Orientation and number of blocks influence structure and self-assembly of protein copolymersJennifer S. Haghpanah1, Carlo Yuvienco1, Peter J. Baker1, Hanna Barra1, Deniz E. Civay2, Sachin Khapli1, Natalya Voloshchuk1, Mukta Asnani1, Susheel K. Gunasekar1, Murugappan Muthukumar2,

and Jin Kim Montclare1,3and Jin Kim Montclare1,3

Chemical & Biological Sciences, Polytechnic Institute of NYU, Brooklyn, NY, 112011

Polymer Science and Engineering Department, University of Massachusetts, Amherst, MA 010032

Department of Biochemistry, SUNY Downstate Medical Center, Brooklyn, NY, 11203 3

Abstract1 Circular Dichroism (CD)of Homopolymers

74 Cloning of Block Co-Polymers 10 Dynamic Light Scattering (DLS)of Block Polymers

The requirement for smart biomaterials to change in macromolecularstructure in response to external stimuli necessitates the design ofcontrollable modes of self-assembly. Driven by this need andinspired by the natural self-assembly of proteins, we describe thebiosynthesis and characterization of three block polymers that consistof a b-spiral elastin-mimetic polypeptide (E) and the -helical coiled-coil region of cartilage-oligomeric matrix protein (C). These proteins,synthesized as the block sequences – EC, CE, and ECE – werechosen for their distinct structures, functions, and modes of self-assembly. For these fusion constructs we demonstrate that the blockorientation and the number of repeated blocks of the two proteinmotifs play a significant role in their self-assembly on the micro- andmacroscale Our results provide insight into the future development

of Homopolymers

5

-4

-3

-2

-1

0

1

2

3

4Elastin (E)

30

-20

-10

0

10

20

30

40

50

[θ] m

rw(x

103

deg

· cm

2· d

mol

-1)

COMPcc (C)

1 Kbp

500 bp

EC CE ECE L1 L2

of Block PolymersMean Volume %

EC4C 55C

CE4C 55C

ECE4C 55C

200

300

400

500

600

700

Radius (n

m)

• Solution becomes cloudy• Scattering is greatly reduced

200

300

400

500

600

700

Radius (n

m)

200

300

400

500

600

700

macroscale. Our results provide insight into the future developmentof smart biomaterials with emergent properties.

8 CD of Block PolymersCartilage Oligomeric Matrix Protein Coiled-Coil (C)

2

-5190 200 210 220 230 240 250

-30190 200 210 220 230 240 250

10EC

10CE

10ECE

5 Protein Block Polymers

•Different structure vs. EC•Random-like

0

100

0 10 20 30 40 50Temperature (°C)

0

100

0 10 20 30 40Temperature (°C)

0

100

0 10 20 30 40Temperature (°C)

Curcumin binding after 90 min ATR binding after 90 min

Wavelength, nm

11 Binding of Polymers to Curcumin & All-Trans Retinol

655545353025

Temperature Scale, °C

OH

OH

HO

1,25-dihydroxyvitamin D3 O

OH

all-trans retinoic acid

O

OH

all-trans retinoic acid

O

OH

cyclohexane

-14-12-10

-8-6-4-2024680

190 200 210 220 230 240 250

[θ] m

rw(x

103

deg

· cm

2· d

mol

-1)

-14-12-10

-8-6-4-2024680

190 200 210 220 230 240 250Wavelength, nm

-14-12-10

-8-6-4-2024680

190 200 210 220 230 240 250

-6-4-202468

10

03de

g · c

m2

· dm

ol-1

)

EC + Vitamin D3

-6-4-202468

10CE + Vitamin D3

-6-4-202468

10ECE + Vitamin D3

MRGSH6GSKPIAASA–Elastin–LEGSELA(AT)6AACG–COMPcc–LQA(AT)6AVDLQPS

MRGSH6GSACELA(AT)6AACG–COMPcc–LQA(AT)6AVDKPIAASA–Elastin–LEGSGTGAKL

MRGSH6GSKPIAASA–Elastin–LEGSELA(AT)6AACG–COMPcc–LQA(AT)6AVDKPIAASA–Elastin–LEGSGTGAKL

•More -helical at 4Cstructure at low T•Shift to -helical

25

50

75

100

125

150

175

200

225

RFU

5

10

15

20

25

30

35

40 CH3CH3CH3

CH3CH3

OHH3CO

HO

OOCH3

OH

O

HO

CH3

CH3

CH3

CH3

CH2

2015104

Conclusions & Future Work

• Our studies with CD and DLS suggest that the

12

elaidic acid

V. N. Malashkevich, R. A. Kammerer, V. P. Efimov, T. Schulthess, and J. Engel, Science, (1996) 274, 761-765.

Elastin (E)3

-14-12-10

-8

190 200 210 220 230 240 250

[θ] m

rw(x

1

-14-12-10

-8

190 200 210 220 230 240 250Wavelength, nm

-14-12-10

-8

190 200 210 220 230 240 250

Elastin = [(VPGVG)2VPGFG(VPGVG)2]5VPCOMPcc = DLAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDASG

6 9 Comparison of Melting Curves in the Presence and Absence of Vitamin D3

25 KDa

37 KDaECE CE ECL

0EC CE ECE

0EC CE ECE

Protein Constructs

22,555

Protein Purification & MALDI Analysis

EC0.9

1EC

0.91

CE

0.91

ECE

physicochemical behaviors of EC and CE constructs,compositionally similar macromolecules, are different.

• Further comparison with ECE shows that the number ofblocks contributes to modes of self-assembly taking place.

• We will continue to study rheological properties of theconstructs.

• We hope these block co-polymers will provide proteinengineers with an extra level of control for drug delivery.These will also serve as novel scaffolds for tissue engineers.

•Added thermal stability – less random conformation – in the presence of vitamin D3 for EC

•Presence of additional E domain increases Tm – diblocks vs. triblock

•Enhanced cooperativity in the presence of vitamin D3 for ECE

•The influence of the small molecule on the polymer structure and assembly is dependent

on the block orientation and composition

Expected Molecular Weights

34.2 22.6 22.4m/z

m/z

22,572

33,882

CE

ECE

00.10.20.30.40.50.60.70.8

0 10 20 30 40 50 60 70 80 90

% F

olde

d ‐VD3

+VD3

00.10.20.30.40.50.60.70.8

0 10 20 30 40 50 60 70 80 90Temperature, °C

00.10.20.30.40.50.60.70.8

0 10 20 30 40 50 60 70 80 90

Protein  Theoretical (Da) 

Actual(Da) 

EC 22379 22555

CE 22656 22572

ECE 34173 33882

ACKNOWLEDGEMENTSWE WOULD LIKE TO THANK POLYTECHNIC UNIVERSITY START-UP FUNDS, THE OTHMER INTITUTE, THE WECHLER

AWARD, AIR FORCE OFFICE OF SCIENTIFIC RESEARCH, SOCIETY OF PLASTIC ENGINEERS, ACS CHEMISTRY INSITUTE, ACS ENVIORNMENTAL CHEMISTRY DIVISION, UNILEVER, THE NATIONAL SCIENCE FOUNDATION GK-12

FELLOWS GRANT DGE-0741714

Li, B.; Alonso, D. O.; Daggett, V., The molecular basis for the inverse temperature transition of elastin. J Mol Biol 2001, 305, (3), 581-92. m/z