Orientation and number of blocks influence structure and...
Transcript of Orientation and number of blocks influence structure and...
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
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500
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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
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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
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190 200 210 220 230 240 250
-6-4-202468
10
03de
g · c
m2
· dm
ol-1
)
EC + Vitamin D3
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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
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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
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elaidic acid
V. N. Malashkevich, R. A. Kammerer, V. P. Efimov, T. Schulthess, and J. Engel, Science, (1996) 274, 761-765.
Elastin (E)3
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-8
190 200 210 220 230 240 250
[θ] m
rw(x
1
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-8
190 200 210 220 230 240 250Wavelength, nm
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-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
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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