Phage Display Selected T59 Peptide Binding Characterization to PPyCl

1. Phage Display Selected T59 Peptide Binding Characterization to PPyCl Archit B. Sanghvi The University of Texas at Austin Department of Biomedical Engineering

2. synthetic material Motivation: Promoting Bioactivity Soft Tissue Engineering Drug Delivery Bioreactor Systems

3. De novo synthesis Covalent modification Physical adsorption • polylactic acid-polylysine • QuirkR. (2001) Biomaterials , • 22: 865-872. • protein self-assembly • Zhang S.(1999) Reac. & Func. • Polym., 41:91-102. • amine functional groups • Xiao S.J. (2000) Langmuir., 14(19): • 5507-5516. + + + Copolymerization • Limitations: • complex chemical processing • alteration of bulk polymer properties • Limitations: • weak hydrophobic • interactions • non-specific binding • Limitations: • complex chemistries • need existing functional groups Current State-of-the-Art

4. Advantages: • Control orientation • No changes to bulk property • High affinity binding • Ease of application • Applied to various biomaterials • including existing FDA approved polymers Polymer-Specific Surface Modification Peptide isolation using phage display: Yellow  peptide motifs Red  cell adhesion molecules Green  growth factors

5. A 10 mm 10 mm B left  right: T59 peptide on PPyCl, PPyPSS, and PS T59 peptide binds to PPyCl and not to PPyPSS or to polystyrene (PS). T59 Phage/Peptide Binding to PPyCl- left  right: T59 phage on PPyCl, random phage on PPyCl

6. Functional Cell Applications T59-GRGDS on PPyCl promotes PC12 cell adhesion. Negative control --serum-free condition T59-GRGDS -- serum-free condition 10 mm

7. Characterization of T59 Peptide Binding to PPyCl • Mechanism of binding • Role of specific amino acids • Mechanical/adhesive strength • Compliance properties (unbinding force) • Binding affinity (Ka) • Quantify stability

8. His Arg Asp Surface Coverage Analysis: Fluorescamine Assay • Reacts with freea-amino groups (NH2-C-COOH) • Evaluate bound T59 peptide (variants) using std. curve. analysis Fluorescamine Assay

9. Identifying Critical Amino Acids of T59 Peptide in Binding PPyCl BT59  GGGGGG-LDYFVI RBT59  GGGGGG-IVFYDL T59D  THRTSTLGYFVI T59HR  TGGTSTLDYFVI T59  THRTSTLDYFVI RT59  IVFYDLTSTHRT FT59  THRTST-GGGGGG n = 6

10. Evaluating the Role of Asp (8) in T59 Peptide Binding to PPyCl Input Conc. = 15 mM n = 6

11. Varying pH Effects T59 Peptide Binding to PPyCl n = 6 pKa of Asp R - group = 3.86

12. Characterization of T59 Peptide Binding to PPyCl • Mechanism of binding • Role of specific amino acids • Mechanical/adhesive strength • Compliance properties (unbinding force) • Binding affinity (Ka) • Quantify stability

13. Why are Adhesive Interactions Important? • Critical for cell function • Survival • Migration • Proliferation • Differentiation http://www.neuro.wustl.edu/neuromuscular/lab/adhesion.htm

14. What is an AFM? • Measures topography with a force probe (x,y,z dimensions) • Lateral resolution  0.5 nm • Vertical resolution  0.1 nm • Operates by measuring attractive or repulsive forces • Components • Cantilever-mounted tip • Piezoelectric micropositioner • Cantilever deflection sensor • Feedback micropositioner http://stm2.nrl.navy.mil/how-afm/how-afm.html#General%20concept http://www.asylumresearch.com/Products/Mfp3D/Mfp3D.shtml

15. Force Curve Analysis:Contact Mode AFM • Basic principle • Sharp probe (tip) raster-scans over the sample • Deflections are detected by optical methods • Air, high vacuum, and liquid • Control of forces used to study mechanical properties • Tip modification • Immobilization of receptor (T59 peptide) http://www.molec.com/index_what_is_afm.html Biotin-BSA Streptavidin Biotin-T59 PPyCl

16. Anatomy of a Force Curve Praster, C.B. et al. Probing nano-scale forces with the atomic force microscope. 1995, App. Notes. • Measures deflections off of the free end of the cantilever • FN = kNΔz • High force sensitivity (10-5 nN) • High dynamic range (0.0001-5000 nN)

17. -500 -600 pN -700 -800 -900 50 0 -50 -100 -150 -200 -250 nm 600 400 200 pN 0 -200 -400 -600 -200 -400 -600 -800 nm AFM Force Plots:T59-PPyCl & Streptavidin-Biotin surface binding approach T59 functionalized AFM tip approach to PPyCl surface ~128 pN retraction unbinding force Biotin functionalized AFM tip approach to streptavidin surface ~309 pN

18. Comparison of Adhesive Forces n = 10 spots/sample and 30 readings/spot Published Data biotin-streptavidin = 340 pN ± 120 Lee, G. et al. Langmuir 1994, 10. covalent bond (Au-S) = 1400 pN ± 300 Grandbois, M. et al. Science 1999, 283. oligonucleotide base pair (20) = 1520 pN Lee, G. et al. Science 1994, 266.

19. Conclusion • PPyCl-specific peptide sequence identified (“T59”) • Qualitative/quantitative demonstration of T59 phage/peptide binding • Functional application: T59-GRGDS modified PPyCl • Characterization of T59 peptide binding to PPyCl • Involvement of Asp (8) in binding PPyCl • Strength of binding (adhesion force)

20. synthetic material Motivation: Promoting Bioactivity Soft Tissue Engineering Drug Delivery Bioreactor Systems

21. Acknowledgements • Dr. Wolfgang Frey (BME, UT @ Austin) • John Mendenhall and Dr. Klaus Linse-Institute for Cellular and Molecular Biology (ICMB) • Dr. Yang-Ming Sun - Texas Materials Institute (TMI) • John Wright - IGERT • Funding - National Science Foundation and Gillson Longenbaugh Foundation

22. http://www.bme.utexas.edu/faculty/schmidt