., 1000 Kamoshidacho, Aoba-ku, Yokohama , Japan Full Methods (online version) Preparation of peptide/nucleotide droplets All chemicals were purchased from Sigma and used without further purification; oligo- and polylysines were received as the bromide salt unless otherwise stated. Milli-Q water (18.2 MΩ cm) was used to prepare all aqueous solutions. Typically, micro-droplet dispersions were prepared at room temperature by adding an aqueous solution of sodium adenosine 5 / - triphosphate (ATP) ( mm) to the same volume of an aqueous solution of oligo-α-llysine (OLys) peptides with molecular weights of 0.5-2k or 1-5kDa corresponding to chain lengths of 2-10 and 5-24 monomer units, respectively, and a monomer concentration between 0.4 and 50 mm, depending on the molar ratio used. The ph was adjusted to 8 by addition of a small amount of aqueous 1 M NaOH. Similar preparations were carried out with ATP but using poly-α-l-lysine (PLys) with a molecular weight of 24 kda (ca. 115 monomer units) in place of the short chain peptides. Other nucleotides including AMP, ADP, CTP, GTP, UTP, TTP, datp or dctp were also used to prepare peptide/nucleotide droplets. In general, the peptide/nucleotide droplets were separated and collected from the aqueous suspension as a bulk phase by centrifugation at 5,000 rpm for 15 min. Turbidimetric measurements The influence of nucleotide concentration, ph and temperature on the formation of peptide/nucleotide droplet suspensions was investigated by monitoring the changes in absorption intensity (turbidity) at a fixed wavelength, λ = 500 nm. Most data were recorded at room temperature using a UV/Vis spectrometer (Perkin Elmer Lambda25 UV/VIS Spectrometer) equipped with a Peltier temperature controller (Perkin Elmer PTP-6). The ATP concentration dependence of droplet formation was measured for a 4 mm OLys or PLys NATURE CHEMISTRY 1

2 solution at ph 8 by progressively increasing the nucleotide concentration from 0.4 mm by addition of small amounts of a 10 mm nucleotide solution until the turbidity value (100-%T, where %T is transmittance) reached 100. A small amount of 10 mm peptide solution was also added simultaneously to offset the decrease in concentration caused by addition of the nucleotide solution. The following nucleotides were used: AMP, ADP, ATP, CTP, GTP, UTP, TTP, datp or dctp. The PLys concentration dependence over a range of 1 to 5 mm was also investigated at ph 8 using increasing ATP concentrations from 0 to 1.2 mm. The ph dependence of droplet formation between ph values of 0.8 to 12 was measured for solutions containing 4 mm PLys and 4 mm nucleotide (AMP, ADP, ATP, CTP, UTP, TTP and GTP) using 1M HCl and 1M NaOH aqueous solutions. The ph dependence of droplet formation was also performed with 10 mm OLys and ATP. Droplet imaging Coacervate samples of OLys/ATP or PLys/ATP droplets were prepared from equal volumes of 20 mm solutions and imaged using optical microscopy (DMI3000B, Leica). The dispersion of coacervate droplets was maintained at 60 C for about 30 min prior to observation at room temperature. Methylene blue and Nile red staining of the droplet interiors was used to enhance the image contrast, determine the molecular weight dependence of the number-averaged diameter, and confirm sequestration of these dyes into the peptide/nucleotide compartments. Light scattering measurements All measurements were performed using a Malvern Zetasizer Nano-ZS instrument equipped with an internal Peltier temperature controller. Formation of OLys/ATP coacervate droplets was confirmed at ph 8 by dynamic light scattering (DLS) measurements on a 4 mm OLys solution by increasing the [ATP] with 10 mm ATP solution (10 mm OLys solution was used to maintain constant [OLys]). Thermal stability of the coacervate droplets from 25 to 90 ºC was confirmed by DLS measurements on dispersions that contained 4 mm PLys or OLys and 4 mm ATP or CTP at ph 8. The ph stability of OLys/ATP coacervate droplets was also confirmed by DLS measurements of a 10 mm OLys (0.5-2k or 1-5kDa) and 10 mm ATP with small quantities of 4 M HCl/NaOH used to adjust the ph. NATURE CHEMISTRY 2

3 Structural studies Structural studies of the PLys/ATP coacervates prepared at ph 8 from mixing equal volumes of 50 mm ATP and 50 mm PLys solutions were undertaken using small angle X-ray scattering (SAXS). The coacervate phase was isolated by centrifugation at 5,000 rpm for 15 min and then samples were prepared for SAXS by injecting the sample into a thin glass capillary (cross-sectional diameter = 1.5 mm). SAXS measurements were conducted using a small angle scattering apparatus operating at 40 kv, 0.2 ma (Cu K radiation, Å) with a graphite monochromator) using a two dimensional position-sensitive detector (200 mm square, manufactured by Molecular Metrology) located at 0.8 m from the sample. The magnitude of the observed scattering vector Q ranged from 0.04 to 0.4 Å -1. Circular dichroism measurements Circular dichroism (CD) spectroscopy was undertaken to confirm the presence of peptide secondary structures in coacervate droplets prepared from 50 mm PLys (24 kda; HCl salt, Sigma) and 50 mm ATP. The chloride salts were used to reduce the level of anion adsorption in the CD experiments. All CD measurements were performed at 25 ºC using a JASCO J-815 spectropolarimeter fitted with a Peltier temperature controller. Coacervate droplets prepared at a variety of ph values were measured by isolating the bulk phase by centrifugation at 5,000 rpm for 15 min, and then placing the viscous material between two quartz plates to make a thin liquid coacervate film. CD spectra of PLys aqueous solutions were also measured. For this, 4 mm PLys aqueous solution was adjusted to the desired ph value using small quantities of 1M NaOH and then 200 μl was inserted into a 1 mm path length quartz cuvette for measurement. Composition and density studies PLys/ATP coacervates were prepared at [PLy] = 50 mm and [ATP] = 50 mm, at ph 8 and then the coacervates were collected as a bulk phase by centrifugation at 5,000 rpm for 15 min. The water content of the bulk coacervate phase was evaluated from the weights of the coacervate before and after air-drying in a convection oven (MOV-112F, SANYO) at 65 o C for 48 hr and subsequent cooling in a desiccator. This procedure was repeated 3 times and the average value was calculated. The PLys/ATP molar ratio of the coacervate phase prepared at [PLys] = 25 mm and [ATP] = 6.25, 12.5, 25 or 50 mm ([PLys]/[ATP] molar ratio = 4, 2, 1 or NATURE CHEMISTRY 3

4 0.5) at ph 5 and 8 were determined by measurement of the free and total ATP concentration using UV-Vis absorption spectroscopy (Perkin Elmer Lambda25). Droplets were isolated by centrifugation at 5,000 rpm for 15 min to produce a coacervate bulk liquid phase, and the ATP concentration in the aqueous clear supernatant ([ATP] S ) was evaluated from the intensity of the optical absorbance at 259 nm using the molar extinction coefficient of ATP ( M -1 cm -1 ). The number of ATP molecules binding per monomeric unit of the peptide (β) was obtained from the relationship, was the total ATP concentration. ([ATP] T [ATP] )/[PLy], where [ATP] T S Droplet assembly/disassembly by ph cycling using CO 2 / NH 3 gas bubbling A turbid dispersion of peptide/nucleotide droplets prepared at [PLys] = [ATP] = 10 mm and ph 8 was adjusted to ph 12,5 by addition of 1M NaOH to produce a clear solution of disassembled droplets. Carbon dioxide gas was then bubbled through the solution until the ph had dropped to a value of 7.2 and turbidity was re-established due to re-assembly of the peptide/nucleotide droplets. Subsequent bubbling of NH 3 gas through the solution produced a clear solution at ph = 11.5 and disassembly of the protocells by deprotonation of the PLys side chains. Cycles of sequential NH 3 and CO 2 gas bubbling, which resulted in concomitant growth and decay of the protocells, could be maintained provided that that the increasing ionic strength associated with the presence of ammonium carbonate was reduced intermittently by dialysis. For this, 2 ml of the gas-treated PLys/ATP solution was then mixed with 4 ml Milli-Q water in a centrifugal filter tube (Amicon Ultra-4, molecular weight cut-off 3kDa, Millipore) and centrifuged until the volume of the mixture was reduced to the original volume (2 ml) of the PLys/ATP solution. Use of NaOH in the initial step of the cycle was required to limit the buffering effect of [NH + 4 ] ions during changes in ph. Partitioning of dye molecules and metal nanoparticles 200 μl of the supernatant equilibrium solution phase of a PLys/ATP coacervate droplet dispersion, prepared by mixing equal volumes (750 μl) of equimolar (50 mm) solutions of ATP and PLys at ph 8, was removed post-centrifugation and replaced with 200 μl of a 1 mm aqueous solution of methylene blue (cationic, pk a >12), fluorescein (anionic, pk a = 6.4) or anionic porphyrin derivatives (chlorophyllin, protoporphyrin IX). Similar experiments were undertaken but with an aqueous solution of polysaccharide-coated inorganic nanoparticles (dextran-fe 3 O 4 (Dex-Fe 3 O 4 ), carboxymethyl-dextran (CMDex)-Fe 3 O 4, or NATURE CHEMISTRY 4

5 CMDex-Co 3 O 4 or Dex-Co 3 O 4, ph = 5) or citrate-coated gold nanoparticles added in place of the organic dye. Partitioning of gold nanoparticles in droplets prepared using oligolysine/atp droplets with a peptide molecular weight of 0.5-2k was also investigated by TEM. In each case, the droplets were separated from the equilibrium solution by centrifugation at 5,000 rpm for 15 min, and the concentrations of the added solute/nanoparticles in the coacervate and bulk aqueous phase determined by optical absorbance measurements at 674 nm (methylene blue), 645 nm (chlorophyllin), 470 nm (protoporphyrin IX), 375 nm (Dex-Fe 3 O 4 and CMDex-Fe 3 O 4 nnaoparticles) 403 nm (Dex- Co 3 O 4 and CMDex-Co 3 O 4 nanoparticles) or 543 nm (citrate-stabilized gold nanoparticles). Solute/nanoparticle concentrations in the coacervate phase were determined by disassembling the peptide/atp complex by adding 10 µl of the isolated coacervate to 1 ml of 0.5 M NaCl aqueous solution, followed by UV-vis spectroscopic analysis of the clear solution taking into account the dilution ratio. The partition coefficient (K) was determined as K = C COA /C s, where C COA and C S were the concentrations of the solute or nanoparticles in the coacervate and equilibrium solution phases, respectively. Partitioning of hexokinase The partition constant (K) for the sequestration of HK into the peptide/nucleotide droplets was determined using separate enzymatic rate assays that were carried out on the equilibrium aqueous solution phases isolated by centrifugation from a mixture containing PLys (24kDa), ATP and HK. The concentration of HK in the external aqueous phase ([HK] out ) was determined from the enzymatic reaction rate and the volume of the continuous phase (V out ). Given the total number of units of HK added to the system ([HK] total V total ), K was determined from the relationships; K = [HK] in /[HK] out, and [HK] in = ([HK] total V total [HK] out V out )/V in where [HK] in and V in are the concentration of HK in the coacervate droplet and the total volume of the coacervate phase. Spectroscopic analysis was undertaken on the equilibrium water phases rather than by direct measurement on the coacervates because of the background turbidity associated with the peptide/nucleotide droplets. Furthermore, equilibrium water phases rather than pure water NATURE CHEMISTRY 5

6 were used to determine the value of [HK] total in order to calibrate against any changes in the reaction rates due to the presence of ATP and trace levels of aqueous PLys. In each case, the ATP and PLys concentrations were set at a constant value so that differences in reaction rate correlated directly with changes in the concentration of HK. The value for [HK] out was determined as follows. A solution containing 40 mm ATP and ~1 units/ml HK (1 unit = 1 µmole/min) was prepared by mixing equal volumes of aqueous solutions containing 80 mm ATP (ph 8, adjusted with 1M NaOH) or ~2 units/ml HK. 750 μl of the mixture was added to the same volume of an aqueous solution of PLys (40 mm, 24 kda) to form a coacervate phase (V total = 1500 µl). The equilibrium aqueous solution phase and coacervate droplet phase were isolated by centrifugation at 5,000 rpm for 15 min. About 15 μl of the coacervate phase was isolated (V in = 15 µl and V out = 1485 µl). The equilibrium water phase was diluted with the same volume of water to give a final enzyme concentration equivalent to half of [HK] out, and then 50 µl was added into a buffered reaction mixture containing excess levels of substrate (glucose), co-factors ([Mg(ATP)] 2-, β- NADP) and glucose 6-phospate dehydrogenase (G-6-PDH) (Table 1), and the time dependence of the concentration of the product (β-nadph) in the continuous water phase determined by monitoring the absorption at 340 nm. The corresponding slope of the plot was used to determine the rate of HK-catalysed glucose phosphorylation in the equilibrium water phase. Experiments to establish the linearity between reaction rate and [HK], as well as the value of [HK] total were also undertaken. A solution containing 40 mm ATP was prepared by mixing equal volumes of an aqueous solution of 80 mm ATP and water. 750 μl of the mixture was added to the same volume of an aqueous solution of PLys (40 mm, 24 kda) to form a coacervate phase. The equilibrium aqueous solution phase was isolated by centrifugation and then diluted with the same volume of ~0.5 unit/ml HK solution, which was prepared from ~2 units/ml HK solution (as used above), to give a final enzyme concentration (0.25 units/ml) equivalent to half of [HK] total in the experiment undertaken to determine [HK] out as described above. Alternatively, the equilibrium aqueous solution phase was isolated by centrifugation and then diluted with the same volume of ~0.25 units/ml HK solution to provide a further concentration-dependent data set to assess the linearity of the system. In both cases, 50 µl of the HK-containing solution was added into a buffered reaction mixture as described above. NATURE CHEMISTRY 6

7 Table 1. Composition of reaction mixture Solution Volume / ml Total concentration a) 50 mm Tris buffer, ph 7.8 at 25 o C mm b) 555 mm D-Glucose mm c) 40 mm ATP at ph mm d) 500mM MgCl mm e) 14 mm -NADP mm f) ~ 120 U/mL G-6-PDH 0.02 ~ 1 U /ml Dielectric constant measurements Preferential sequestration of the water insoluble dye, Nile red, into the peptide/atp droplets was used to estimate the dielectric constant ( ) of the protocell interior. Droplets containing Nile red molecules were prepared by mixing 750 μl of 50 mm ATP aqueous solution with an equal amount of 50 mm PLys aqueous solution and 20 µl of a concentrated Nile red/dmso (dimethyl sulfoxide) solution (~2 mm). The ph value was adjusted to 3, 5, 7, 9 or 11 by addition of 1 M HCl or NaOH. The coacervate phase was isolated by centrifugation at 5,000 rpm for 15 min and then examined using UV-Vis reflectance spectroscopy (Lambda 35 with integrating sphere RSA-PE-20, Perkin Elmer). The dielectric constant of the droplet interior was estimated from the absorbance peak position and compared with those for Nile red in dodecane (500 nm, = 2), DMSO (552 nm, = 47.2), and for micromolar concentrations of Nile red in water (591 nm, = 80) assuming a linear relationship between dielectric constant and absorption wavelength. Intra-droplet self-assembly of porphyrin aggregates Preferential sequestration of protoporphyrin IX into the peptide/atp droplets was used to investigate the possibility of producing supramolecular organic assemblies within the protocell interior. PLys/ATP coacervate droplets containing protoporphyrin IX were prepared by mixing 200 μl of a 1 mm aqueous solution of the porphyrin with 200 μl of a coacervate solution prepared by mixing equal volumes (750 μl) of equimolar (50 mm) solutions of ATP and PLys. The ph was adjusted to 4, 8 or 10 by addition of 1 M HCl or NaOH and the porphyrin-containing droplets isolated by centrifugation at 5,000 rpm for 15 min and then examined using UV-Vis reflectance spectroscopy. NATURE CHEMISTRY 7

8 Nanoparticle-mediated catalysis Nanoparticle-mediated peroxidase reactions [A. Asati et al. Angew. Chem. Int. Ed., 48, (2009)] were undertaken by sequestration of CMDex-Co 3 O 4 nanoparticles into PLys/ATP droplets prepared by mixing 750 µl of 50 mm ATP aqueous solution with an equal amount of 50 mm PLys aqueous solution and 100 µl of CMDex-Co 3 O 4 nanoparticle aqueous dispersion (7.2 mg/ml). The ph value was adjusted to 5 by addition of 1 M NaOH. The kinetics of the nanoparticle-mediated peroxidation were monitored in PLys/ATP droplets suspended in 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) aqueous solution. The samples were prepared by mixing 2.4 ml of citrate-buffered (ph 5) 0.04 mm ABTS solution with 100 µl of a coacervate aqueous dispersion containing 50 mm PLys and 100 µl of 40 mm ATP solution ([PLy] = 1.9 mm and [ATP] = 1.5 mm in the mixed solution). The increase in absorbance at 410 nm as a function of the elapsed time after addition of 20 µl of CMDex-Co 3 O 4 nanoparticle aqueous dispersion (7.2 mg / ml) was recorded by UV-Vis spectroscopy. The intensity data were corrected for changes in turbidity associated with the complexation of the oxidized ABTS species with PLys. (No background changes were observed in the CMDex-Co 3 O 4 -free or PLys-free systems). For this, the increase in the background absorption was determined from measurements at an absorbance wavelength of 472 nm. The contribution of the turbidity change in the absorbance time profile at 410 nm was eliminated assuming that the total turbidity change at 410 nm was 0.12 (same as that at 472 nm) and the rate of turbidity change was proportional to the concentration of product (oxidized ABTS; molar extinction coefficient = 25.5 x 10 3 M -1 cm -1 ). The absorbance at 410 nm after the elimination of turbidity contribution (Abs*(t)), was estimated by the measured absorbance value (Abs(t)) and a relationship of Abs ( t 8min) Abs ( t 0) 0.12 Abs *( t) ( Abs ( t) Abs ( t 0)) Abs ( t 0) Abs ( t 8min) Abs ( t 0) (Abs(t = 0) = and Abs (t = 8min) = 1.418, so [Abs(t = 8min) Abs(t=0)- 0.12]/[ Abs(t = 8min)-Abs(t=0)] = This equated to 62.7 % of the absorbance change at 410 nm being assigned to the ABTS oxidization and 37.3 % assigned to the turbidity change due to the formation of complexes between oxidized ABTS and PLys. Confirmation that peroxidase activity was specifically associated with the coacervate phase was established as follows. A nanoparticle-containing coacervate phase prepared was prepared in water and isolated by centrifugation at 5,000 rpm for 15 min. The equilibrium aqueous phase was then replaced with 0.04 mm ABTS citrate-buffered (ph 5) aqueous NATURE CHEMISTRY 8

9 solution. Oxidation of ABTS specifically within the bulk phase of the centrifuged coacervate phase was confirmed by the colour change of the coacervate phase from brown (colour of CMDex-Co 3 O 4 nanoparticle) to blue-green colour (oxidized ABTS). Enzymatic reactions in coacervate droplets Determination of the activity of the ATP-dependent enzyme hexokinase (HK) in the presence and absence of PLys in aqueous solution, as well as in the presence of PLys/ATP coacervate droplets, was carried out by using a coupled assay with an excess of glucose-6-phosphate dehydrogenase (G-6-PDH) according to the reaction scheme; Hexokinase Glucose ATP Glucose- 6 - phosphate ADP, G-6-PDH Glucose- 6 - phosphate β - NADP 6 - Phospho- D - Gluconate β - NADPH. Enzyme activity in the presence of peptide/nucleotide droplets was undertaken using the following solution composition (Table 2): 19 mm triethanolamine buffer (ph = 7.8 at 25 o C); 211 mm D-glucose; 0.72 mm ATP at ph 8 (by addition of 1 M NaOH solution); 1.9 mm PLys; 5.8 mm MgCl 2 ; 1.1 mm β-nicotinamide adenine dinucleotide phosphate, oxidized form (β-nadp); ~1 unit G-6-PDH from baker's yeast. All enzymatic reactions were carried out at 25 o C with a total concentration (Units/mL) of G-6-PDH x50 times higher than that of HK such that glucose phosphorylation was the rate determining step in the coupled assay. The above mixture was left for 3 min and then HK from Saccharomyces cerevisiae was added to start the reaction (composition of HK in the reaction mixture ~0.02 units/ml). The increase in absorbance at 340 nm as a function of the elapsed time after HK addition was recorded by UV-Vis spectroscopy, and the data converted to a time profile of β-nadph production using the molar extinction coefficient of β-nadph (6.22 x 10 3 M -1 cm -1 ). Background turbidity changes associated with possible complexation between tetra-anionic β-nadph and PLys were accounted for by determining the general increase in the background absorption during the enzyme reaction. A linear relationship between the amplitude of the increase in background signal as a function of wavelength, was derived from the least square fitting to the absorbance values in the wavelength region from 500 to 600 nm. The contribution of the total turbidity change in the absorbance time profile at 340 nm was 0.37 at 3 min. Thus, the absorbance at 340 nm after the elimination of turbidity contribution (Abs*(t)) assuming that the rate of turbidity change was proportional to the concentration of NATURE CHEMISTRY 9

10 product (β-nadph), was estimated by the measured absorbance value (Abs(t)) and a relationship of Abs ( t 3min) Abs ( t 0) 0.37 Abs *( t) ( Abs ( t) Abs ( t 0)) Abs ( t 0) Abs ( t 3min) Abs ( t 0) (Abs(t = 0) = and Abs (t = 3min) = 1.310, so [Abs(t = 3min) Abs(t=0)- 0.37]/[ Abs(t = 3min)-Abs(t=0)] = This equated to 62.2 % of the absorbance change at 340 nm being assigned to the β-nadph production and 37.8 % assigned to the background absorption change. Studies were also undertaken under low turbidity conditions to obtain more accurate values of the reaction rates using the following solution composition prior to the addition of HK as described above (Table 3): 19 mm triethanolamine buffer (ph = 7.8 at 25 o C); 211 mm D-glucose; 0.36 mm ATP at ph 8 (by addition of 1 M NaOH solution); 2.8 mm PLy; 5.7 mm MgCl 2 ; 1.1 mm β-nicotinamide adenine dinucleotide phosphate, oxidized form (β- NADP); ~1 units/ml G-6-PDH from baker's yeast. Table 2. Composition of reaction solutions for coacervation effect observation Solution With coacervates / ml Without coacervates / ml a) 50 mm Tris buffer, ph 7.8 at 25 o C 1 1 b) 555 mm D-Glucose 1 1 c) 19 mm ATP at ph d) 50 mm Polylysine e) Distilled water f) 100 mm MgCl g) 14 mm -NADP h) ~ 120 U/mL G-6-PDH Table 3. Composition of reaction solutions for pre-coacervation effect observation Solution with coacervates / ml without coacervates / ml a) 50 mm Tris buffer, ph 7.8 at 25 o C 1 1 b) 555 mm D-Glucose 1 1 c) 19mM ATP at ph d) 50 mm Polylysine e) Distilled water f) 100mM MgCl g) 14 mm -NADP h) ~ 120 U/mL G-6-PDH NATURE CHEMISTRY 10

11 Supporting Information: Figures Figure S1. Plots showing the turbidity increase associated with the formation of PLys (4 mm)/nucleotide coacervate droplets. Nucleotides: ATP (closed circles), datp (open squares), ADP (closed diamonds), GTP (closed squares), UTP (open circles), TTP (open triangles), CTP (closed triangles), dctp (open diamonds), and AMP (crosses, far right panel). Figure S2. Dynamic light scattering (DLS) data for coacervate droplets prepared from 4 mm oligo-lys (0.5-2kDa, 2-10 monomer units) and various ATP concentrations at 0.15 mm (circles), 0.5 mm (squares), 1 mm (triangles) and 4 mm (diamonds). Main plots show correlation data with particle size distribution (by intensity) viewed in top right inset. NATURE CHEMISTRY 11

12 Figure S3. DLS data showing size and temperature stability of coacervate droplets. Measurements were taken at 25 ºC (open circles), after heating to 90 ºC (open squares), or after cooling from 90 ºC to 25 ºC (closed circles). Droplets were prepared from PLys (24kDa) with ATP (a) or CTP (b), oligo-lys (1-5kDa) with ATP (c) or CTP (d), oligo-lys (0.5-2kDa) with ATP (e) or CTP (f). All concentrations = 4 mm. NATURE CHEMISTRY 12

13 Figure S4. Plots showing concentration of ATP inside ([ATP coac ], closed circles) and outside ([ATP super ], open circles) PLys/ATP droplets as a function of the total ATP concentration ([ATP total ]) at a [PLys] = 25 mm and ph values of 5 (left panel) and 8 (right panel). Figure S5. (Left panel) Turbidity measurements showing ph stability range for PLys/nucleotide droplets. [PLys] = [nucleotide] = 4 mm. Nucleotides: ATP (closed circles), datp (formation only; open squares), ADP (closed diamonds), GTP (closed squares), UTP (open circles), TTP (open triangles), CTP (closed triangles), dctp (formation only; open diamonds) and AMP (crosses). (Right panel) Plot of DLS relative scattering intensities (closed symbols) and number average diameter (open circles) for oligolysine/atp droplets showing stability field across a range of ph values. Peptides; kda, 2-10 monomer units (squares), (1-5 kda), 5-24 monomer units (circles). Disassembly of the droplets occurs at ph values of and 2-3 in both cases. NATURE CHEMISTRY 13

14 Figure S6. Images showing the stability of a PLys/ATP coacervate in the presence of increasing concentrations of NaCl. Coacervate droplets were produced from equal amounts of 50 mm PLys and ATP (at ph 8), and the phase isolated using centrifugation. NaCl (5 M) solution was added to the droplets to give final [NaCl] of (i) 100 mm, (ii) 167 mm, (iii) 333 mm and (iv) 500 mm. Except for at 500 mm, arrows indicate the presence of a coacervate/aqueous phase interface in the centrifuged samples, confirming droplet stability under these conditions. Figure S7. Guinier plot for the SAXS profile of PLys/ATP coacervate. Solid line shows the linear least-squares fitting results in the Guinier region (QR g <1). NATURE CHEMISTRY 14

15 Figure S8 Optical images of PLys/ATP droplets prepared at [PLys] = [ATP] = 25 mm at ph 8 (unless otherwise noted) in the presence of various solutes and nanoparticles, and after centrifugation. From left to right; citrate-stabilized gold nanoparticles (5nm, Sigma), dextran-coated magnetite (Dex-Fe 3 O 4, ph = 5), carboxymethyldextran-coated magnetite (CMDex-Fe 3 O 4, ph =5), Dex-Co 3 O 4 (ph =5), CMDex-Co 3 O 4 (ph = 5), protoporphyrin IX, copper chlorophyllin, fluorescein, and methylene blue. In each case the PLys/ATP droplets are sedimented into a bottom layer and the lower density continuous water phase is observed as the upper layer. Colouration within the PLys/ATP coacervate layer is indicative of preferential sequestration into the membrane-free micro-compartments. Figure S9. UV-vis spectra of protoporphyrin IX in aqueous solution (open triangles) or in PLys/ATP droplets (filled triangles) at ph 8 after centrifugation. The spectra are essentially identical, and show a split Soret band at 350 and 460 nm, and Q bands at 540, 590, and 640 nm, corresponding to supramolecular porphyrin aggregates. The absorption band intensities for the porphyrin-containing PLys/ATP sample are augmented at lower wavelengths by the background turbidity. NATURE CHEMISTRY 15

16 Figure S10. Time profile for of ABTS oxidation (absorbance at A 410 nm ) in the presence ( ) and absence ( ) of PLys/ATP droplets at ph 5. The correction for background turbidity is shown (dotted line).. Figure S11. (Left) Time profile of absorbance at 340 nm (left) corresponding to increase in the concentration of β-nadph in the presence ( ) and absence ( ) of PLy/ATP coacervate droplets at ph 8. Dotted line shows the estimated absorbance profile after the elimination of turbidity contribution (see Methods). (Right) Time profile of β-nadph concentration in the presence ( ) and absence ( ) of PLys/ATP under conditions of reduced turbidity. The β- NADPH concentration was derived from optical absorbance changes at 340 nm using the molar extinction coefficient of β-nadph (6.22 x 10 3 M -1 cm -1 ). The reaction rates under conditions of low turbidity were 21 and 10 µm min -1 in the presence and absence of the droplets, respectively, which gave the same 2 : 1 ratio as determined using turbidity corrections. See Methods for composition of reaction solution used. NATURE CHEMISTRY 16

17 Figure S12. Partitioning of HK into Plys/ATP droplets. Enzymatic rate assay for the equilibrium aqueous phase isolated from a HK-containing PLys/ATP droplet dispersion ( ). Similar assays for samples prepared by addition of known amounts of HK to the continuous water phase obtained after isolation from coacervate dispersions were also undertaken with final HK concentrations of ~0.25 units/ml ( ) and ~0.125 units/ml ( ). Reaction rates estimated from the corresponding slopes were 4.8 µm min -1 ([HK] = units/ml), 5.7 µm min -1 ([HK] = units/ml) and 2.9 µm min -1 ([HK] = units/ml), respectively. [HK] out and [HK] total were calculated from the [HK] values obtained for plot ( ) and plot ( ) (~0.5 units/ml HK) with a dilution factor of (= (ml) (total volume of reaction mixture) / (ml) (volume of added HK solution) 2 (factor of dilution before addition of HK solution into the reaction mixture)), which gave [HK] out = 0.49 units/ml and [HK] total = 0.58 units/ml, respectively. The partition coefficient (K) of HK between PLys/ATP coacervate droplet and equilibrium solution phase was calculated using the relationships; K = [HK] in /[HK] out, and [HK] in = ([HK] total V total [HK] out V out )/V in with V total = ml, V out = ml and V in = ml. This gave a value of K = 20, indicating that extensive sequestration of HK into the PLys/ATP droplets. NATURE CHEMISTRY 17