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Engineered Protein Biomaterials

Engineered Protein Biomaterials. Jin Kim Montclare Polytechnic Institute of NYU Department of Chemical and Biological Sciences. March 22, 2010. Protein Interactions: Intra- and Extracellular Matrix. Functional role. Structural role. In vivo incorporation of unnatural amino acids.

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Engineered Protein Biomaterials

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  1. Engineered Protein Biomaterials Jin Kim Montclare Polytechnic Institute of NYU Department of Chemical and Biological Sciences March 22, 2010

  2. Protein Interactions: Intra- and Extracellular Matrix Functional role Structural role

  3. In vivo incorporation of unnatural amino acids

  4. Amino acid analogs incorporated into E. coli proteins

  5. Histone acetyltransferase tGcn5 C-terminus specificity N-terminus affinity 10 Phe’s distributed Question: How does fluorinated phenylalanines influence stability, function and selectivity of tGcn5? Wagner G. et al.Nature1999, 400, 86; Poux, A. N. and Marmorstein R. Biochemistry 2003, 42, 14366; Treivel, R.C., Li, F.-Y. and Marmorstein, R. Anal. Biochem.2000,287, 319.

  6. Histone Acetyltransferases (HATs) Histone Transcription factor p53 Histone acetylation or Transcription factor acetylation-->birth defects, cancers, heart disorders

  7. Variants with altered substrate specificity H3 Kcat/km (mM-1s-1) p53 Kcat/km (mM-1s-1) Two variants pFF-F112R and pFF-112R-Q114L exhibit a preference for p53 with no measurable activity for target histone H3. K. Mehta

  8. Selectivity switch by integrating chemical and genetic diversity pFF- F112R X pFF- F112R Histone pFF- F112R Transcription factor p53 Combination of unnatural amino acid incorporation with mutagenesis modulates substrate specificity.

  9. Protein Interactions: Intra- and Extracellular Matrix Functional role Structural role

  10. Biopolymer Synthesis Synthetic Polymer (PMMA) Proteins (PLGAs) Chain length, sequence and stereochemistry • Well defined secondary structures • Mono-dispersity [(AG)xEG]n Krejchi et.al. Science, 1994, 265, 1427 I. Chen and B. Gao Anal. Chem., 1997, 69, 4399

  11. Block polymers of 2 different self-assembling domains (SADs) Integrate signaling molecules Tunable smart materials Control binding of small molecules: delivery Control temperature-dependent assembly Orientation of SADs influence Structure and assembly Understand self-assembly, control hierarchical organization

  12. Cartilage oligomeric matrix protein coiled coil (COMPcc) Comprised of homopentamer of coiled coils Hydrophobic pore 7.3 nm long and 0.2-0.6 nm diameter Binds the hormone 1,25-dihydroxy (vitamin D3) Vitamin D3 Ozbeck, S., Engel, J., Stetefeld, J. EMBO J.2002,21, 5960.

  13. Elastin polypeptide Comprised of pentapeptide repeat (GVPXP)n Exhibits lower critical solution temperature (LCST) depends on identity of X and number of repeats Urry, D.W. and Parker, T.M. J. Muscle Res Cell Mot. 2002, 23, 543.

  14. Secondary structure and stability characterization EC CE ECE 6.9oC -11.6oC 1.3oC The orientation of fusion does make a difference on overall structure of di-blocks The number of blocks play an important role in overall conformation and temperature dependent behavior of block polymers Influence of vit D on the polymer structure and assembly is dependent on block orientation and composition J. Haghpanah, C. Yuvienco

  15. TEM Analysis of Block Polymers: Particle-Fiber Switch ECE CE EC 31.5-39.2 nm 33.8-40.1 nm 26.9-29.8.1 nm EC and ECE look to have similar features with slightly larger sizes when compared to CE, consistent with DLS While ECE is larger in molecular weight, the article sizes are slightly smaller than EC. J. Haghpanah, E. Roth

  16. Block Polymer Binding of ATR and Ccm via Fluorescence CE: binds best to ATR and Ccm indicating importance of N-terminal C domain ECE and EC: additional C-terminal E domain improves binding J. Haghpanah, H. Barra

  17. Microrheology of Block Polymers 9.6 mg/ mL EC CE ECE 4.8 mg/ mL EC: elastic, CE: viscous and ECE: viscoelastic--orientation and block number important J. Haghpanah, R. Tu

  18. Future collaborations

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