Impact of Fluorinated Amino Acids on Artificial Protein Block Copolymers of Two Self-assembling Domains Carlo Yuvienco 1 , Jennifer S. Haghpanah 1 , Richard Hwang and Jin Kim Montclare 1,2 Chemical & Biological Sciences, Polytechnic Institute of NYU, Brooklyn, NY, 11201 1 Department of Biochemistry, SUNY Downstate Medical Center, Brooklyn, NY, 11203 2 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 Abstract The requirement for smart protein-derived biomaterials to change in macromolecular structure in response to external stimuli necessitates the design of controllable modes of self-assembly. The recent advances in unnatural amino acid incorporation enables the integration of chemical diversity into such proteins, further expanding the level of control and materials properties. In particular, fluorinated amino acids have been `of the biomaterials. Specifically, we have incorporated para-fluorophenylalanine and trifluoroleucine into three block polymers that consist of a β-spiral elatin-mimetic protein (E) and an α-helical coiled-coil region of cartilage-oligomeric matrix protein (C). These proteins, synthesized as the block sequences – EC, CE, and ECE – are chosen for their distinct structures, functions, and modes of self-assembly. We demonstrate successful incorporation of the non-natural amino acids as well as characterization emphasizing their structural and functional distinction relative to the non-fluorinated constructs. 1 Expression 7 Conclusions & Future Work • Successful incorporation of pFF into elastin/COMPcc fusions •Difference in melting curves apparent in fluorinated constructs •Increase in CE cooperativity, demonstrating a dependence on block orientation when comparing fluorinated proteins •Increase in ECE cooperativity but decrease in T m , suggesting an accelerated hydrophobic effect •We will continue to incorporate other non-canonical aa into the fusions, including trifluoroLeu, photoLeu and p-azidoPhe •Microscopy studies of protein solutions to investigate supramolecular assembly structures 8 Purification of pFF Constructs OH OH HO 1,25-dihydroxyvitamin D3 O OH all-trans retinoic acid O OH all-trans retinoic acid O OH elaidic acid cyclohexane Cartilage Oligomeric Matrix Protein Coiled-Coil (C) 2 V. N. Malashkevich, R. A. Kammerer, V. P. Efimov, T. Schulthess, and J. Engel, Science, (1996) 274, 761-765. Suat Ozbek, Jürgen Engel, Jörg Stetefeld, EMBO J 2002, 21, 5960-8. B. Li, V. Daggett, J Muscle Res Cell Motil 2002, 23, 561-73. Bumjoon Kim, Ashutosh Chilkoti, J Am Chem Soc 2008, 130, 17867-73. Elastin (E) 3 -14 -12 -10 -8 -6 -4 -2 0 2 4 6 8 10 190 200 210 220 230 240 250 [θ] mrw (x 10 3 deg · cm 2 · dmol -1 ) EC -14 -12 -10 -8 -6 -4 -2 0 2 4 6 8 10 190 200 210 220 230 240 250 Wavelength, nm CE -14 -12 -10 -8 -6 -4 -2 0 2 4 6 8 10 190 200 210 220 230 240 250 ECE 4 5 Protein Block Polymers MRGS H 6 GSKPIAASA –Elastin–LEGSELA(AT) 6 AACG –COMPcc–LQA(AT) 6 AVDLQPS MRGS H 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 Elastin = [(VPGVG) 2 VPGFG(VPGVG) 2 ] 5 VP COMPcc = DLAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDASG 6 •Different structure vs. EC •More a-helical at 4C 9 Comparison of Melting Curves of Wild-type and Fluorinated Constructs •Negligible effect on melting temperature for EC and CE diblocks •Notable shift in T m for ECE triblock •Enhanced cooperativity of ECE with incorporated fluorinated Phe •Though melt temperatures are the same as wild-type, the CE shows an increase in transition cooperativity versus the EC diblock upon incorporation 10 EC 4C 55C CE 4C 55C •Random-like structure at low T •Shift to a-helical 11 65 55 45 35 30 25 20 15 10 4 Temperature Scale, ° C Hydrophobic pore 73 Å long 2-6 Å wide Self-assembles into a pentameric bouquet-like structure α-Helical secondary structure 10.9 kDa (Val/Ile)-Pro-Xaa-Yaa-Gly Non-canonical Amino Acids Residue-specific incorporation of non-natural amino acids 740 790 840 m/z 740 790 840 m/z 740 790 840 m/z 750 770 790 810 m/z L pre 19aa wt pFF pre 19aa wt TFL p-fluoroPhe trifluoroLeu 25 kD 20 kD 15 kD 37 kD 50 kD 0.66 0.60 1.78 1.56 0.86 1.44 2.56 1.74 OD 600 25 kD 20 kD 15 kD 37 kD 50 kD 0.60 0.66 1.90 1.58 0.68 1.20 2.34 1.76 OD 600 37 kD 25 kD 20 kD 50 kD 75 kD 0.60 0.64 1.72 1.26 0.84 0.94 2.00 1.66 OD 600 -14 -12 -10 -8 -6 -4 -2 0 2 4 6 8 10 190 200 210 220 230 240 250 [θ] mrw (x 10 3 deg · cm 2 · dmol -1 ) EC pFF -14 -12 -10 -8 -6 -4 -2 0 2 4 6 8 10 190 200 210 220 230 240 250 Wavelength, nm CE pFF -14 -12 -10 -8 -6 -4 -2 0 2 4 6 8 10 190 200 210 220 230 240 250 ECE pFF L pre 19aa wt pFF pre 19aa wt TFL p-fluoroPhe trifluoroLeu L pre 19aa wt pFF pre 19aa wt TFL p-fluoroPhe trifluoroLeu •Small-scale(5 mL) and large-scale (100 mL) expressions of •Phe auxotrophic AFIQ cells •Leu auxotrophic LAM1000 cells •Residual natural amino acids removed prior to induction with IPTG by washing with 2 cycles of washing with 0.9% NaCl •All lanes in gels below normalize to OD 600 =1.00 •Purification of whole cell lysate under denatured conditions (6M urea) •Co 2+ affinity chromatrography using a 5mL HiTrap IMAC FF column (GE) •Gels showing elution fractions from purification •MALDI-TOF analysis confirming non-canonical amino acid incorporation EC pFF CE pFF ECE pFF 37 kD 25 kD 20 kD 50 kD 75 kD 25 kD 20 kD 15 kD 37 kD 50 kD 25 kD 20 kD 15 kD 37 kD 50 kD 768 Da 768 Da 750 Da 772 Da EC wt 768 Da 750 Da Relative Intensity CD of pFF Constructs Tang, Y. et al. Fluorinated Coiled-Coil Proteins Prepared In Vivo Display Enhanced Thermal and Chemical Stability. Angew. Chem. Int. Ed. Engl 40, 1494-1496(2001). [1]A James Link, David A Tirrell, Methods 2005, 36, 291-298. mRNA Cells washed of residual Phe AFIQ Phe auxotroph E. coli Introduction of non-canonical amino acid upon induction •Incorporated non-canonical amino acids have been shown to enhance structural stability some proteins •Photo-crosslinking possible with the incorporation of p-azidoPhe •We hypothesize that the incorporation of fluorinated amino acids into our fusion sequences will result in an alteration of temperature- sensitive behavior •Separate incorporation of p-fluoroPhe and trifluoroLeu •Different wavelength scan •Initial structure at 4C 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 10 20 30 40 50 60 70 80 90 Fraction Folded EC pFF and wt p-fluoroPhe wild-type 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 10 20 30 40 50 60 70 80 90 Temperature (°C) CE pFF and wt 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 10 20 30 40 50 60 70 80 90 ECE pFF and wt T m = 47C 33C T m = 44C 45C T m = 33C 34C •Temperature-sensitive aggregation and self-assembly •Coupling to other functional domains has been shown to affect both aggregation and temperature- sensitivity •Focus of potential use lies in drug (small molecule) delivery and release Lower criticial solution temperature (LCST) – from liquid to solid by adding heat Binding small molecules