Protein Function in Nanobioconjugates

1. Protein Function in Nanobioconjugates CHEM 570 March 27, 2008 Jennifer A. Jamison Matthews Research Group

2. Outline • Nanobioconjugates • Definition • Motivation: Why do we care? • My Research • Objective • Introduction to Proteins • Model Sytem • Overview of Gel Electrophoresis • Stability and Stoichiometry • Application: Epitope-Mapping • Conclusions

3. - - - - - - - - - - - - What are Nanobioconjugates? • A biomolecule (protein, DNA, RNA, etc.) attached to a nanomaterial Electrostatically Covalently + + + + S S + S + - - S S + + - S - + S + S + + +

4. - - - - - - - - - - - - What are Nanobioconjugates? • A biomolecule (protein, DNA, RNA, etc.) attached to a nanomaterial Electrostatically Covalently + + + + S S + S + - - S S + + - S - + S + S + + +

5. - - - - - - - - - - - - What are Nanobioconjugates? • A biomolecule (protein, DNA, RNA, etc.) attached to a nanomaterial Electrostatically Covalently + + + + S S + S + - - S S + + - S - + S + S + + +

6. Why Study Nanobioconjugates? • Hybrid materials = Hybrid properties • Exploit nano properties in the bio applications • Fluorescence, magnetism, etc. • Use bio properties to affect nano synthesis • Self-assembly, nano-patterning

7. Applications • Drug Delivery • Electron Microscopy Tags • Sensing/Detection • MRI Contrast Agents • Consumer Products • Etc.

8. Applications • Drug Delivery • Electron Microscopy Tags • Sensing/Detection • MRI Contrast Agents • Consumer Products • Etc. These applications could change the way we practice medicine!

9. Example Application: Breast Cancer Detection Breast carcinoma cells, with green QDots With R. Drezek (Rice University)

10. Objective To understand and manipulate protein-nanoparticle interactions Electrostatic Non- Specific What governs these types of interactions? Targeted Covalent

11. Objective To understand and manipulate protein-nanoparticle interactions Electrostatic Non- Specific What governs these types of interactions? Targeted Covalent

12. Objective To understand and manipulate protein-nanoparticle interactions Electrostatic Non- Specific What governs these types of interactions? Targeted Covalent

13. Quick Intro to Proteins…. Amino Acid www.wikipedia.org

14. Quick Intro to Proteins…. Amino Acid Chains of amino acids make up proteins…. …)∞ ∞(… www.wikipedia.org

15. Quick Intro to Proteins…. Amino Acid Chains of amino acids make up proteins…. Amino Acid #2 Amino Acid #1 …)∞ ∞(… www.wikipedia.org

16. Due to non-covalent interactions, proteins fold into complex structures…..

17. Representations of 3D Protein Structures Ribbon “Linguini” Ball and Stick Space-filling

18. My Research

19. MW = 17 kDa Isoelectric point ≈ 7.0 MW ≈ 153 kDa Isoelectric point ≈ 6.1-8.5 My Model Nanobioconjugate System *4.4 nm **15 nm Myoglobin Immunoglobin G Gold Nanocrystal MW ≈ 1,000 kDa Isoelectric point ≈ 5.5 14 nm *Kent, M.S.; Yim, H.; Sasaki, D.Y.; Langmuir2004,20, 2819-2829 **Hainfeld, J.F.; Powell, R.D. Journal of Histology & Cytochemistry2000, Vol. 48(4): 471-480 http://www.umass.edu/microbio/rasmol/igg_w.gif

20. Solution of HAuCl4•H2O 10-15% size distribution Particles > 10 nm in diameter Trisodium Citrate Synthesis of Aqueous Gold Nanoparticles Dispersion of Gold Nanoparticles +++ AuCl2- +++ AuCl2- AuCl2- AuCl2- Au AuCl2- AuCl2- AuCl2- AuCl2- +++

21. Synthesis of Nanobioconjugates + = or Gold Nanoparticles Unstable  Stable  But, how do we know if the proteins are actually attached? How do we determine the number of proteins on the surface of the nanoparticles?

22. Flocculation Assay Add 1% sodium chloride to nanobioconjugates with varying protein concentrations…. No protein / No NaCl No protein + NaCl Protein + NaCl …when the particles are completely covered by the protein, they will not change colors

23. 0.00 0.02 0.10 0.20 0.38 0.57 0.95 1.40 1.90 [protein] Flocculation Assay No Protein No NaCl Increasing Protein Concentration

24. 0 0.1 0.3 0.5 0.7 0.9 1 0 0.4 1.6 2.4 3.2 4 Quantify Flocculation Assays Ultra-Violet/Visible Spectroscopy [IgG] (uM) [myoglobin] (uM) control control

25. We have determined stability and quantified the number of proteins. Now, what about protein function?

26. Gel Electrophoresis • Separates proteins based on size… First, proteins are unfolded and charged…. Sodium Dodecyl Sulfate (SDS)

27. Gel Electrophoresis

28. Gel Electrophoresis

29. Gel Electrophoresis

30. Gel Electrophoresis

31. Gel Electrophoresis

32. Gel Electrophoresis

33. Gel Electrophoresis

34. Gel Electrophoresis

35. Gel Electrophoresis

36. Gel Electrophoresis

37. Gel Electrophoresis - + Apply a Voltage

38. Gel Electrophoresis - + Apply a Voltage

39. Gel Electrophoresis - Large Proteins + Small Proteins Apply a Voltage

40. Electrophoresis: Confirmation of IgG Orientation IgG bands either missing or too light to detect! myoglobin IgG IgG first Myo first

41. Nanobioconjugates for Epitope-Mapping

42. Objective To understand and manipulate protein-nanoparticle interactions Electrostatic Non- Specific What governs these types of interactions? Targeted Covalent

43. Objective To understand and manipulate protein-nanoparticle interactions Electrostatic Non- Specific What governs these types of interactions? Targeted Covalent

44. Definitions Antibody Antigen

45. Definitions Paratope Epitope Antibody Antigen

46. Definitions Paratope Epitope Antibody Antigen We actually don’t know the epitope of Myoglobin!

47. Epitope-Mapping • Studying the interactions of antibodies with specific regions of protein antigens • Very expensive and time-consuming!

48. Epitope-Mapping • Studying the interactions of antibodies with specific regions of protein antigens • Very expensive and time-consuming! • Why should anyone care? • Development of new vaccines & diagnostics

49. Can we use nanobioconjugates to determine the relative position of the epitope? ?

50. Can we use nanobioconjugates to determine the relative position of the epitope? ?