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 ElectrostaticallyCovalently - - - - - - - - - - - - - - - - + + + + + + + + + + + + + S S S S S S S S

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

5. What are Nanobioconjugates? A biomolecule (protein, DNA, RNA, etc.) attached to a nanomaterial ElectrostaticallyCovalently - - - - - - - - - - - - - - - - + + + + + + + + + + + + + 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 Non- Specific Targeted Electrostatic Covalent What governs these types of interactions?

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

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

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

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

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

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

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

18. My Research

19. My Model Nanobioconjugate System MW = 17 kDa Isoelectric point ≈ 7.0 http://www.umass.edu/microbio/rasmol/igg_w.gif **15 nm*4.4 nm *Kent, M.S.; Yim, H.; Sasaki, D.Y.; Langmuir 2004,20, 2819-2829 **Hainfeld, J.F.; Powell, R.D. Journal of Histology & Cytochemistry 2000, Vol. 48(4): 471-480 MW ≈ 153 kDa Isoelectric point ≈ 6.1-8.5 14 nm MW ≈ 1,000 kDa Isoelectric point ≈ 5.5 Myoglobin Immunoglobin G Gold Nanocrystal

20. Synthesis of Aqueous Gold Nanoparticles Solution of HAuCl 4 H 2 O Trisodium Citrate Dispersion of Gold Nanoparticles 10-15% size distribution Particles > 10 nm in diameter +++ Au AuCl 2 -

21. Synthesis of Nanobioconjugates Gold Nanoparticles + Unstable  Stable = or 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…. …when the particles are completely covered by the protein, they will not change colors No protein / No NaClNo protein + NaCl Protein + NaCl

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

24. Quantify Flocculation Assays Ultra-Violet/Visible Spectroscopy 010.10.30.50.70.9 01.643.22.40.4 control [IgG] (uM)[myoglobin] (uM)

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

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

27. Gel Electrophoresis

37. + - Apply a Voltage Gel Electrophoresis

38. + - Apply a Voltage Gel Electrophoresis

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

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

41. Nanobioconjugates for Epitope- Mapping

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

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

44. Definitions AntibodyAntigen

45. Definitions Antibody Paratope Antigen Epitope

46. Definitions Antibody Paratope Antigen Epitope 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. ?

51. ?

52. ? One mutant’s interaction with the gold nanoparticles should occlude the epitope region and either prevent IgG from binding or decrease its binding affinity in comparision to the other mutants.

53. Myoglobin Mutants 4 myoglobin mutants: K63C, H81C, E105C, and G121C

54. Myoglobin Mutants K63CH81C E105C G121C WT WT alone IgG alone 4 myoglobin mutants: K63C, H81C, E105C, and G121C

55. Myoglobin Mutants K63CH81C E105C G121C WT WT alone IgG alone 4 myoglobin mutants: K63C, H81C, E105C, and G121C K63C mutant does not interact as well with IgG (near epitope?)

56. Summary & Conclusions Nanobioconjugates – Nanomaterial + Biomolecule – Have both nano and bio properties – Stability can be quantified – Stoichiometry can be evaluated Epitope-Mapping – Nanobioconjugates provide an easier and cheaper way of determining epitopes.