Stepwise Mechanism of Temperature-Dependent Coacervation of the

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Stepwise mechanism of temperature-dependent coacervation of the elastin-like peptide analog dimer, (C(WPGVG)) 3

2

Daiki Tatsubo, Keitaro Suyama, Masaya Miyazaki, Iori Maeda, and Takeru Nose Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b01144 • Publication Date (Web): 01 Feb 2018 Downloaded from http://pubs.acs.org on February 1, 2018

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Biochemistry

1

Stepwise mechanism of temperature-dependent

2

coacervation of the elastin-like peptide analog

3

dimer, (C(WPGVG)3)2

4

Daiki Tatsubo a, Keitaro Suyama b, Masaya Miyazaki c, Iori Maeda d, and Takeru Nose a,b,*

5

a

6

Fukuoka, 812-8581, Japan.

7

b

8

c

9

and Technology (AIST), Tosu, Saga, 841-0052, Japan.

Department of Chemistry, Faculty and Graduate School of Science, Kyushu University,

Faculty of Arts and Science, Kyushu University, Fukuoka, 819-0395, Japan.

Advanced Manufacturing Research Institute, National Institute of Advanced Industrial Science

10

d

11

Fukuoka 820-8502, Japan.

12 13 14 15 16

Manuscript Correspondence: Prof. Takeru Nose Tel and Fax: +81-92-802-6025 e-mail: [email protected]

17

KEYWORDS

18

Elastin, Elastin-like peptide, Self-assembly, Coacervation, Intrinsic disordered protein,

19

Secondary structure

Department of Bioscience and Bioinformatics, Kyushu Institute of Technology, Iizuka,

ACS Paragon Plus Environment

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Biochemistry 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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Page 2 of 49

Abstract

2

Elastin-like peptides (ELP) are distinct, repetitive, hydrophobic sequences, such as

3

(VPGVG)n, that exhibit coacervation— the property of reversible, temperature-dependent self-

4

association and dissociation. ELPs can be found in elastin and have been developed as new

5

scaffold biomaterials. However, the detailed relationship between their amino acid sequences

6

and coacervation properties remain obscure because of the structural flexibility of ELPs. In this

7

study, we synthesized a novel, dimeric ELP analog (H-C(WPGVG)3-NH2)2, henceforth

8

abbreviated to (CW3)2, and analyzed its self-assembly properties and structural factors as

9

indicators of coacervation. Turbidity measurements showed that (CW3)2 demonstrated

10

coacervation at a much lower concentration than its monomeric form and another ELP. In

11

addition, the coacervate held water-soluble dye molecules. Thus, potent and distinct coacervation

12

was obtained with a remarkably short sequence of (CW3)2. Furthermore, fluorescence

13

microscopy, dynamic light scattering, and optical microscopy revealed that the coacervation of

14

(CW3)2 was a stepwise process. The structural factors of (CW3)2 were analyzed by molecular

15

dynamics simulations and circular dichroism spectroscopy. These measurements indicated that

16

helical structures primarily consisting of proline and glycine became more disordered at high

17

temperatures with concurrent, significant exposure of their hydrophobic surfaces. This extreme

18

change in the hydrophobic surface contributes to the potent coacervation observed for (CW3)2.

19

These results provide important insights into more efficient applications of ELPs and their

20

analogs, as well as the coacervation mechanisms of ELP and elastin.

21 22

Introduction


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Biochemistry

1

In addition to elucidating the molecular mechanisms of biomacromolecules, controlling

2

their various functions is a crucial issue in biochemistry. The functions of intrinsically disordered

3

proteins (IDPs) have attracted much recent attention [1, 2]. These proteins serve significant roles in

4

cellular and extracellular functions, particularly by participating in molecular recognition and

5

providing mechanical properties [3, 4]. Of these IDPs, elastin, an elastic protein in the extracellular

6

matrix, provides extensibility and condensability to connective tissues in arterial walls, lungs,

7

and skin [5, 6]. Elastin also exhibits temperature-dependent, reversible association and dissociation

8

that results in phase transition characterized by lower critical solution temperature (LCST)

9

behavior

[7]

. This distinct, self-assembly behavior, known as coacervation, is considered

10

important for elastin maturation [8]. Due to this characteristic, elastin-like peptides (ELPs), which

11

contain elastin derived sequence(s), have been recently considered useful in the design of new

12

scaffold materials for developing biomedical products related to drug delivery systems and tissue

13

engineering

14

many other IDPs, ELPs have also been studied as a model peptide

15

mechanism of elastin coacervation is important for its efficient use in applications and for

16

understanding the behaviors of other complex IDPs.

17

[9, 10]

. Furthermore, as the biophysical properties of ELPs are similar to those of [11]

. Thus, elucidating the

The characteristic, hydrophobic, repetitive sequences of elastin, such as (Val-Pro-Gly[8, 12]

18

Val-Gly)n, abbreviated as (VPGVG)n, are thought to control its coacervation

19

studies have examined various ELPs based on the (VPGVG)n sequence by altering its length or

20

substituting the amino acid residues

21

sequences and the coacervation properties of ELPs. The introduction of residues with greater

22

hydrophobicity (Ile, Leu > Val > Ala), higher numbers of hydrophobic amino acids, and longer

23

chain lengths resulted in more potent coacervation properties [14-18]. In contrast, few studies have

[9, 13]

. Previous

, which revealed the relationships between peptide


3

Biochemistry 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

Page 4 of 49

1

focused on ELPs that contain aromatic amino acids, though the significant hydrophobicity of

2

aromatic amino acids would clearly contribute to coacervation. ELPs are disordered, because

3

their repetitive sequences contain many secondary structure breakers, such as glycine and proline

4

residues

5

[6, 17]

6

relationship between the structure and coacervation properties of ELPs has not been completely

7

elucidated.

[16]

. Coacervation of ELPs was assumed to be related to their disordered conformations

. Due to the difficulties in analyzing flexible and disordered secondary structures, the

8

The coacervation of ELPs is attributed to the temperature-dependent changes in

9

interpeptide hydrophobic interactions. ELPs are hydrated in clathrate-like water molecules, [8, 21, 22]

10

which shield the interpeptide hydrophobic interactions at low temperatures

11

hypothesized that ELPs assemble via hydrophobic interactions after the collapse of the clathrates

12

at high temperatures [8, 23-25]. In addition, the hydrophobic domains of ELPs are presumed to be

13

dominated by recurring β-turns

14

important for interpeptide interaction formation

15

that the structures of ELPs fluctuate between folded and disordered depending on the

16

temperature

17

not been clarified. Thus, further structural analyses of ELPs in conjunction with self-assembly

18

measurements to detect interpeptide interactions are required for elucidating the general

19

coacervation mechanism of ELPs.

[26, 27]

[26-30]

. It has been

, and it has been suggested that the β-structures are [31, 32]

. However, previous studies have revealed

. The essential structural factors for regulating the coacervation of ELPs have

20

Short ELPs with a strong coacervation properties have been developed to investigate the

21

mechanism of coacervation. Previous studies have indicated that a sufficiently high repetition

22

number (n > 40) is required for coacervation of ELPs such as (VPGVG)n

23

investigate the structure of such a long peptide chain—more than 200 amino acid residues—

[33, 34]

. It is difficult to


4

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Biochemistry

1

without a fixed conformation. Our previous studies have demonstrated that synthetic

2

hydrophobic oligomers of H-(IPGVG)n-NH2, H-(FPGVG)n-NH2 [Fn], and H-(WPGVG)n-NH2 [Wn]

3

[34-36]

4

respectively). It was demonstrated that the increased molecular hydrophobicity enhanced ELP

5

coacervation. Furthermore, we recently reported that dimeric analogs of F5, which were

6

synthesized via introduction of disulfide bonds at N-terminal additional Cys residues, showed

7

strong coacervation properties at lower temperatures and concentrations than the monomer

8

Therefore, short ELPs synthesized by disulfide bond-mediated dimerization allows for

9

convenient structural analyses and simultaneous measurements of coacervation compared to long

10

ELPs. Thus, the dimeric elastin-derived peptides were assumed to be useful models for

11

elucidating the mechanism of coacervation due to their short length and strong coacervation

12

activity.

exhibit coacervation at significantly lower repetition numbers (n = 7, 5, and 3,

[37]

.

13

In the present study, we synthesized the novel, dimeric elastin-derived peptide analog

14

(H-C(WPGVG)3-NH2)2, abbreviated as (CW3)2, and investigated its self-assembly properties, dye

15

uptake ability, and the structural features important for coacervation. It was demonstrated that

16

the short, elastin-derived peptide analog did not require specific, ordered secondary structures for

17

its coacervation. Moreover, the increase in exposed hydrophobic surface area associated with the

18

structural shift mediated interpeptide interactions. This study revealed new information on the

19

relationship between the structure and function of short ELPs, and will be useful for further

20

elucidating and controlling the biophysical functions of elastin, ELPs, and other

21

biomacromolecules.

22


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Biochemistry 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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Materials & Experimental details

2

Chemicals

3

Page 6 of 49

Fmoc-amino acids and Rink Amide MBHA resin LL (100–200 mesh) were purchased

4

from

Merck

KGaA.

(Darmstadt,

Germany).

2-(1H-benzotriazole-1-yl)-

1,1,3,3-

5

tetramethyluronium hexafluorophosphate (HBTU) and 1-hydroxybenzotriazole (HOBt) were

6

purchased from Kokusan Chemical Co., Ltd. (Tokyo, Japan). 8-Anilino-1-naphthalenesulfonic

7

acid (1,8-ANS) was purchased from Sigma-Aldrich Co. (St. Louis, MO). Water for sample

8

preparation was purified by Milli-Q Integral 3 (Merck Millipore, Billerica, MA). Other reagents

9

for peptide synthesis were purchased from Watanabe Chemical Industries Ltd. (Hiroshima,

10

Japan). Solvents for peptide synthesis and other reagents were also obtained from commercial

11

suppliers and used without further purification.

12 13 14

Synthesis of the monomer peptide A building block of the dimer H-C(WPGVG)3-NH2 [CW3] was synthesized by following [37]

15

the synthesis method in our previous study

16

433A peptide synthesizer (Applied Biosystems, Foster city, CA) using the 0.1 mmol scale solid-

17

phase method by Fmoc chemistry. Rink Amide MBHA resin LL (100–200 mesh, 285 mg) was

18

used as the solid phase support. After deresination by a reagent cocktail containing

19

trifluoroacetic acid and 1,2-ethanedithiol, the peptide was purified by reversed phase-high

20

performance liquid chromatography (RP-HPLC). The peptide was obtained as a colorless

21

powder (40.5 mg, 25.2 µmol, 25.2%). The purity and molecular weight was confirmed by ultra-

. In brief, the substrate was synthesized by ABI


6

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Biochemistry

1

performance liquid chromatography–mass spectrometry (UPLC–MS). Further detailed synthesis

2

protocols are described in the Supporting Information.

3 4

H-C(WPGVG)3-NH2.

Retention time = 1.907 min. MS (ESI) m/z, calculated for C78H104N20O16S

5

: 805.94 ([M + 2H]2+) , found 805.96.

6 7

Synthesis of the dimeric peptide

8

The dimeric peptide (CW3)2 was synthesized by aerobic oxidation of purified CW3.

9

CW3 (6.49 mg, 4.03 µmol) was dissolved in 1 mg/mL ammonium bicarbonate/60% acetonitrile

10

aqueous solution (649 µL) [37]. This solution was stirred overnight at 5 °C by a rotator, ACR-100

11

(AS ONE Co., Osaka, Japan), in shake mode. The resulting mixture was purified by RP-HPLC.

12

The peptide was obtained as a colorless powder (5.88 mg, 1.82 µmol, 90.3%). The purity and

13

molecular weight were confirmed by UPLC–MS, with the method described in Supporting

14

Information. The molecular weight of the dimeric peptide was also strictly determined by matrix-

15

assisted laser desorption ionization (MALDI)–time of flight (TOF)–MS using a mass

16

spectrometer VoyagerTM DE-PRO (PerSeptive Biosystems Inc., Framingham, MA).

17

(H-C(WPGVG)3-NH2)2. Retention time = 2.438 min. MS (ESI) m/z, calculated for

18

C156H206N40O32S2 : 1073.59 ([M + 3H]3+), found 1073.46 (Figure S1). MS (MALDI-TOF) m/z,

19

calculated 3218.75 ([M + H]+), found 3218.58 (Figure S2).

20


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Page 8 of 49

Turbidity measurement

2

The temperature-dependent coacervation of (CW3)2 was evaluated using a JASCO V-

3

660 spectral photometer, (JASCO Co., Tokyo, Japan). Solutions of (CW3)2 were prepared at

4

0.050, 0.50, and 1.0 mg/mL in pure water. Turbidity measurements were collected at 400 nm

5

while increasing or decreasing the temperature by 0.2°C/min (for the 0.50 and 1.0 mg/mL

6

samples) or 0.5°C/min (for the 0.05 mg/mL samples) from 5°C. Each concentration was

7

measured at least three times. Coacervation was determined by the phase transition temperature

8

(Tt), which is temperature at which the turbidity of the solution reaches half the maximum value

9

[37]

10

. Reversibility was defined as the percent decrease in solution turbidity after cooling to 5°C

(which took at least 8 h) to the increase in absorbance after increasing the temperature [38].

11 12

Fluorescence measurement

13

The fluorescence of (CW3)2 was measured using 1,8-ANS and was conducted on a FP-

14

8500 Fluorescence Spectrometer (JASCO Co.). Fluorescence intensity of the peptide solution

15

was measured between 400–700 nm with excitation wavelength of 370 nm. The emission of the

16

peptide solution was measured at various concentration of 0.003–0.5 mg/mL in pure water

17

containing 50 µM 1,8-ANS

18

10 °C from 5 °C to 25 °C. The measurement of each concentration was performed at least three

19

times.

[39]

, and it was measured by increasing temperature at an interval of

20 21

Dynamic light scattering (DLS) analysis


8

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Biochemistry

1

The distribution of the particle size in (CW3)2 solution was analyzed by DLS

2

measurement using Zetasizer nano ZS (Malvern Instruments Ltd., Worcestershire, UK) in a

3

measurement cell ZEN0112 (Malvern Instruments Ltd.). The peptide sample solution was

4

diluted to a concentration of 1.0 mg/mL in pure water and filtered using the Millex®-LG (Merck

5

Millipore) filter before measurement. DLS analysis was performed by increasing temperature at

6

10 °C intervals from 5 °C to 65 °C. Measurement duration was selected automatically. Protein

7

(dataset: refractive index, 1.450; absorption, 0.001) was used as the material, and water (dataset:

8

refractive index, 1.330; viscosity, 0.8872) was chosen as the dispersant. Attenuation was selected

9

automatically. The measurement of each concentration was performed at least three times.

10 11

Optical microscopy

12

The morphologies of the coacervates of (CW3)2 in the presence and absence of a water-

13

soluble dye, Ponceau S (Sigma-Aldrich Co.) was observed by bright-field microscopy at 180X

14

magnification using a BIOREVO BZ-9000 (KEYENCE Co., Osaka, Japan) equipped with a

15

PlanApo VC 60X oil objective (digital zoom = 3X; Nikon Co., Tokyo, Japan). The peptide

16

solution was diluted to 1 mg/mL in pure water and applied to a glass slide. Sample imaging was

17

performed at 25°C or after heating to 60°C on a hot plate for 30 s.

18

Coacervates containing dye was prepared as follows: 1.0 mg/ml (CW3)2 solution was

19

prepared with 25 mM Ponceau S at 4°C. The mixture was heated to 60°C for 1 h and then

20

centrifuged (14,000 rpm, 2 min). The resulting precipitate was washed with hot water (60°C).

21

Bright-field microscopy was performed at 400X magnification using a Leica DM IL LED (Leica

22

Microsystems CMS GmbH, Wetzlar, Germany) equipped with HI PLAN 40X oil objective


9

Biochemistry 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

Page 10 of 49

1

(Leica Microsystems CMS GmbH) and an HC PLAN 10X eyepiece (Leica Microsystems CMS

2

GmbH).

3 4

Molecular dynamics (MD) simulation

5

Computational experiments were performed using the software package Discovery

6

Studio 4.0 (Accelrys, Inc., San Diego, CA). All calculations were performed using DELL

7

PRECISION™ T3610 Workstation (Dell Inc., Round Rock, TX). MD simulations of (CW3)2

8

and W3 were performed at 278 K, 294 K, 310 K, 326 K, and 343 K. These models were

9

subjected to “Standard Dynamics Cascade” protocol, which implements a series of energy

10

minimization and molecular dynamics steps with the CHARMM force field. The simulation

11

parameters were set as described in a previous study [37]. Further detailed synthesis protocols are

12

described in the Supporting Information.

13 14

Circular dichroism (CD) measurement

15

CD measurement was performed in a 1.0 mm path-length cuvette using a JASCO J-725

16

spectropolarimeter (JASCO Co.). (CW3)2 was dissolved in pure water at 0.1 mg/mL. Spectra of

17

the sample solution were measured from 190 to 260 nm at various cell temperatures between 5

18

°C and 65 °C. All spectra of the peptide solution were obtained by subtracting the solvent spectra

19

obtained under the same conditions and smoothing with Savitzky–Golay smoothing filters. The

20

measurement was performed two times.

21


10

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Biochemistry

1

Results and Discussion

2

Synthesis of the peptide

3

The monomeric peptide CW3 was successfully synthesized by a conventional solid

4

phase peptide synthesis procedure and then dimerized by aerobic oxidation in 60% acetonitrile

5

aqueous solution with 1 mg/mL ammonium bicarbonate. The dimerization was not successful in

6

pure water owing to the production of insoluble CW3 precipitate. Therefore, acetonitrile aqueous

7

solution was used to dissolve the peptide deposition. The purity and molecular weight of each

8

peptide were confirmed by RP-UPLC-MS and MALDI-TOF-MS (Figures S1 and S2). The

9

synthetic yield of the dimeric peptide was approximately 90%. In summary, we found that the

10

reaction conditions were suitable to obtain a dimeric short elastin-like peptide with a high yield.

11 12

Coacervation of the dimeric peptide

13

The temperature-dependent coacervation of the synthetic peptide, (CW3)2, was

14

evaluated by measuring the turbidity of aqueous peptide solutions at various concentrations. To

15

quantitatively evaluate coacervation, Tt and reversibility were calculated from the change in

16

turbidity [37, 38]. Our previous study showed that at least 30 mg/mL of W3 monomer was required

17

to demonstrate turbidity [36]. In comparison with the W3 monomer, (CW3)2 showed coacervation

18

at significantly lower concentrations of 0.50 and 1.0 mg/mL (Figure 1 and Table 1), which

19

indicated that dimerization significantly enhanced the coacervation of the W3 monomer.

20

Similarly, this trend has been observed in dimeric peptides of F5

21

required a concentration of at least 10 mg/mL and a longer chain length than the W3 dimer to

22

exhibit coacervation. Thus, it was established that (CW3)2 possessed a stronger coacervation

[37]

. However, F5 dimers


11

Biochemistry 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

Page 12 of 49

1

propensity than the F5 dimer. Peptides that exhibit properties at low concentrations and having

2

short chain lengths can be advantageous for analytical experiments and applied research [34-37].

3

Thus, we hypothesized that (CW3)2 could be used to elucidate the mechanism of coacervation.

4

We concluded that dimeric peptides demonstrate distinct characteristics from their linear peptide

5

analogs that have longer peptide chains. Specifically, (CW3)2 showed enhanced coacervation

6

while maintaining high water solubility, whereas the linear 6-mer analog of (WPGVG),

7

C(Cyc)W6, had low water solubility and irreversible self-assembly (Figures S3 and S4). Thus,

8

ELP dimers are an effective method for obtaining peptide analogs that possess strong

9

coacervation abilities and are easy to handle.

10

In this study, (CW3)2 showed hysteresis during coacervation. In the turbidity assay

11

profile, the two turbidity curves of (CW3)2 obtained while heating and cooling the sample did

12

not coincide. This disagreement between the heating and cooling processes could indicate that

13

aggregates formed during heating remained stable while cooling to temperatures below the Tt.

14

However, the turbidity of the dimer solution gradually lowered when the temperature reached

15

below 10°C with some additional time. Finally, almost complete reversibility was observed after

16

maintaining low temperature for a several hours (5°C for at least 8 h). This hysteresis was also

17

observed for the F5 dimer

18

244) that contains repeats of exons 20–24 from the human aortic elastin gene

19

previous studies determined that the aggregation and dissociation rates of the ELPs disagreed

20

with each other. Similar to these reports, the dissociation of (CW3)2 progressed more slowly

21

than the aggregation. These results suggested that hysteresis during coacervation of (CW3)2 was

22

caused by differences in the association and dissociation rates.

23

Figure 1 and Table 1

[37]

and other ELPs, such as (APGVGV)n, (VPAVG)n, and (EP20– [39-41]

. These


12

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Biochemistry

1 2 3

Existence of invisible initial microcoacervates The self-assembly behavior of (CW3)2 was examined by the use of the fluorescent [42]

4

probe 1,8-ANS

. The fluorescence analysis showed that the fluorescence intensity increased

5

when the concentration of (CW3)2 solution was higher than 0.03 mg/mL (Figure 2A). This

6

increase in the fluorescence intensity at a particular peptide concentration indicated that 1,8-ANS

7

penetrated into the hydrophobic peptide assemblies. These results showed that the peptides

8

required a critical concentration to form assemblies and suggested that the initial

9

microaggregates may be generated above the boundary concentration.

10

The effect of temperature on the critical concentration of (CW3)2 was also investigated.

11

The analysis was only performed at relatively low temperatures (5 °C, 15 °C, and 25 °C),

12

because the fluorescence intensity was significantly decreased at higher temperatures. The

13

critical concentrations were analyzed by the two-line method

14

0.05 mg/mL, 0.032 ± 0.05 mg/mL, and 0.033 ± 0.05 mg/mL at 5 °C, 15 °C, and 25 °C,

15

respectively (Figures 2B and S5). Therefore, a temperature-dependent critical concentration was

16

not observed between 5 °C and 25 °C. However, these observed critical concentrations were

17

significantly lower than the concentrations at which the peptide solution became turbid, which

18

suggested that the formation of initial invisible microaggregates occurred at a low temperature

19

and at a concentration at which coacervation was apparently not observed. Consequently,

20

fluorescence analysis has revealed a novel morphology of (CW3)2 that could not be observed by

21

conventional turbidity measurements.

[42]

and were calculated as 0.031 ±

22


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1

Page 14 of 49

Figure 2

2 3

Size distribution of the microaggregates

4

The coacervation behavior of (CW3)2 was also analyzed by DLS analysis at various

5

temperatures in the range from 5–65 °C. The observed distribution of the hydrodynamic diameter

6

of the peptide aggregates is shown in Figure 3. In this analysis, (CW3)2 showed a broad peak at

7

approximately 100 nm between 5 °C and 25 °C. Moreover, DLS analysis of the turbidity showed

8

that the submicrometer aggregates did not induce apparent phase transition behavior.

9

Furthermore, the size of the aggregates increased with an increase in temperature (Figure 3).

10

Because light scattering and turbidity are dependent on the particle size, the growth of the

11

aggregates would imply coacervation. Based on the DLS analysis, we suggested that the

12

coacervation of (CW3)2 probably follows a stepwise process in which the generation of

13

microaggregates is followed by coacervate maturation triggered by the increase in temperature.

14 15

Figure 3

16 17

Observation of the coacervate morphology

18

The optical microscopy analysis of (CW3)2 solution was performed to study the

19

morphology of the aggregates at different temperatures. Microscopic images of the peptide

20

solution were captured at 25 °C and after heating to 60 °C (Figure 4). At 25 °C, several dozen

21

distorted and non-spherical microaggregates were observed (Figure 4A). Microaggregates of


14

Page 15 of 49 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

Biochemistry

1

(CW3)2 were formed below the phase transition temperature. Although microaggregates

2

between 0.2 µm and 0.8 µm in diameter were hardly observed in solution, microaggregates

3

between 0.8 µm and 1.0 µm in diameter, which were also observed in the DLS measurement,

4

were commonly observed (Figure S6). After heating, the number of spherical coacervates was

5

significantly increased at 60 °C. Microaggregates between 0.8 µm and 1.0 µm in diameter were

6

also predominantly observed (Figures 4B and S6). Owing to the limited resolution of the optical

7

microscope, smaller microaggregates (approximately 200 nm or less) were hardly observed;

8

therefore, the measurements were not as specific as those obtained in DLS and the size

9

distribution did not necessarily agree with DLS. In this study, the distribution trend changed

10

between 200 nm and 1000 nm; an increase in the distribution of large aggregates in optical

11

microscopy (Figure S6) was similar to that observed in the DLS measurement (Figure 3), but a

12

change in the dominant distribution between two temperatures was not clearly observed in

13

optical microscopy. Consequently, it was suggested that the increment in turbidity during

14

coacervation was derived from the proliferation of the aggregates observed in microscopy and

15

the increase in the distribution of large aggregates, which could be also observed in DLS. The

16

morphological changes in the peptide aggregates affected the coacervation behavior. Because

17

these results were consistent between fluorescence and DLS analyses, the hypothesis of the

18

stepwise coacervation process was strongly supported.

19

Furthermore, the ability to hold water-soluble dye was observed in the coacervate of

20

(CW3)2. As shown in Figure S7, the dye uptake of the (CW3)2 coacervate was observed at 60°C

21

and was also stable during observation at 25°C. The uptake ability of exogenous molecules such

22

as drugs and the stability of the coacervate at low temperatures might be useful while developing


15

Biochemistry 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

Page 16 of 49

1

ELP biomaterials possessing temperature-sensing capabilities, such as matrices for drug delivery

2

systems or tissue engineering.

3 4

Figure 4

5 6

Estimation of the structural properties of the dimeric peptide by MD simulation

7

The analysis of the structural properties of the dimeric peptide was valuable to

8

determine the intermolecular events of self-assembly. Thus, the conformational behavior of W3

9

and (CW3)2 in the solution was estimated by MD simulation at various target temperatures. In

10

addition, the structural parameters of these peptides were analyzed from the trajectories of the

11

MD simulation.

12

A proportion of the representative secondary structures in the peptides was calculated

13

from the trajectory. The representative structures of W3 and (CW3)2 at varying temperatures are

14

shown in Figures 5 and S8. The secondary structures were determined by using the method of

15

Kabsch and Sander

16

structures (Table 2). The ratios of each secondary structure on a residue were also estimated. The

17

turn structure frequently appeared in each PGV sequence and was present in the middle of the

18

building block. The sheet structures were formed in VG and WP sequences, which were present

19

at the either sides of the turns (Figure S9). These peptides exhibited a well-folded structure and

20

lower Rg between 278 K and 310 K. As shown in Figure 5, accumulated turn structures were

21

frequently observed in the dimer at 273 K. In addition, the consecutive secondary structure sheet-

[43]

. The peptides primarily showed turns, sheets, and random-coiled


16

Page 17 of 49 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

Biochemistry

1

turn-sheet was frequently observed at 294 K and 310 K. However, the proportion of these

2

secondary structures decreased with increased simulation temperature. A random coiled structure

3

was predominantly observed at 326 K and 343 K. These results suggested that the dimeric

4

peptide possessed a tendency to alter the ordered structure observed at low temperatures to a

5

disordered structure at higher temperatures.

6 7

Figure 5 and Table 2

8 9

The Rg and total solvent-accessible surface area (SASA) value were calculated to

10

describe the manner in which the structural changes of the peptide chain were associated with

11

temperature change (Table 2). These values increased gradually with an increase in temperature.

12

Notably, the changes in the Rg and SASA values of the dimeric peptide were significantly higher

13

than those of the monomer, which suggested that the dimeric peptide experienced a remarkable

14

change in structure with increases in temperature. The SASA value of the side chain on each

15

residue was also calculated in the trajectory at various simulation temperatures (Figure 6).

16

Although the SASA value of each residue tended to increase with an increase in temperature, the

17

change in the dimer was more significant than that in the monomer. In particular, the SASA

18

values of hydrophobic residues, such as valine and tryptophan, were markedly altered in the

19

dimeric peptide. As the SASA value of the monomer did not change drastically, it was plausible

20

that the molecular surface of the monomeric peptide was exposed in both ordered or disordered

21

states. In contrast, it has been suggested that the hydrophobic surface of the dimeric peptide

22

would be embedded when the peptide dissolves in water, which implied that the estimated


17

Biochemistry 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

Page 18 of 49

1

molecular structure of the dimeric peptide preserved its water solubility by effectively

2

embedding hydrophobic residues into the interface between single peptide chains. The increase

3

in SASA value with a corresponding increase in temperature indicated that the secondary

4

structures could collapse and the folded structure of the dimeric peptide shifted to an extended

5

structure. Because the extended structure of (CW3)2 was not water dispersible, the molecules of

6

(CW3)2 assembled to form hydrophobic intermolecular interactions via the hydrophobic surfaces

7

of the peptides. This drastic change in the hydrophobic surface may contribute toward the potent

8

coacervation property of (CW3)2. Furthermore, these results may indicate that coacervation

9

occurred primarily as a result of the exposure of the hydrophobic surface of the peptides and not

10

as a result of the formation of a particular structure.

11 12

Figure 6

13 14

The structural transitions of the dimeric peptide are temperature-dependent

15

Since the conformational change of (CW3)2 was temperature-dependent, its structure

16

was further analyzed by circular dichroism spectroscopy. A reversible spectrum shift was

17

observed during heating and cooling (Figure 7). The spectrum showed a minor negative band at

18

225 nm, a positive band at 220 nm, and a prominent negative band at 198 nm. As the

19

temperature increased, the intensity of these bands decreased; this result was similar to the CD

20

spectra of the W3 monomer observed in our previous study

21

exhibited conformational changes similar to the monomer. Conversely, it was considered that the

22

structural features of the peptide were not altered by dimerization. In addition, this spectral

[36]

. Thus, the dimeric peptide


18

Page 19 of 49 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

Biochemistry

1

change was similar to that observed during disordering of collagen helices or polyproline helix II

2

structures

3

based on proline at low temperature and was converted to a disordered structure during heating.

[44-46]

. Thus, it was suggested that the dimeric peptide formed a helix-like structure

4 5

Figure 7

6 7

Plausible mechanism of the stepwise coacervation process of ELP

8

The stepwise coacervation process of the dimeric peptide (CW3)2, with detailed

9

analyses of the morphology after coacervation, is shown in Figure 8. As shown in Figure 2,

10

(CW3)2 exhibited a critical concentration for the generation of initial microaggregates. In

11

addition, these aggregates matured at higher temperatures, showing increased size and

12

proliferation. To the best of our knowledge, we report the first demonstration of these

13

characteristic properties of the ELP analog using the tryptophan residue. Previous studies

14

showed that the hydrophobicity of the residue enhanced the coacervation property of ELP

15

34-35]

16

residue affected the morphology of the coacervates. Hydrophobic amino acids are abundantly

17

contained in tandem repeats of elastin. Therefore, the morphology of elastin could be regulated

18

by an ingenious combination of hydrophobic amino acid residues.

[14-18,

. In addition to these studies, the present study revealed that the hydrophobicity of the

19 20

Figure 8

21


19

Biochemistry 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

1

Page 20 of 49

Insights into the coacervation mechanism of the dimeric peptide

2

In this study, we demonstrated novel insight into the relationship between the structure

3

and coacervation property of ELP. The hydrophobicity of (CW3)2 was extremely potent; the

4

peptide formed microaggregates, even at low concentration and temperature. Previous studies

5

have suggested that ELP exhibit coacervation via collapse of the clathrate water

6

Therefore, we considered that the strong hydrophobicity of the dimeric peptide effectively

7

destabilized the hydrating water. Consequently, the potent coacervation property of (CW3)2 that

8

resulted from the hydrophobic interpeptide interaction occurred during the early stages of

9

aggregate formation (Figure 8). Previous studies have suggested that the manner of the structural [30, 47, 48]

[8, 21-25]

.

10

shift of ELP was dependent on the peptide chain length

11

that longer ELP exhibited stronger coacervation property and were likely to show ordered

12

structures at high temperatures [47, 48]. However, the results of this study clearly indicated that the

13

short dimeric elastin-like peptide (CW3)2 possessed a potent coacervation property and that the

14

structure was disordered at high temperatures. Thus, this study showed that the expression of

15

coacervation property did not necessarily require long peptide chain length or specific ordered

16

structure at high temperatures, but required potent hydrophobicity and soluble structures at low

17

temperatures. Therefore, we believe that this study offered new insights into the self-assembly

18

mechanism of ELP, based on the results of hydration, dispersion, and stereostructural analyses of

19

the hydrophobic molecule. Research on IDP, including ELP, has gained momentum in recent

20

years [1-4, 6, 11] and the applications of ELP have been fervently studied [9, 10, 13]. The present study

21

on ELP appears to provide insights with respect to its structure and coacervation properties that

22

would assist the design of more efficient ELP for industrial applications and support future

23

research on IDP.

. Moreover, it was considered


20

Page 21 of 49 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

Biochemistry

1 2

Conclusion

3

In this study, we conducted turbidity measurements, fluorescence microscopy, dynamic

4

light scattering (DLS), molecular dynamics (MD) simulation, and circular dichroism (CD)

5

spectroscopy on the novel, model, elastin-derived, dimeric peptide (H-C(WPGVG)3-NH2)2. The

6

results revealed that the hydrophobicity of the residues regulated the morphology of the

7

aggregates. Moreover, it was shown that the dimeric peptide exhibited strong coacervation and

8

ability to uptake dye, despite its low molecular weight and disordered structure at high

9

temperatures. It was also suggested that its coacervation was dependent on the hydrophobicity

10

and structure solubility at low temperatures rather than the high molecular weight compounds or

11

formation of any specific ordered structures at high temperatures. In conclusion, these findings

12

elucidated the coacervation mechanism of the short-length ELP dimer and demonstrated the

13

potential for the future development of short coacervatable peptide-based biomaterials.

14 15

Supporting Information.

16

The supporting information includes detailed experimental procedures regarding peptide

17

synthesis and molecular dynamics simulations. This section also includes the following

18

supporting figures and a table: identification of (CW3)2 by ultra-performance liquid

19

chromatography (UPLC) (Figure S1), matrix-assisted laser desorption ionization-time of flight

20

spectrometry (MALDI-TOF) (Figure S2), structure of C(Cys)W6 (Figure S3), coacervation of

21

C(Cys)W6 (Figure S4), dynamic light scattering results (Figure S5), optical microscopy (Figure

22

S6 and S7), molecular dynamics simulations results (Figures S8, S9 and Table S1), and


21

Biochemistry 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

Page 22 of 49

1

identification of C(Cys)W6 by UPLC (Figure S10) and MALDI-TOF (Figure S11). This

2

information is available free of charge via http://pubs.acs.org.

3 4

Corresponding Authors

5

Prof. Takeru Nose

6

e-mail: [email protected]

7 8

Funding Sources

9 10

This work was supported by JSPS KAKENHI Grant Number JP26550068 and JP17K20066, and research fund by Urakami Foundation.

11 12 13

Notes The authors declare no competing financial interests.

14 15 16 17 18

Acknowledgment We thank Dr. Ayami Matsushima (Associate Professor of Kyushu university) for equipment use and her help in CD spectra measurement.

19 20

Abbreviations


22

Page 23 of 49 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

Biochemistry

1

1,8-ANS, 8-anilino-1-naphthalenesulfonic acid; CD, circular dichroism; CHARMm,

2

chemistry at Harvard macromolecular mechanics; DLS, dynamic light scattering; ELP, elastin

3

derived polypeptides; Fmoc, 9-fluorenylmethyloxycarbonyl; GBSW, generalized born with

4

simple

5

hexafluorophosphate; HOBt, 1-hydroxybenzotriazole; HPLC, high performance liquid

6

chromatography; IDPs, Intrinsic disordered proteins; LCST, lower critical solution temperature;

7

MALDI, matrix-assisted laser desorption/ionization; MD, molecular dynamics; MS, mass

8

spectrometry; Rg, radius of gyration; RP, reverse phase; SASA, solvent-accessible surface area;

9

TOF, time of flight; Tt, phase transition temperature; UPLC, ultra performance liquid

10

switching;

HBTU,

2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium

chromatography

11


23

Biochemistry 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

Page 24 of 49

1

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4029.

6

[48] Zhao, B., Li N. K., Yingling, Y. G., Hall, C. K. (2016) LCST Behavior is Manifested in a

7

Single Molecule: Elastin-Like polypeptide (VPGVG)n, Biomacromolecules 17, 111-118.

8


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Biochemistry 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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Page 30 of 49

Figures & Tables

2

3 4 5

Figure 1. Turbidity measurement of (CW3)2.

6 7

Turbidity changes associated with heating (solid line) and cooling (dashed line) were shown.


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Biochemistry

1

2 3 4 5 6 7 8

Figure 2. Fluorescent measurement at various concentration of (CW3)2 with 1,8-ANS. (A) Fluorescence spectra of 50 µM 1,8-ANS were measured at 5 °C and various concentration of peptide. (B) Critical concentration of (CW3)2 was determined from maximum fluorescence intensity of the spectra by the two-line method. Assays were repeated six times and the critical concentration was calculated with the mean SE.


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Biochemistry 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

Page 32 of 49

1 2 3

Figure 3. Particle size distribution of (CW3)2 at various temperature.

4 5

Micro-aggregation of the peptide grew up its size with increasing temperature.


32

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Biochemistry

1 2 3

Figure 4. Phase-contrast microscopy images of (CW3)2.

4 5 6

(A) The images were obtained at room temperature (25 °C) and (B) after heating at 65 °C. An aqueous solution of the peptide was prepared at concentration of 1.0 mg/mL.


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Biochemistry 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

Page 34 of 49

1 2 3

Figure 5. Representative structures of (CW3)2 estimated by MD simulation.

4 5 6 7

The structure at (A) 278.0 K, (B) 294.0 K, and (C) 343.0 K were shown. The dimeric peptide shifted disordered structure by increasing temperature. Each secondary structure was indicated as follows, light blue: sheet, green: turn, and white random.


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1 2 3 4 5

Biochemistry

Figure 6. SASA of side chain at each residue of (CW3)2. The SASA values of monomer (A) and dimer (B) were obtained from trajectory analyses of the MD simulation.


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Biochemistry 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

Page 36 of 49

1 2 3

Figure 7. CD spectra of (CW3)2 at various temperature.

4 5 6

The spectra were measured from 5 °C to 65 °C with increasing temperature. Inset shows spectra with decreasing temperature.


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1 2 3 4 5 6 7

Biochemistry

Figure 8. Images of structural feature and stepwise coacervation model of (CW3)2. The morphology is changed stepwise. The dimeric peptide would be dissolved in water as microaggregation over critical concentration, by taking soluble structure. However, heating would cause destabilization of hydration and maturing of coacervation.


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Biochemistry 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

1 2

3 4 5 6 7

Page 38 of 49

Table 1. Properties in turbidity measurement of (CW3)2. Concentration

Tt [˚C]

Maximum OD400

Reversibility (%)

1.0 mg/mL

38.5 ± 1.5

0.63 ± 0.03

97.9 ± 1.0

0.50 mg/mL

40.3 ± 1.5

0.41 ± 0.03

99.1 ± 0.8

0.050 mg/mL

N. D.

0.032 ± 0.016

N. D.

Assays at a concentration of 1.0 mg/mL and 0.5 mg/mL were repeated seven times. The assay at concentration of 0.050 mg/mL was repeated three times. Data were shown with the mean ± standard error. N. D., not determined.


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1 2

Biochemistry

Table 2. Structural properties of (C(WPGVG)3)2 obtained from MD simulation. Temperature Rg (Å) (K) 278.0 9.09 ± 0.02 294.0 9.29 ± 0.07 310.0 10.81 ± 0.15 326.0 14.68 ± 0.18 343.0 15.10 ± 0.16

3 4 5 6 7

Total SASA Random coil (Å2) (%) 2357.3 ± 7.2 75.6 ± 0.5 2392.8 ± 11.9 61.2 ± 0.4 2656.6 ± 21.7 75.6 ± 0.6 3230.2 ± 21.5 85.3 ± 0.7 3274.6 ± 18.2 84.5 ± 0.7

Turn (%) 23.1 ± 0.5 20.7 ± 0.2 19.4 ± 0.4 13.1 ± 0.6 12.5 ± 0.5

Sheet (%) 1.2 ± 0.3 17.9 ± 0.5 4.9 ± 0.5 1.3 ± 0.2 2.9 ± 0.4

The properties were obtained from trajectories of the MD simulation with the mean SE. Contents of secondary structure were calculated as ratios of the residue recognized as each secondary structure by the discovery studio to the number of all residues.


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Biochemistry 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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TOC Graphic (For Table of Contents Use Only)

2

3 4 5

Title: Stepwise mechanism of temperature-dependent coacervation of the elastin-like peptide

6

analog dimer, (C(WPGVG)3)2

7

Authors: Daiki Tatsubo, Keitaro Suyama, Masaya Miyazaki, Iori Maeda, and Takeru Nose

8 9 10


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Biochemistry

Figure 1. Turbidity measurement of (CW3)2. Turbidity changes associated with heating (solid line) and cooling (dashed line) were shown.

51x31mm (300 x 300 DPI)


Biochemistry 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

Figure 2. Fluorescent measurement at various concentration of (CW3)2 with 1,8-ANS. (A) Fluorescence spectra of 50 µM 1,8-ANS were measured at 5 °C and various concentration of peptide. (B) Critical concentration of (CW3)2 was determined from maximum fluorescence intensity of the spectra by the two-line method. Assays were repeated six times and the critical concentration was calculated with the mean SE.

51x14mm (300 x 300 DPI)


Page 42 of 49

Page 43 of 49 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

Biochemistry

Figure 3. Particle size distribution of (CW3)2 at various temperature. Micro-aggregation of the peptide grew up its size with increasing temperature.

51x31mm (300 x 300 DPI)


Biochemistry 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

Figure 4. Phase-contrast microscopy images of (CW3)2. (A) The images were obtained at room temperature (25 °C) and (B) after heating at 65 °C. An aqueous solution of the peptide was prepared at concentration of 1.0 mg/mL.

51x14mm (300 x 300 DPI)


Page 44 of 49

Page 45 of 49 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

Biochemistry

Figure 5. Representative structures of (CW3)2 estimated by MD simulation. The structure at (A) 278.0 K, (B) 294.0 K, and (C) 343.0 K were shown. The dimeric peptide shifted disordered structure by increasing temperature. Each secondary structure was indicated as follows, light blue: sheet, green: turn, and white random.

51x14mm (300 x 300 DPI)


Biochemistry 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

Figure 6. SASA of side chain at each residue of (CW3)2. The SASA values of monomer (A) and dimer (B) were obtained from trajectory analyses of the MD simulation.

51x14mm (300 x 300 DPI)


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Biochemistry

Figure 7. CD spectra of (CW3)2 at various temperature. The spectra were measured from 5 °C to 65 °C with increasing temperature. Inset shows spectra with decreasing temperature.

51x31mm (300 x 300 DPI)


Biochemistry 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

Figure 8. Images of structural feature and stepwise coacervation model of (CW3)2. The morphology is changed stepwise. The dimeric peptide would be dissolved in water as micro-aggregation over critical concentration, by taking soluble structure. However, heating would cause destabilization of hydration and maturing of coacervation. 51x31mm (300 x 300 DPI)


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Biochemistry

TOC Graphic (For Table of Contents Use Only) Title: Stepwise mechanism of temperature-dependent coacervation of the elastin-like peptide analog dimer, (C(WPGVG)3)2 Authors: Daiki Tatsubo, Keitaro Suyama, Masaya Miyazaki, Iori Maeda, and Takeru Nose

34x13mm (300 x 300 DPI)


Stepwise Mechanism of Temperature-Dependent Coacervation of the

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