Protein Engineered Triblock Polymers Composed of Two SADs

Subscriber access provided by Kaohsiung Medical University

Protein Engineered Triblock Polymers Comprised of Two SADs: Enhanced Mechanical Properties and Binding Abilities Andrew J. Olsen, Priya Katyal, jennifer s haghpanah, Matthew B. Kubilius, Ruipeng Li, Nicole L Schnabel, Sean C. O'Neill, Yao Wang, Min Dai, Navjot Singh, Raymond S. Tu, and Jin Kim Montclare Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01259 • Publication Date (Web): 15 Mar 2018 Downloaded from http://pubs.acs.org on March 21, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

Protein Engineered Triblock Polymers Comprised of

2

Two SADs: Enhanced Mechanical Properties and

3

Binding Abilities

4 5 6 7 8 9

Andrew J. Olsen,† Priya Katyal,† Jennifer S. Haghpanah,† Matthew B. Kubilius,‡ Ruipeng Li,∂ Nicole L. Schnabel, † Sean C. O’Neill,‡ Yao Wang, † Min Dai,† Navjot Singh,† Raymond S. Tu,‡ Jin Kim Montclare†┬∫¥* †

Chemical and Biomolecular Engineering Department, New York University Tandon School of Engineering, 6 Metrotech Center, Brooklyn, NY 11201, United States

10 11

‡

12

∂

13 14

┬

15 16 17 18 19 20

Chemical Engineering Department, 160 Convent Avenue, City College of New York, New York, NY 10031, United States National Synchrotron Light Source II, Brookhaven National Laboratory, Upton, USA

Biochemistry Department, SUNY Downstate Medical, 450 Clarkson Avenue, Brooklyn, New York 11203, United States

∫

Chemistry Department, New York University, 100 Washington Square East, New York, NY 10003, United States

¥

Biomaterials Department, New York University College of Dentistry, 433 First Ave, New York, NY 10010, United States

21

ABSTRACT: Recombinant methods have been used to engineer artificial protein triblock

22

polymers comprised of two different self-assembling domains (SADs) bearing one elastin (E)

23

flanked by two cartilage oligomeric matrix protein coiled-coil (C) domains to generate CEC. To

24

understand how the two C domains improve small molecule recognition and the mechanical

ACS Paragon Plus Environment

1

Biomacromolecules 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 36

1

integrity of CEC, we have constructed CL44AECL44A, which bears an impaired CL44A domain that

2

is unstructured as a negative control. The CEC triblock polymer demonstrates increased small

3

molecule binding and ideal elastic behavior for hydrogel formation. The negative control

4

CL44AECL44A does not exhibit binding to small molecule and is inelastic at lower temperatures,

5

affirming the favorable role of C domain and its helical conformation. While both CEC and

6

CL44AECL44A assemble into micelles, CEC is more densely packed with C domains on the surface

7

enabling the development of networks leading to hydrogel formation. Such protein engineered

8

triblock copolymers capable of forming robust hydrogels hold tremendous promise for

9

biomedical applications in drug delivery and tissue engineering.

10 11

KEYWORDS: protein engineering, hydrogel, coiled-coil, elastin, biomaterials, triblock

12

copolymer

13 14 15

INTRODUCTION

16

Engineered protein hydrogels are an excellent resource for biomaterials development

17

since they possess controllable pore sizes, flexible morphologies, and tunable mechanical

18

properties1-3. Manipulation of the protein hydrogel secondary structure can accommodate diverse

19

biological properties and enable associated applications4. Hydrogels containing proteins with

20

extensive β-character have been primarily designed for stimuli-response1. In particular, elastin-

21

like polypeptides (E) exhibit a hydrophobic collapse into a β-turn from a soluble random coil

22

above their transition temperature (Tt) via adjustments in temperature, pH, or salt concentration5-

23

8

. Alternatively, α-helical constructs such as leucine zippers9-12, four-helix bundles13, and the


2

Page 3 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

cartilage oligomeric matrix protein coiled-coil14-18 have been attractive for development of

2

hydrogels because of their remarkable mechanical properties19 as well as their ability to bind to

3

other molecules including DNA20 and drugs21.

4

Hydrogels consisting of E can yield thermoresponsive biomaterials with mechanical

5

properties appropriate for drug delivery22, gene therapy23, 24, and tissue mimicry25. Samples

6

containing E that self-assemble into micelle forming hydrogels exhibit enhanced mechanical

7

properties including “self-healing” due to the reversible nature of the non-covalent crosslinks

8

formed by metal coordination 26. Similar E micelles functionalized with a radionuclide have

9

been successful for forming hydrogels capable of targeted intratumoral radiation therapy27. Silk-

10

elastin-like proteins (SELPs) retain the mechanical durability of silk and the stimuli responsive

11

nature of elastin. SELPs exhibit tunable properties including: irreversible gel formation at body

12

temperature; controlled drug delivery upon covalent modification, an observation of trans-cis

13

isomerization by protonated Schiff Base22; and controlled gene transfection by entrapping

14

adenoviral gene carriers into the hydrogel matrix23, 24.

15

Coiled-coils also have been employed in the development of protein hydrogels1, 4. Their

16

self-association has been exploited to design protein block polymers; they have been engineered

17

at the ends of protein polymers flanking a randomly structured mid-block9, 13, 28-31. Hydrogels

18

containing coiled-coils demonstrate morphology changes upon shifts in pH, temperature,

19

denaturant, and protein polymer concentration9, 32, 33. Since coiled-coils in nature possess the

20

ability to bind DNA34 and hydrophobic molecules16, 21, they have been used for cargo delivery.

21

The association of coiled-coil end blocks flanking a water soluble randomly structured midblock

22

in protein triblock polymers at concentrations of approximately 5-7% w/v have shown the

23

capacity to form hydrogels as well as present the ability to bind and release small molecule


3


Page 4 of 36

1

drugs9, 28, 29. Evidence indicates that the coiled-coil end blocks are essential for gelation through

2

their intrinsic ability to form multimers9. The length and charge of the midblock can affect the

3

mechanical character of the hydrogels28. Furthermore, protein triblocks equipped with the

4

integrin-binding domain, arginine-glycine-aspartic acid (RGD), have been developed as cell

5

binding scaffolds30, 35-37.

6

Our lab has developed protein block polymers comprised of the elastin-like polypeptide

7

domain (E) and the coiled-coil derived from cartilage oligomeric matrix protein (C). These two

8

different self-assembling domains (SADs) have been stitched together in three general

9

combinations CE, EC and ECE14, 17, 18, 38. Herein, we develop a triblock co-polymer, CEC

10

capable of assembling into a thermoresponsive hydrogel with improved small molecule binding

11

properties. The CEC triblock polymer exhibits reversible folding and unfolding as well as small

12

molecule recognition and binding by 2.7 fold. As assessed by using rheology, CEC exhibited

13

elastic behavior ideal for hydrogel formation at relatively low protein polymer concentrations,

14

similar to other triblock polymers bearing coiled-coil domains9, 39. These attributes of CEC are

15

due to the helical conformation and self-assembly of the C domain.

16 17

MATERIALS AND METHODS

18

Chemicals. Hot Start PFU Ultra, dNTP’s, denatured/ non-denatured methanol, denatured/ non-

19

denatured ethanol, sucrose, glucose, trizma base, ampicillin, chloramphenicol, isopropyl β−D-1-

20

thiogalactopyranoside (IPTG), thiamine, curcumin (ccm), calcium chloride, cobalt chloride,

21

ammonium chloride, tryptone, magnesium sulfate, imidazole, urea, sodium phosphate

22

(monobasic and dibasic), ammonium chloride, sodium chloride, potassium chloride, sodium

23

dodecyl sulfate (SDS), ethylenediaminetetraacetic acid (EDTA), HEPES, and potassium


4

Page 5 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

phosphate monobasic were purchased from Fisher Scientific. Yeast extract was purchased from

2

Acros Organics. 30% acrylamide /bis solution was attained from Bio-Rad. HiTrap IMAC FF 5

3

mL columns were obtained from GE life sciences and Ni-NTA beads were purchased from either

4

Sigma Aldrich or Qiagen. DMSO, tri-fluoroacetic acid, HPLC grade acetonitrile, ammonium

5

persulfate were from Sigma Aldrich. Snake Skin pleated dialysis tubing, sinapinic acid, Micro

6

bicinchoninic acid (BCA) and BCA protein assay kits were attained from Thermo Scientific.

7

The 96 well assay plates were purchased from Corning. Zip-Tip, 0.22 µm filters, 3 KDa and 30

8

KDa Amicon Ultra-15 centrifugal filter units were acquired from Millipore. T4 ligase was

9

purchased from New England Bio-labs. Restriction enzymes Bam HI, Hind III and Kpn I were

10

obtained from Roche.

11 12

Biosynthesis. Cloning of the constructs was carried out as described previously16, 17. Using

13

PQE30/CE as the template, a single-alanine mutant in the C domain was generated using the

14

primers L44A – 5’-CCAACGCGGCGGCGCAGGACGTCGTG-3’ and the reverse complement

15

16

16

GCATGGGTACCGGATCCGGTGACCTGGCGCCG-3’ and Hind III 5’-

17

GCATGAAGCTTATTAAGCTTACCAGACGCGTC-3’ using PQE9/C and PQE9/CL44A. The

18

amplified genes C and CL44A were restricted simultaneously with PQE30/CE and PQE30/CL44AE

19

using Kpn I and Hind III enzymes. The C and CL44A restricted inserts were gel purified and then

20

ligated into the purified, restricted PQE30/CE and PQE30/CL44AE using T4 ligase enzyme at 16 ˚C

21

for 48 hrs to produce PQE30/CEC, PQE30/CL44AECL44A, respectively. Sequences were confirmed

22

via sequencing by MWG Operon.

. The C and CL44A genes were amplified with the following primers: Kpn I 5’-


5


Page 6 of 36

1

DNA including PQE30/CEC, PQE30/CE, PQE30/CL44AECL44A, and PQE30/CL44AE were

2

transformed into Escherichia coli (E. coli) AFIQ strain40, which has the ability to express the

3

lac1q repressor40, 41. Minimal M9 media comprised of 0.5 M Na2HPO4, 0.22 M KH2PO4, 0.08 M

4

NaCl, 0.18 M NH4Cl, 1g L-1 of 20 amino acids (pH 9.0), 0.4 % wt. vol.-1 glucose, 0.2 mg mL-1

5

ampicillin, 0.1 mM CaCl2, 1 mM MgSO4, 0.35 mg mL-1 chloramphenicol and 0.35 mg mL-1

6

vitamin B was employed for protein expression. Cells were cultured at 37 ˚C, 350 rpm for 6

7

hours until the OD600 was 1.0. The cells were centrifuged and the pellets were re-suspended in

8

ice cold 0.9% NaCl, centrifuged and repeated. The washed cell debris was re-suspended in M9

9

minimal media bearing 0.2 mg mL-1 IPTG. Expression was induced for 3 hours at 37 ˚C and 350

10

rpm. After harvesting, cell pellets were stored at -80 ˚C until they were prepared for purification.

11

Both C and CL44A were expressed using XL1-Blue cells. A single colony of PQE9/C and

12

PQE9/CL44A were cultured in 1L Luria Broth containing 0.2 mg mL-1 ampicillin at 37 ˚C at 250

13

rpm until the OD600 was 1.0, and subsequently induced with 0.2 mg mL-1 IPTG. After incubation

14

for 3 hours at 37 ˚C at 250 rpm, cells were harvested. Pellets were stored at -80 ˚C until the time

15

of purification.

16 17

Purification. Bacterial pellets bearing CEC, CL44AECL44A, CE and CL44AE were thawed at 4 ˚C

18

for approximately 2 hours. After they were thawed completely, they were subjected to osmotic

19

shock42 via re-suspending in ice-cold sucrose buffer into 10:1 expression volume to re-

20

suspension volume containing 50 mM HEPES, 20% sucrose, 1 mM EDTA pH 7.9, followed by

21

centrifugation. The sucrose buffer facilitated the removal of the periplasmic material in E. coli

22

including metallophores that could potentially bind to the Co2+ column42,

23

supernatant from the sucrose wash was complete, 40 mL of ice cold 5 mM MgSO4 was added to

43

.

Once the


6

Page 7 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

the pellet, re-suspended and incubated on ice for 10 min. The suspension was centrifuged. After

2

discarding the supernatant, the pellet was re-suspended into 10:1 expression volume to lysis

3

buffer A composed of 50 mM sodium phosphate buffer dibasic (Na2HPO4), 6 M urea (CH4N2O),

4

and 20 mM imidazole, sterile filtered, pH 8.0.

5

probe sonicator (Qsonica, Inc., Newtown, CT) with a 50% amplitude and a pulse of 5 seconds on

6

and 25 seconds off for 2 minutes. The lysed sample was centrifuged and the supernatant was

7

loaded onto and purified by an IMAC HighTrap FF Co2+ column (GE Life Sciences), using

8

ÄKTA FPLC purifier (G.E. Healthcare, Piscataway, NJ). Prior to loading the supernatant, the

9

column was charged with 3 mL of 0.5 M sterile filtered CoCl2 and equilibrated with buffer A at

10

4 ˚C. Bound proteins were then eluted with a gradient of 2.4% (15 mL), 4.8% (15 mL), and 100

11

% (60 mL) buffer B (50 mM Na2HPO4, 6 M Urea (CH4N2O), and 500 mM imidazole sterile

12

filtered pH 8.0). Purity of 95% or higher was confirmed on 12% SDS PAGE stained with

13

Coomassie blue bearing the precision plus ladder (Bio-Rad) and analyzed on the Image Quant

14

(GE Life Sciences) (Figure 1, Figure S1).

15

Lysis was achieved using a Q500 ultrasonic

Cell pellets bearing C and CL44A were thawed at 4 ˚C and re-suspended into 10:1

16

expression volume to ice cold lysis buffer comprised of 0.1 M NaH2PO4, 8.0 M urea CH4N2O

17

and 10 mM trizma base (HOCH2)3CNH2, sterile filtered, pH 8.0. The re-suspended cells were

18

stored at -80 ˚C for 30 min and then thawed at 4 ˚C for lysis. After the freeze thawing cycle was

19

complete, the solution was centrifuged. At the same time, the Ni-NTA beads were re-suspended

20

in ice cold lysis buffer (0.1 M NaH2PO4, 8.0 M urea CH4N2O and 10 mM trizma base

21

(HOCH2)3CNH2, sterile filtered, pH 8.0) in a 1:10 v/v ratio and centrifuged for 30 min at 4000

22

rpm at 4 ˚C on the Allegra-25R Centrifuge (Beckman Coulter). After centrifugation was

23

complete, the supernatant from the beads was decanted and the lysate containing protein was


7


Page 8 of 36

1

poured onto the beads. The C and CL44A were allowed to bind to the beads at 4 ˚C with the

2

constant rotation using a Labquake Shaker/Rotator (Bernstead Thermolyne, Dubuque, IA) for 3

3

hours. The mixture was centrifuged at 1000 rpm for 10 min at 4 ˚C on the Allegra-25R

4

Centrifuge (Beckman Coulter) and the supernatant was decanted after the protein of interest was

5

bound to the beads. Lysis buffer (0.1 M NaH2PO4, 8.0 M urea CH4N2O and 10 mM trizma base

6

(HOCH2)3CNH2, sterile filtered, pH 8.0) was added to the beads. The beads mixture was poured

7

onto the 5 mL polypropylene column (Pierce), and the flowthrough was collected. The beads

8

were then washed with 15 mL of wash buffer (0.1 M NaH2PO4, 8.0 M urea CH4N2O and 10 mM

9

trizma base (HOCH2)3CNH2, sterile filtered, pH 7.0, 6.3 and 6.1) at each pH. Once washes were

10

collected, the protein was eluted with 5 mL of the elution buffer (0.1 M NaH2PO4, 8.0 M urea

11

CH4N2O and 10 mM trizma base (HOCH2)3CNH2, sterile filtered, pH = 5.9, 5.5 and 4.9) at

12

various pH’s. Once the purification was complete, the fractions were run on 12% SDS PAGE

13

and analyzed on the Image Quant (GE Life Sciences) (Figure S1).

14

CEC, CL44AECL44A, CE and CL44AE were concentrated and purified further using the 3

15

KDa and 30 KDa molecular weight cut-off Amicon centrifugal filters (Millipore). Proteins were

16

then dialyzed against cold water at 4 ˚C, making sure that the ratio of protein to dialysis media

17

was 1.5:500 v/v with 10 buckets across 2 days. By contrast, C and CL44A, were either stepwise

18

dialyzed with 4.0 M, 2.0 M, and 1.0 M urea into 100 mM phosphate buffer, pH 8.0 using the

19

ratio of protein to dialysis media of 1.5:500 v/v over 6 bucket changes across 1 day or against

20

cold water at 4 ˚C fridge making sure that the ratio of protein to dialysis media was 1.5:500 v/v

21

over 10 bucket changes across 2 days.

22

For experiments conducted in 1x phosphate buffered saline (PBS) pH 7.4, CEC and

23

CL44AECL44A were dialyzed step-wise using the following: 4.0 M urea in PBS, 2.0 M urea in


8

Page 9 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

PBS, 1.0 M urea in PBS, followed by 3 buckets containing PBS, pH 7.4. Concentration was

2

assessed via PierceTM BCA Protein Assay Kit (Thermo Scientific) using bovine serum albumin

3

(BSA) standards in PBS, pH 7.4.

4

Proteins that were dialyzed into water were then lyophilized down to dryness for 1-2 days

5

and re-suspended in distilled water (RNase/ DNase free) for Micro-BCA (Pierce) analysis. For

6

the C and CL44A, proteins were dialyzed into 100 mM phosphate buffer pH 8.0, and were assessed

7

via PierceTM Micro-BCA Protein Assay Kit (Thermo Scientific) using BSA standards in 100 mM

8

phosphate buffer at pH 8.0.

9 10

Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI MS). Lyophilized

11

protein sample was re-suspended into 0.1% TFA solution at a concentration of 10 mg mL-1 and

12

sonicated on a Branson 2210 sonicator (Abott Park, Illinois) for at least 30 min at room

13

temperature. Sinapinic acid was dissolved in 50% acetone with 0.1% TFA at a concentration of

14

20 mg mL-1 and sonicated for 30 min at room temperature. After centrifugation at 14,000 rpm

15

for 10 minutes, the supernatant was employed as the matrix. The proteins were purified via zip-

16

tip (Millipore, Billerica, MA) eluted with 75% HPLC grade acetonitrile and 0.1% TFA.

17

Approximately, 1 µL of the 10 mg mL-1 sample was then mixed with 1 µL of matrix for the

18

Bruker Maldi-TOF/TOF UltraflexXtreme MS Spectrometer (Bruker Daltonics) (Billerica, MA).

19

1 µL of sample was spotted on a 24 × 16 target plate made of ground steel (Bruker Daltonics)

20

(Billerica, MA) and was left overnight to dry. Samples were then calibrated with RNaseA in the

21

linear mode with a laser power of 80 equipped with a high energy pulse of > 85 µJ at 1 kHz on

22

the Bruker Maldi-TOF/TOF UltraflexXtreme MS Spectrometer (Bruker Daltonics, Billerica,

23

MA), and molecular weights of proteins were determined using UltrafleXtreme software.


9


Page 10 of 36

1 2

UV-Vis Spectroscopy. Solutions for CEC and CL44AECL44A were prepared at 0.5 mg mL-1 in

3

PBS, pH 7.4. Samples were loaded into a type 21 quartz cuvette with a 10 mm path length

4

(BuckScience, Los Angeles, CA) covered with 20 µL mineral oil. Proteins were characterized

5

for their temperature transition (Tt) behavior using a UV-Vis Cary-50 (Varian Inc., Cary, NC)

6

equipped with a TC125 temperature regulator (Quantum Northwest, Liberty Lake, WA).

7

Samples were heated at a rate of 0.5 ˚C min-1 from 20 ˚C to 80 ˚C. At least 3 sample preps from

8

different batches of expression were prepared and the average data was plotted to acquire the

9

average inflection point in the absorbance curve44.

10 11

Rheology. Lyophilized CEC and CL44AECL44A were re-suspended at a concentration of 5% wt.

12

vol.-1 using 20 mM NaOH solution followed by the addition of 10 times PBS solution to correct

13

the pH back to 7.4. Curcumin (ccm) bound samples were prepared by incubating the samples at a

14

5:1 ccm to protein molar ratio for 7 hours prior to lyophilization, allowing the ccm to reach

15

equilibrium with the protein. Samples were re-suspended as above. The rheological properties of

16

the protein solution were determined using a stress-controlled rheometer (ARES-G2, TA

17

Instruments, New Castle, DE). The rheometer was equipped with 8 mm diameter parallel plates

18

with a 0.2 mm geometry gap. Oscillatory frequency sweeps were used to determine the storage

19

moduli (Gʹ) and the loss moduli (Gʺ) as a function of frequency from 0.1-1 Hz with a strain of

20

1%. Measurements were carried out at 25 ˚C and 37 ˚C for each in triplicate.

21 22

Small Angle X-Ray Scattering (SAXS). SAXS was performed at CMS beamline in NSLS II,

23

Brookhaven National Lab. A 0.2 × 0.2 mm sized beam with a wavelength of 0.918Å and wide


10

Page 11 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

bandpass (0.7%) was generated using a double-bounce multilayer monochromator. The samples,

2

including buffer solution, were transferred into 1 mm dia. glass capillaries. The scattering

3

patterns were collected by a Pilatus 2M detector placed 3.03m away from the samples. The

4

exposure time was 30 s for all samples.

5 6

Circular Dichroism (CD). Proteins at a final concentration of 4 µM in 10 mM PB pH 8.0 were

7

analyzed on the J-815 CD Spectrometer bearing a PTC-423S Peltier temperature system (Jasco,

8

Easton, MD). Samples were loaded into a Helma quartz cuvette with 1 mm path length. Raw

9

data was subtracted from the phosphate buffer background and passed through a Savitsky-Golay

10

function with a smoothing width of 1117, 45. Wavelength scans from three different sample preps

11

at temperatures (5 ˚C, 25 ˚C, 45 ˚C, 65 ˚C, 85 ˚C and 95 ˚C) were acquired and the data was then

12

converted to molar residue ellipticity [θ]mrw using the following equation: [] =

10 × × ×

13

where l is the pathlength in cm, CM is the molar concentration of the protein, and r is the total

14

number of residues46, 47. Secondary structures were investigated further via CONTIN/LL to

15

determine the amount of helical, unordered, strand and turns 48-50. A low normalized root mean

16

square deviation (NRMSD) with similar structures before and after analysis indicated a

17

“goodness” of fit was achieved51.

18

The melting temperature of CEC and CL44AECL44A was assessed by monitoring the signal

19

at 222 nm from 10-85 ˚C at a ramp rate of 0.5 ˚C min-1 using 10 µM CEC and CL44AECL44A in 10

20

mM PB pH 8.0 and 1x PBS, pH 7.4. The signal was processed using a Savitsky-Golay function

21

with a smoothing width of 13 and converted to [θ]mrw as above. Scans were collected for 3

22

different sample preparations.


11


Page 12 of 36

1

Fluorescence. Lyophilized proteins were dissolved in 100 mM PB pH 8.0 at 4 ˚C to a final

2

concentration of 9.5 µM16. Ccm was prepared in HPLC grade methanol to a final concentration

3

of 10 mM and was used as stock to prepare 100 µM ccm in 100 mM PB pH 8.0. A final

4

concentration of 50 µM ccm containing 0.5% of HPLC grade methanol for proteinccm complex

5

was employed. Fluorescence readings were obtained in triplicate after 360 minutes of incubation

6

at 25, 45 and 65 ˚C for 5 min16, 18 on a SpectraMax Plus M2 instrument (Sunnyvale, CA).

7

Samples were excited at 420 nm and the emission spectra were assessed from 450 nm – 600

8

nm52.

9

For dissociation constant (KD) determination, the final concentration of protein was 4 µM

10

and the ccm concentrations ranged from 16 µM to 0.5 µM in 10 mM PB pH 8.0 with less than

11

0.17% methanol16, 18, 53. Samples were equilibrated at room temperature for 7 hours and

12

subsequently excited at 420 nm and the emission spectra were collected from 450 nm to 600 nm.

13

After subtraction of background, all samples exhibited a maxima near 500 nm. KD’s, were

14

determined via fitting binding curves with Sigma Plot (San Jose, CA) using the equation Fb = Fs

15

[L]/(KD + [L])14, 18, 53, where Fb is the signal from the bound ligand, [L] is the ligand

16

concentration, and Fs is the plateau signal at saturation point14, 18, 53. Data was obtained in

17

triplicate.

18 19

Dynamic Light Scattering (DLS). DLS experiments were performed on the Zetasizer Nano

20

Series ZS90 (Malvern Instruments, Worcestershire, UK) with the following settings: material

21

protein with a refractive index 1.450 and absorption 0.001, dispersant PBS with a viscosity of

22

0.8882 cP and refractive index of 1.330. In a disposable low volume cuvette, measurements were

23

carried out in triplicate using three different preparations of 0.5 mg mL-1 CEC and CL44AECL44A


12

Page 13 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

in PBS pH 7.4. Samples were passed through a 0.2 µm syringe filter before each measurement,

2

then covered with 50 µL of mineral oil, and sealed with parafilm prior to measurement. The Z-

3

average diameter and the derived count rate were recorded and were carried out in at least 3

4

preparations of each protein.

5 6

Critical Micelle Concentration (CMC). CMC experiments were conducted using 5-

7

dodecanoylaminofluorescein (5-daf) as the fluorescent probe54. A 10 nM stock of 5-daf in

8

methanol was prepared, and 20 µL aliquots were prepared in loosely capped, clear threaded vials.

9

The vials were covered to protect from light and were placed in a vacuum chamber for at least 6

10

hours. A filtered (0.2 µm) 0.1 mg mL-1 stock of CEC and CL44AECL44A in PBS, pH 7.4 was

11

prepared and diluted using freshly filtered (0.2 µm) PBS to the following concentrations: 0.03,

12

0.025, 0.0225, 0.02, 0.0175, 0.0150, 0.0125, 0.01, 0.0075, and 0.005 mg mL-1. The 5-daf film in

13

the vials was re-suspended using each concentration of protein to a final concentration of 1 nM

14

of the 5-daf. Equilibration of the protein with the 5-daf was carried out for 24 hours at room

15

temperature with shaking at 80 rpm. Each mixture was dispensed in a 96-well solid black, round

16

bottom polystyrene microplate in 100 µL volumes. Using a FlexStation 3 plate reader (Molecular

17

Devices, Sunnyvale, CA), the polarization was recorded using the following settings: excitation

18

of 485 nm, emission of 528 nm with a cutoff of 515 nm, and grating (G) factor of 1.0 for

19

endpoint measurements. The instrument determined the polarization by collecting the

20

fluorescence intensities in perpendicular and parallel of the excitation plane though the following

21

equation54, 55: = 1000 ×

( − ( × ( + ( ×


13


Page 14 of 36

1

where mP is the polarization, parallel is the fluorescence intensity parallel to the excitation plane,

2

perpendicular is the fluorescence intensity perpendicular to the excitation plane, and G is the

3

instrument-derived grating factor for the fluorescent probe alone. The polarization values were

4

plotted as a function of protein concentration. The CMC was determined by the intersection of

5

two lines at which a substantial increase in the fluorescence intensity observed. These

6

experiments were repeated in triplicate with 3 different preparations of protein.

7 8

Zeta Potential. Characterization of protein surface charge was performed on the Zetasizer Nano

9

Series ZS90 (Malvern Instruments, Worcestershire, UK) with the following settings: material

10

protein with a refractive index 1.450 and absorption 0.001, dispersant water with a viscosity of

11

0.9308 cP and refractive index of 1.330. Samples of CEC or CL44AECL44A were prepared with a

12

concentration of 0.5 mg mL-1 in water, pH 6.5. A volume of 800 µL was deposited into a

13

disposable folded capillary cell DTS1070 (Malvern Instruments, Worcestershire, UK), and 10

14

measurements, conducting 20 runs each with a 2 second delay between each measurement, were

15

carried in duplicate for each sample at room temperature. This was repeated for 3 preparations of

16

CEC or CL44AECL44A each.

17 18

Transmission electron microscopy (TEM). The morphologies of CEC and CL44AECL44A

19

proteins were examined using a Phillips CM-12 transmission electron microscope. The protein

20

samples were prepared in 1x PBS, pH 7.4 at 0.5 mg mL-1. 2 µL of samples were applied on

21

Formvar/carbon-coated copper 400 mesh grids and incubated for 60s and blotted with Whatman

22

filter paper. The samples were rinsed twice with 5 µL of milli-Q water and negatively stained


14

Page 15 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

with 2 µL of 1% uranyl acetate solution. The excess stain was blotted and the grids were

2

incubated at room temperature for 5 minutes. ImageJ was used to measure the particle size56.

3 4

Multiangle Light Scattering (MALS). The molecular weight of the protein particles formed by

5

CEC and CL44AECL44A was determined by MALS57, 58. Measurements were carried out using a

6

DAWN-HELEOS II detector (Wyatt Technology Corporation, Santa Barbara, CA) equipped

7

with an Optilab rEX differential refractometer (Wyatt Technology Corporation, Santa Barbara,

8

CA) and Agilent Technologies 1200 Series HPLC pump and UV detector (Agilent Technologies,

9

Santa Clara, CA). The refractive index increment (dn/dc) and the UV extinction coefficient (ε)

10

were determined by using a series varying concentrations of sample for both CEC and

11

CL44AECL44A. Concentrations of 0.5 mg ml-1 were used for determining the molecular weight of

12

CEC and CL44AECL44A. Measurements were collected in triplicate. Analysis of the data was

13

carried out using the Astra software (Wyatt Technology Corporation, Santa Barbara, CA).

14

Statistical Analysis. The statistical significance of the results were analyzed using an unpaired

15

two-tailed student’s t-test in Prism 7 (GraphPad Software, La Jolla, CA).

16 17

RESULTS

18

Rationale. To test our hypothesis that adding an extra C end block would improve mechanical

19

integrity and small molecule binding, we generated CEC from the parent protein CE. As a

20

negative control, we produced CL44AECL44A and CL44AE, since CL44A has been previously shown to

21

impair structure, self-assembly and binding15, 16. The amino acid sequences of CEC and

22

CL44AECL44A are illustrated in Figure 1A. All proteins were overexpressed in E. coli, purified and


15


Page 16 of 36

1

confirmed via SDS-PAGE (Figure 1B). Since elastin like proteins have been shown previously to

2

run slightly higher on SDS-PAGE because of the hydrophobicity associated with the E domain59-

3

62

4

Table S1).

5

Turbidity and Mechanical Integrity. Prior to assessing the mechanical integrity of the protein

6

block polymers, the inverse transition temperatures (Tt’s) of CEC and CL44AECL44A, were

7

determined. As block polymer CE has been previously demonstrated to exhibit phase separation

8

as a function of temperature with a single Tt14, 17, we anticipated an alteration upon addition of a

9

C-terminal end block. The CEC triblock demonstrated two Tt’s at 57.23 ± 2.12 ˚C and 72.23 ±

10

4.75 ˚C, respectively (Figure 2A). While the CL44AECL44A negative control also exhibited two

11

Tt’s at 48.21 ± 2.32 ˚C and 64.85 ± 4.32 ˚C, respectively (Figure 2A), it revealed significantly

12

lower Tt values relative to CEC. The first transition (Tt1) of CL44AECL44A was 9.02 ˚C lower

13

(**, p < 0.01) than CEC, while the second transition (Tt2) of CL44AECL44A was 7.38 ˚C than

14

CEC.

15

, the molecular weights were further verified via MALDI-TOF analysis (Figure 1B, Figure S2,

The mechanical properties were assessed via rheology through analysis of the storage

16

modulus (Gʹ) and the loss modulus (Gʺ). At a concentration of 5% wt. vol.-1, CEC and

17

CL44AECL44A were elastic and viscous at 25 ˚C, respectively (Figure 2B). When the temperature

18

was raised from 25 ˚C to 37 ˚C, the Gʹ increased for CEC from 50.2 Pa to 231.7 Pa for

19

maximum values for each, stabilizing the elastic network. Notably, at 37 ˚C, the Gʹ value of

20

CL44AECL44A exceeded that of the Gʺ in the linear region, forming a gel (Figure 2B, Table S2).

21

Addition of the small molecule curcumin (ccm) led to an overall increase in the elastic nature

22

for both CEC and CL44AECL44A as demonstrated by the increase in the magnitude of Gʹ to 427.1

23

Pa and 110.3 Pa, respectively (Figure 2C, Table S2). Similar to the protein samples without


16

Page 17 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

ccm, CEC and CL44AECL44A containing ccm demonstrated an increase of 1350.5 Pa and 286.2

2

Pa in Gʹ, respectively, upon heating from 25 ˚C to 37 ˚C (Figure 2C, Table S3).

3 4

Structure and Thermodynamics. To gain insight into the impact of the C domain end block on

5

protein block polymer secondary structure, temperature-dependent wavelength scans via circular

6

dichroism (CD) were performed (Figure 3, Table S4). Previously, the CE diblock revealed an

7

overall random and β-structure at low temperatures that red-shifted to a single minima

8

illustrating β-turn conformation at elevated temperatures (Figure S3A, S4A, Table S5, S6)17. The

9

CEC triblock was largely α-helical at lower temperatures and exhibited increase in unordered

10

structure with increasing temperatures (Figure 3A, S4B Table S4, S6). The α-helical

11

conformation was predominantly dictated by two C domains, leading to a random conformation

12

at elevated temperatures. The reversibility of the protein polymer conformation was assessed by

13

investigating the structure upon cooling after heat treatment. While CEC was able to regain its

14

original structure at 4 µM, the parent CE was unable to return to the starting structure, exhibiting

15

hysteresis (Figures 3A, S3A, S4A-B, Tables S4-S6). The negative control, CL44AECL44A,

16

exhibited unordered and β-turn conformation that did not significantly change as the temperature

17

was increased (Figure 3B, S4C, Table S4, S6). In addition, the parent CL44AE diblock exhibited

18

a similar conformation trend with a random and β-turn starting structure that upon heating

19

remained similar (Figure S3B, S4D Table S5, S6).

20

The thermostability was assessed for the proteins by monitoring the signal at 222 nm as a

21

function of temperature. In order to obtain sufficient signature for the melting studies, the protein

22

concentration was increased to 10 µM. The CEC triblock exhibited two transitions in 10 mM PB

23

with a Tm at 36.0 ± 3.6 ˚C and 69.3 ± 2.4 ˚C, respectively (Figure 3C, Table S7). The presence


17


Page 18 of 36

1

of two C end blocks led to two discernable melts. By contrast, CL44AECL44A possessed a single

2

transition with a Tm of 61.9 ± 3.0 ˚C (Figure 3C, Table S7).

3

Small Molecule Binding. As the C domain has been demonstrated to encapsulate small

4

molecules including ccm15, 16, an agent with anticancer63, anti-alzheimer64, and antihypertrophic65

5

properties, we explored binding to the proteins via fluorescence. The CEC triblock demonstrated

6

the best binding of 195.03 ± 43.60 RFU (Figure 4A). This was followed by CE, which exhibited

7

a loss of 22.74 RFU indicating that the addition C-terminal C end block improved the binding

8

capacity (Figure 4A). The CL44AECL44A and CL44AE illustrated binding to ccm with 104.58 ±

9

10.14 RFU and 88.48 ± 11.571 RFU, respectively (**, p < 0.0025) (Figure 4A). Both negative

10

control proteins possessed poor binding when compared to either CEC or CE (****, p < 0.001

11

for CE and CL44AE and ***, p < 0.0002 for CEC and CL44AECL44A). The binding ability was

12

further assessed as a function of ccm concentration in order to determine dissociation constants

13

(KD)14, 18, 53. The CEC triblock possessed a KD of 2.13 ± 0.52 µM, while CL44AECL44A exhibited

14

no discernable increase in fluorescence as a function of ccm concentration (Figure 4B). Thus a

15

KD was not determined for CL44AECL44A. The parent CE diblock demonstrated a previously

16

reported KD of 5.7 ± 1.1 µM18, suggesting that the addition of the C-terminal end block improved

17

ccm binding by 2.7 fold. While appending an extra C domain enhanced small molecule

18

recognition from determination of KD, mutating the C domains to CL44A abolished the binding

19

affinity.

20

The binding was assessed as a function of elevated temperatures in order to gain insight

21

into the temperature dependent behavior observed in the mechanical properties. As already noted

22

at 25 ˚C, CEC had the best binding profile, followed by CE, then C and CL44AECL44A (Figure

23

4A). Importantly, these proteins exhibited an almost 2x better affinity for ccm, relative to their


18

Page 19 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

L44A mutant counterparts (Figure 4A). At 65 ˚C, an analogous trend was observed with the

2

exception of C L44AECL44A, which revealed an increase in binding of 338.25 ± 3.69 RFU (Figure

3

4A). Surprisingly, CL44AEC L44A showed increased binding nearing that of the CEC parent, but

4

statistically different (****, p < 0.0001). For CEC, a large increase in binding was observed from

5

25 to 45 ˚C, while CL44AECL44A demonstrated a large increase in binding from 45 to 65 ˚C

6

(Figure 4A). The counterparts C, CL44A, CE and CL44AE showed moderate changes (increases/

7

decreases) in binding with respect to temperature. Thus, appending an extra C domain to the CE

8

constructs allowed for an improvement in binding with increasing temperature.

9

Supramolecular Assembly Analysis. Both CEC and CL44AECL44A demonstrated the formation

10

of particles as characterized by dynamic light scattering (DLS). Particle sizes were analyzed as a

11

function of temperature. From 20 ˚C to 35 ˚C, CEC exhibited a consistent average diameter of

12

58.9 ± 2.9 nm, with statistically significant increase from the initial size occurring after 36 ˚C

13

(**, p > 0.0038) (Figure 5A). However, particles formed by CL44AEC L44A, demonstrated an

14

average diameter of 53.9 ± 1.5 nm (Figure 5B) from 20 ˚C to 30 ˚C and subsequent statistically

15

significant increase in size (*, p < 0.0390). Notably, the particle size of CL44AEC L44A increased

16

more rapidly than that of CEC. The derived count rate, which demonstrates the rate of

17

aggregation of the protein particles, exhibited a similar trend in which the CL44AEC L44A particles

18

aggregated at a higher rate (Figure 5C, D). Both CEC and CL44AEC L44A illustrated a steady

19

increase in aggregation at 58.7 ± 1.2 ˚C and 56.5 ± 0.5 ˚C, respectively, upon which

20

sedimentation occurred. The observed particle diameter was much larger for CL44AEC L44A than

21

for CEC at elevated temperatures with a high degree of dispersity as evidenced by the large

22

standard deviations (Figure 5A, B).


19


1

Page 20 of 36

To assess whether these particles are indeed forming micelles, 5-dodecanoyl amino

2

fluorescein (5-daf) was employed as the molecular probe54. The critical micelle concentrations

3

(CMCs) as determined by increase in the fluorescence polarization (Figure 6) were 1.10 ± 0.25 ×

4

10-2 mg mL-1 and 0.94 ± 0.11 × 10-2 mg mL-1 for CEC and CL44AEC L44A, respectively (Table 1).

5

The decrease in the fluorescence polarization can be attributed to formation of pre-micellar

6

aggregates resulting in a disparity in intensities66. While both exhibited similar CMC values, the

7

molecular weights of the particles as determined by MALS differed with 2218.5 ± 300.5 kDa for

8

CEC and 856.1 ± 144.0 kDa for CL44AEC L44A (***, p < 0.0009). Upon deriving the number of

9

monomers (Nagg) from the molecular weight (Table 1), the CEC particles demonstrated 2.6×

10

11

more monomer assemblies than CL44AEC L44A, indicating a more densely packed particle. The particle size and morphology for CEC and CL44AEC L44A were further characterized

12

by transmission electron microscopy (TEM). CEC demonstrated spherical particles with an

13

average size of 28.5 ± 6.4 nm while CL44AEC L44A showed irregular-shaped particles having an

14

average size of 30.6 ± 5.3 nm (Figure 7). While the overall size measured by TEM was smaller

15

compared to DLS likely due to dried state of TEM samples, the data confirmed the micellar

16

particles.

17

To gain insight into the surface charge of the CEC and CL44AEC L44A micelles, zeta

18

potential experiments were conducted. Previously, the C and E domains alone were assessed for

19

surface charge; C carried a surface charge while E was nearly neutral67. Both micelles exhibited

20

negative values of -28.5 ± 0.4 mV for CEC and -22.6 ± 1.0 mV for CL44AEC L44A (**, p

Protein Engineered Triblock Polymers Composed of Two SADs

Recommend Documents