Viscoelastic forces can affect the dynamics of pattern formation during phase separation in polymeric materials. We programmed an artificial protein hydrogel to undergo viscoelastic phase separation above a critical temperature. Highly dynamic phase patterns that coarsened under the influence of viscoelastic stresses spontaneously emerged in these gels. Local oxidative crosslinking promoted by mild photobleaching could be used to initiate phase separation locally, enabling the creation of non-equilibrium patterns that evolved under the influence of surface tension and viscoelastic stresses to yield dynamic structures of controlled size and shape

Rapp, Peter B.

Silverman, Bradley R.

English

Caltech Authors - Main

 
 
1 
 
 
 
 
 
Supplementary Materials for: 
 
Viscoelastic phase patterning in artificial protein hydrogels 
 
Peter B. Rapp, Bradley R. Silverman 
 
correspondence to:  peter.rapp@yale.edu 
 
 
This PDF file includes: 
 
Materials and Methods 
Figs. S1 to S4 
Tables S1 
Captions for Movies S1 to S4 
Supplementary References 
 
Other Supplementary Materials for this manuscript includes the following:  
 
Movies S1 to S4 
 
 
 
 
2 
 
Materials and Methods 
Protein expression and purification. Plasmids encoding the artificial proteins were 
transformed into BL21 or BL21 (DE3) chemically competent E. coli. After overnight 
culture, cells were inoculated (inoculation ratios of 1:50 – 1:100) into 1 L flasks containing 
Terrific Broth (TB) supplemented with 100 – 200 mg ml-1 ampicillin. Cells were grown to 
an OD600 of 0.8 – 1.0 and then induced with 1 mM final concentration of isopropyl β-D-1 
thiogalactopyranoside (IPTG). After 4-5 h, bacterial cultures were harvested by 
centrifugation for 5-10 min at 10,000g, followed by lysis with 8 M urea. Cell lysates were 
freeze-thawed at least once before being subject to high-power tip sonication for 
homogenization (50 mL of lysate from a 1 L culture was typically treated with 30 – 50 W 
for 10 min in 0.5 s pulses). Homogenized lysate was clarified by high-speed centrifugation 
(>30,000g for 1 h) and then subject to standard His-tag purification over Ni-NTA agarose 
beads (Qiagen) under denaturing conditions (8 M urea). 
 
Protein dialysis and refolding. Denatured, purified protein (ca. 1 mg/mL starting in  
50 – 100 mL 8 M urea plus phosphate buffer) was dialyzed against 4 L of distilled water 
at 4 °C. The water was changed repeatedly (5 – 6X) over the course of several days. 
Typically, protein precipitation in the dialysate was used as the dialysis endpoint, after 
which point the aqueous suspensions were lyophilized. This procedure, which was used for 
all of the experiments reported here, routinely gave gels that displayed cloud points near 
40 °C (cf. Fig. 1C). Notably, subtle changes to the refolding procedure (e.g., changes in 
dialysis temperature, starting buffer identity or dialysate exchange frequency) could both 
raise and lower the observed cloud-point temperature of PEP networks above or below  
40 °C. For example, frequent dialysate exchange of purified protein starting in urea plus 
Tris buffer gave protein batches that formed networks with undetectable cloud-points over 
the examined range (25 – 95 °C). We infer that the observed network LCST is not a simple 
function of the E midblock alone, but depends also on the folded state of the P endblocks, 
as well as on noncovalent interactions among E midblocks. 
 
Hydrogel preparation. 100 mM phosphate buffer (pH 6.5 – 7.4) was added to lyophilized 
PEP protein and the suspension was placed on ice for 2 – 4 h to promote gelation. 
Fluorescent hydrogels were prepared by adding low concentrations of labeled PECP to 
normal PEP networks (typically, PECP:PEP mass ratios of 1:50 and 1:100 were used). Dye 
conjugation to cysteine-containing probes was performed as described previously (1).  
  
Rheological analysis of gels. Oscillatory shear rheology was performed on 10% (w/v) PEP 
hydrogels using an ARES-RFS strain-controlled rheometer (TA Instruments) equipped 
with a cone-and-plate geometry. The outer edge of the plate was coated with mineral oil in 
order to minimize evaporation from the exposed gel. Strain sweeps identified a linear 
regime between 0.1 – 10% strain at 10 rad s-1. Frequency sweeps were performed at a fixed 
strain amplitude of 1% between 0.01 and 100 rad s-1. Temperature data was collected at 
1% strain and 10 rad s-1, at 5 °C intervals between 25 °C and 60 °C. 
 
Cloud-point measurements. Protein solutions were prepared at concentrations ranging from 
1 to 10% (w/v) in 100 mM phosphate buffer, pH 6.5 – 7.4. Solutions of PEP above 2 – 3% 
formed viscoelastic gels, whereas below 2% the solutions flowed easily. Solutions and gels 
 
 
3 
 
were loaded between the two halves of a disassembled quartz cuvette. The cuvette was 
assembled by pressing the two halves together, which sandwiched the gel within a 0.1 cm 
thick cavity. Roughly 400 µL of gel was required to fill the cuvette. The absorbance at 650 
nm in response to temperature was monitored continuously on a Cary 100 Bio UV-Vis 
spectrometer equipped with temperature control. Gels were typically heated from 25 °C to 
95 °C, held at 95 °C for 5 min, then cooled to 5 °C. The heating and cooling rates were 
held constant at 1 – 3 °C min-1, with minimal differences observed between the faster and 
slower rates. Prior to cloud-point measurements on the “EC” protein, a 5% solution was 
placed at 4 °C on a rotator plate for 3 – 4 days to promote oxidative crosslinking of the 
thiol groups. 
 
Confocal microscopy, photobleaching and patterning. Imaging of spontaneous pattern 
formation and phase separation in labeled PEP hydrogels was performed on a Zeiss LSM 
880 confocal microscope (488 nm with 10 – 20X objectives) equipped with a “Delta T” 
heated stage programmed to cycle between 25 °C and 50 °C within 60 seconds (Bioptechs, 
Butler PA). Labeled gels were placed on an ITO-coated, thermally conductive Delta T 
culture dish and sealed beneath a coverslip using 120 μm Secure-Seal spacers (Life 
Technologies). Image analysis was performed in MATLAB. FRAP experiments were 
performed on a Zeiss LSM 880 equipped with a 25 mW Argon laser. Recovery curves were 
fit to an effective Fickian diffusion model using the MATLAB function nlinfit (1, 2). 
 
Patterning experiments were performed on a Zeiss LSM 5 Exciter equipped with a 25 mW 
Argon laser (458, 488 and 514 nm) and a 25 mW Diode (405 nm) laser. All laser lines, at 
maximum power, were typically activated during the photobleach. Bleach spot sizes ranged 
from 100 – 2000 µm2. Bleaching of a 500 µm2 (a = 12.5 µm radius) circle at a scan rate of 
4 µm s-1 required roughly 2000 scans to ensure efficient bleaching of fluorescein. 
Bleaching of larger spot sizes (2000 µm2, a = 25 µm) was usually performed at a scan rate 
of 1 µm s-1. Typical bleaching times varied from 2 – 10 min, depending on the size of the 
bleach spot and the total number of scans (2000 – 5000 scans). The total incident power 
emitted from the Argon laser during a typical fluorescein photobleach was measured to be 
~1 mW using a power meter. At 1 mW incident power, the average irradiance (power 
density) was estimated to be 50 W cm-2. After bleaching of the desired pattern, the sample 
was allowed to recover fluorescence for 15 – 30 min. Gels were then slowly heated to 50 
°C at rate of 2 – 3 °C min-1 using a standard heated stage and an aluminum coverslip mount.  
 
Oxidative crosslinking studies. Gels were prepared at 5% in 100 mM phosphate buffer pH 
7, supplemented with 25 µM free fluorescein or Rose Bengal, with or without 100 mM 
NaN3. Gels were sealed between two coverslips, separated by a 0.030 in sheet of PDMS. 
Sample irradiation was performed using a Coherent Innova 70 CW Ar-Ion laser. The total 
incident beam power at 488 nm was fixed at 250 – 300 mW using a circular beam spot 
with a diameter of roughly 0.8 cm. The irradiance (power density) was estimated to be 0.5 
W cm-2. Gels were irradiated for 2 h, then solubilized in 8 M urea and crosslinking was 
assessed by SDS-PAGE under non-reducing conditions. 
 
 
4 
 
 
Fig. S1 
Oscillatory shear rheometry of PEP hydrogels. (A) Frequency sweep of 10% (w/v) gels 
prepared in 100 mM phosphate buffer at a fixed strain amplitude of 1% (25 °C). The 
network behaves as a viscous liquid at low frequencies (G’ < G’’) but transitions to elastic-
dominated behavior at high frequencies (G’ > G’’). This transition occurs at a critical 
frequency ωc, corresponding to the dominant stress relaxation mode of the physical 
network. (B) Storage (G’) and loss (G’’) moduli were measured for a 10% gel at 1% strain, 
10 rad s-1 at various temperatures (n = 2, mean ± SD). Although the viscous loss modulus 
dominates at high temperatures, weak crosslinking is still evident. 
  
25 30 35 40 45 50 55 60
0.1
1
10
100
1000
10000
G' (Pa)
G'' (Pa)
Temperature (C)
G
' 
(P
a
),
 G
'' 
(P
a
)
A B
0.01 0.1 1 10 100
0.1
1
10
100
1000
10000
Frequency (rad/s)
G
',
 G
'' 
(s
tr
e
s
s
, 
P
a
)
G' (Pa)
G'' (Pa)
ωc
 
 
5 
 
 
 
Fig. S2 
Crosslinking affects the LCST of elastin-like proteins. Proteins were dissolved at a 
concentration of 5% in 100 mM phosphate buffer (pH 6.5 – 7.4) and heated to 95 °C at a 
rate 1 °C min-1. The onset of turbidity was monitored at 650 nm. (Top) The predicted LCST 
of E based on its repeat sequence (70 °C) is close to its observed transition temperature (80 
- 90 °C), whereas the transition temperature of gelled PEP is much lower (38 °C). (Bottom) 
The presence of an oxidized thiol (dimerization) in EC depresses its LCST by ~20 °C 
relative to E. 
  
30 40 50 60 70 80 90
Temperature (°C)
E
EC
30 40 50 60 70 80 90
PEP
E
N
o
rm
a
li
z
e
d
 A
b
s
 (

 =
 6
5
0
 n
m
)
 
 
6 
 
 
Fig. S3 
Photobleaching promotes covalent interchain crosslinking and subsequent probe 
enrichment in bleach spots. (A) 5% gels were labeled with green (PECP-fm, 490Ex/525Em) 
and red (PECP-trm, 596Ex/615Em) probes and photobleached (spot radius a = 25 µm, λ488 
bleach). Fluorescence recovery was monitored at 25 °C. Red probes diffused into 
photobleached volumes and remained enriched for several hours. Scale bar = 100 µm. (B) 
(Top) Photobleaching promotes covalent interchain crosslinking by singlet oxygen 
generation. The presence of 100 mM NaN3, a strong singlet oxygen quencher, prevented 
red probe enrichment in 5% gels labeled with red and green probes (λ488 bleach). (Bottom) 
Bulk irradiation of 5% gels (488 nm, 500 mW cm-2) containing 25 μM free fluorescein 
promoted covalent multimer formation of PEP chains. Multimerization was suppressed by 
the presence of 100 mM NaN3. Similar results were obtained replacing fluorescein with 
Rose Bengal, a highly efficient singlet oxygen generator. All scale bars = 100 μM. 
  
PECP-fm
PECP-trm
λ
4
8
8
b
le
a
c
h
t = 0 h 40 h1 h 10 h
0 10 20 30 40
0.9
1.0
1.1
1.2
1.3
Time (h)
N
o
rm
a
li
z
e
d
 F
lu
o
re
s
c
e
n
c
e
PECP-fm PECP-trm
A
- NaN3 + NaN3
PECP-trm
λ488
NaN3
- + +-
- + - +
kDa
38
49
62
188
B
PEP
1 
2 
3 
PECP-trm 
 
 
7 
 
 
Fig. S4 
Anomalous fluorescence recovery behavior above the LCST is poorly fit by standard 
FRAP models. Shown are representative FRAP traces acquired in a 10% PEP gel at 40 °C 
and 50 °C, along with their corresponding fits assuming Fickian diffusion. Imprecision in 
estimating Deff at or above the LCST is attributable both to poor curve fitting as well as to 
the heterogenous character of the phase separated samples (n ≥ 4 per temperature; floating 
bars depict median as well as min/max estimates). 
  
0 10 20 30 40 50 60
0.0
0.2
0.4
0.6
0.8
1.0
Time (min)
N
o
rm
a
li
z
e
d
 F
lu
o
re
s
c
e
n
c
e
25 C
25 C fit
40 C early
40 C early fit
0 10 20 30 40 50 60
0.0
0.2
0.4
0.6
0.8
1.0
Time (min)
N
o
rm
a
li
z
e
d
 F
lu
o
re
s
c
e
n
c
e
25 C
25 C fit
40 C late
40 C late fit
0 10 20 30 40 50 60
0.0
0.2
0.4
0.6
0.8
1.0
Time (min)
N
o
rm
a
li
z
e
d
 F
lu
o
re
s
c
e
n
c
e
25 C
25 C fit
50 C
50 C fit
25 40 40 50
10 -16
10 -15
10 -14
10 -13
D
e
ff
 (
m
2
 s
-1
)
early late
(C)
 
 
8 
 
Table S1 
Plasmids and amino acid sequences for all artificial proteins described in the text. 
Protein coding sequences were confirmed by double-stranded DNA sequencing. Each “P” 
domain is highlighted in blue, and the “E” domain is highlighted in gray. Cysteine residues 
are in red.  
 
Plasmid Protein Molecular Weight (Da) 
pET15b-PEP PEP 32,047 
MKGSHHHHHHHVDGSGSGSGSGSGSGAPQMLRELQETNAALQDVRELLR
QQVKEITFLKNTVMESDASGSGSGSGSGSGSGLDGHGVGVPGVGVPGVGV
PGEGVPGVGVPGVGVPGVGVPGVGVPGEGVPGVGVPGVGVPGVGVPGVG
VPGEGVPGVGVPGVGELYAVTGRGDSPASSAPIATSVPGVGVPGVGVPGE
GVPGVGVPGVGVPGVGVPGVGVPGEGVPGVGVPGVGVPGVGVPGVGVPG
EGVPGVGVPGVGVPGGLLDGSGSGSGSGSGSGAPQMLRELQETNAALQDV
RELLRQQVKEITFLKNTVMESDASGSGSGSGSGSGSGLEMHHHHHHK* 
pET15b-PECP PECP 32,151 
MKGSHHHHHHHVDGSGSGSGSGSGSGAPQMLRELQETNAALQDVRELLR
QQVKEITFLKNTVMESDASGSGSGSGSGSGSGLDGHGVGVPGVGVPGVGV
PGEGVPGVGVPGVGVPGVGVPGVGVPGEGVPGVGVPGVGVPGVGVPGVG
VPGEGVPGVGVPGVGELCYAVTGRGDSPASSAPIATSVPGVGVPGVGVPGE
GVPGVGVPGVGVPGVGVPGVGVPGEGVPGVGVPGVGVPGVGVPGVGVPG
EGVPGVGVPGVGVPGGLLDGSGSGSGSGSGSGAPQMLRELQETNAALQDV
RELLRQQVKEITFLKNTVMESDASGSGSGSGSGSGSGLEMHHHHHHK* 
pQE80L-E E 17,670 
MKGSSHHHHHHVDGHGVGVPGVGVPGVGVPGEGVPGVGVPGVGVPGVG
VPGVGVPGEGVPGVGVPGVGVPGVGVPGVGVPGEGVPGVGVPGVGELYA
VTGRGDSPASSAPIATSVPGVGVPGVGVPGEGVPGVGVPGVGVPGVGVPG
VGVPGEGVPGVGVPGVGVPGVGVPGVGVPGEGVPGVGVPGVGVPGGLLE
WKKM* 
pQE80L-EC EC 17,706 
MKGSSHHHHHHVDGHGVGVPGVGVPGVGVPGEGVPGVGVPGVGVPGVG
VPGVGVPGEGVPGVGVPGVGVPGVGVPGVGVPGEGVPGVGVPGVGELYA
VTGRGDSPACSAPIATSVPGVGVPGVGVPGEGVPGVGVPGVGVPGVGVPG
VGVPGEGVPGVGVPGVGVPGVGVPGVGVPGEGVPGVGVPGVGVPGGLLE
WKKM* 
 
  
 
 
9 
 
Movie S1 
Programmed emergence of a sponge-like morphology at 50 °C during early stage phase 
separation in a 10% PEP hydrogel (related to Fig. 2). Total elapsed time 10 min. Scale bar 
= 100 μm. 
Movie S2 
A global view of spontaneous pattern evolution at 50 °C during late stage coarsening of 
PEP suggests a breakdown of self-similarity and the absence of a characteristic length scale 
(related to Fig. 2). Aqueous phase coarsening by droplet ripening and elastically hindered 
droplet coalescence are also clearly observed. Total elapsed time 16 h. Scale bar = 1 mm. 
Movie S3 
Viscoelastic “snapping” of a late stage coacervate tendril illustrates the persistence of 
viscoelastic coarsening mode at 50 °C (related to Fig. 2). Total elapsed time 1 h. Scale bar 
= 50 μm. 
Movie S4 
Photobleaching enables transient patterning of non-equilibrium phase shapes during phase 
separation onset in a PEP hydrogel (related to Fig. 3). Shown is fluorescence recovery of a 
square photobleached pattern over 30 min, followed by heating of the gel to 50 °C for 1.5 
h. Heating matures the photobleached region into a cylindrically patterned coacervate 
domain. Total elapsed time 2 h. Scale bar = 50 μm. 
 
Supplementary References 
1. P. B. Rapp et al., Analysis and Control of Chain Mobility in Protein Hydrogels. J. 
Am. Chem. Soc. 139, 3796-3804 (2017). 
2. B. L. Sprague et al., Analysis of binding reactions by fluorescence recovery after 
photobleaching. Biophys. J. 86, 3473-3495 (2004). 
 
 


Viscoelastic Phase Patterning in Artificial Protein Hydrogels

https://authors.library.caltech.edu/106758/3/rapp-silverman-phasepatterning-chemRxiv-supp.pdf

Viscoelastic Phase Patterning in Artificial Protein Hydrogels

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