Orientation and number of blocks influence structure and self-assembly of protein copolymers Jennifer S. Haghpanah 1 , Carlo Yuvienco 1 , Peter J. Baker 1 , Hanna Barra 1 , Deniz E. Civay 2 , Sachin Khapli 1 , Natalya Voloshchuk 1 , Mukta Asnani 1 , Susheel K. Gunasekar 1 , Murugappan Muthukumar 2 , and Jin Kim Montclare 1,3 and Jin Kim Montclare 1,3 Chemical & Biological Sciences, Polytechnic Institute of NYU, Brooklyn, NY, 11201 1 Polymer Science and Engineering Department, University of Massachusetts, Amherst, MA 01003 2 Department of Biochemistry, SUNY Downstate Medical Center, Brooklyn, NY, 11203 3 Abstract 1 Circular Dichroism (CD) of Homopolymers 7 4 Cloning of Block Co-Polymers 10 Dynamic Light Scattering (DLS) of Block Polymers The requirement for smart biomaterials to change in macromolecular structure in response to external stimuli necessitates the design of controllable modes of self-assembly. Driven by this need and inspired by the natural self-assembly of proteins, we describe the biosynthesis and characterization of three block polymers that consist of 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 – were chosen for their distinct structures, functions, and modes of self- assembly. For these fusion constructs we demonstrate that the block orientation and the number of repeated blocks of the two protein motifs play a significant role in their self-assembly on the micro- and macroscale Our results provide insight into the future development of Homopolymers 5 -4 -3 -2 -1 0 1 2 3 4 Elastin (E) 30 -20 -10 0 10 20 30 40 50 [θ] mrw (x 10 3 deg · cm 2 · dmol -1 ) COMPcc (C) 1 Kbp 500 bp EC CE ECE L1 L2 of Block Polymers Mean Volume % EC 4C 55C CE 4C 55C ECE 4C 55C 200 300 400 500 600 700 Radius (nm) • Solution becomes cloudy • Scattering is greatly reduced 200 300 400 500 600 700 Radius (nm) 200 300 400 500 600 700 macroscale. Our results provide insight into the future development of smart biomaterials with emergent properties. 8 CD of Block Polymers Cartilage Oligomeric Matrix Protein Coiled-Coil (C) 2 -5 190 200 210 220 230 240 250 -30 190 200 210 220 230 240 250 10 EC 10 CE 10 ECE 5 Protein Block Polymers •Different structure vs. EC •Random-like 0 100 0 10 20 30 40 50 Temperature (°C) 0 100 0 10 20 30 40 Temperature (°C) 0 100 0 10 20 30 40 Temperature (°C) Curcumin binding after 90 min ATR binding after 90 min Wavelength, nm 11 Binding of Polymers to Curcumin & All-Trans Retinol 65 55 45 35 30 25 Temperature Scale, ° C 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 -2 0 2 4 6 8 0 190 200 210 220 230 240 250 [θ] mrw (x 10 3 deg · cm 2 · dmol -1 ) -14 -12 -10 -8 -6 -4 -2 0 2 4 6 8 0 190 200 210 220 230 240 250 Wavelength, nm -14 -12 -10 -8 -6 -4 -2 0 2 4 6 8 0 190 200 210 220 230 240 250 -6 -4 -2 0 2 4 6 8 10 0 3 deg · cm 2 · dmol -1 ) EC + Vitamin D 3 -6 -4 -2 0 2 4 6 8 10 CE + Vitamin D 3 -6 -4 -2 0 2 4 6 8 10 ECE + Vitamin D 3 MRGSH 6 GSKPIAASA–Elastin–LEGSELA(AT) 6 AACG–COMPcc–LQA(AT) 6 AVDLQPS MRGSH 6 GSACELA(AT) 6 AACG–COMPcc–LQA(AT) 6 AVDKPIAASA–Elastin–LEGSGTGAKL MRGSH 6 GSKPIAASA–Elastin–LEGSELA(AT) 6 AACG–COMPcc–LQA(AT) 6 AVDKPIAASA–Elastin–LEGSGTGAKL •More -helical at 4C structure at low T •Shift to -helical 25 50 75 100 125 150 175 200 225 RFU 5 10 15 20 25 30 35 40 CH 3 CH 3 CH 3 CH 3 CH 3 OH H 3 CO HO O OCH 3 OH O HO CH CH CH CH CH 20 15 10 4 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] mrw (x 1 -14 -12 -10 -8 190 200 210 220 230 240 250 Wavelength, nm -14 -12 -10 -8 190 200 210 220 230 240 250 Elastin = [(VPGVG) 2 VPGFG(VPGVG) 2 ] 5 VP COMPcc = DLAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDASG 6 9 Comparison of Melting Curves in the Presence and Absence of Vitamin D 3 25 KDa 37 KDa ECE CE EC L 0 EC CE ECE 0 EC CE ECE Protein Constructs 22,555 Protein Purification & MALDI Analysis EC 0.9 1 EC 0.9 1 CE 0.9 1 ECE physicochemical behaviors of EC and CE constructs, compositionally similar macromolecules, are different . • Further comparison with ECE shows that the number of blocks contributes to modes of self-assembly taking place. • We will continue to study rheological properties of the constructs. • We hope these block co-polymers will provide protein engineers 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 D 3 for EC •Presence of additional E domain increases T m – diblocks vs. triblock •Enhanced cooperativity in the presence of vitamin D 3 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 22.4 m/z m/z 22,572 33,882 CE ECE 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0 10 20 30 40 50 60 70 80 90 % Folded ‐VD 3 +VD 3 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0 10 20 30 40 50 60 70 80 90 Temperature, °C 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0 10 20 30 40 50 60 70 80 90 Protein Theoretical (Da) Actual (Da) EC 22379 22555 CE 22656 22572 ECE 34173 33882 ACKNOWLEDGEMENTS WE 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