1. Observations of Peptide Behavior on the Nanoscale:
Peptide Absorption on Nanophase Al2O3
Elizabeth Ballou, ‘04
Summer Research 2003
Sean Decature, Advisor

2. What is “Nano” Technology?
                                                                                                    
What is “Nano” Technology?
“Nano” refers to things in the range of 10-9 meters
H-bonds can range from 3-5 Å
10 Å = 1 nm
Red blood cells are 20 m, or 2000 nm
Nanotechnology bridges the gap between atomic structure and cellular structure

3. Peptide Absorption on Nanophase Al2O3
Goal: To examine how peptides may undergo changes in conformation when in contact with nanophase ceramics

4. Nanophase Ceramics Have Applications in Prosthetics
Nanophase ceramics are stronger and more dense than their conventional grain sized counterparts.
Uses in orthopedics and dentistry include strengthening the interface between natural bone and prosthetics or implants.

5. Nanophase Materials Bind Biological Materials
How can these surface interactions be understood?

6. Can a Surface Affect Protein Conformation?
OSTEOBLAST
OSTEOBLAST
Can a Surface Affect Protein Conformation?
Vitronectin
RGD
RGD Ca Ca Ca Ca Ca
Nanophase Alumina
Change in conformation, more than change in structure
Conventional Alumina
120 nm grain size
38 nm grain size
Tom Webster, L. Schadler, R. Bizios, R.W. Siegel, Tissue Engineering, vol (2001).

7. Structure of an -Helix
3.6 residues per turn
H-bonding between C=O and N-H every fourth residue

8. Methodology Make Al2O3 substrates
Apply 10 tons of preasure to 38nm grain size Al2O3 powder for 20 minutes
Sinter to 1000˚c for nanophase substrates
Sinter to 1200˚c for conventional substrates
Clean and degrease in acetone and ethanol
Soak in peptide/buffer solution for 24 hours
Measure percent peptide adsorbed using UV-Vis spectrometer

9. Peptide Synthesis Synthesize peptide from amino acids
Cleave from handle, precipitate into ether
Centrifuge and Lyophilize
RP-HPLC
Lyophilize
Exchange in 1.0% PO3- to remove TFA
Examine backbone using CD
Adsorb onto Alumina

10. Circular Dichroism reveals secondary structure of aqueous proteins

11. Peptides with Predictable Structures
4A capped:
Ac-AAAAKAAAAKAAAAKAAAAY-NH2
In a helix, charged lysine side chains will encircle the structure.

12. CD Data: 4A is helical in solution
Add something about CD theory

13. Peptides with Predictable Structures: Sticky K
Ac-KAAAKAAKAAAKAAKAAAKY-NH2
In a helix, charged lysine side chains will be limited to one face of the structure.

14. CD Data: Sticky K forms random coils in solution

15. Peptide Absorption (%)
Measure changes in peptide concentration before and after absorption onto disks using UV-Vis spectrometry.
Data was not consistent.

16. ATR-IR of Peptides on Powdered Al2O3
ATR: Attenuated Total Reflectance

17. IR Peaks are caused by atomic vibrations
Amide I ( cm-1): Due to C=O stretch
Amide II ( cm-1): Due to N-H deformation
Both regions are sensitive to secondary structure

18. Secondary structures can be distinguished by IR spectra
ß-sheet
 -helix
Random coil
Amide I
Amide II

19. Initial ATR-IR Data
Sticky K 4A

20. Improved Resolution 4A
Sticky K

21. Sticky K and 4A: Peptide-Al2O3 Matrix

22. Summary and Further Experiments
4A and Sticky K were observed to undergo a conformation change when adsorbed onto nanophase Al2O3
Continue to examine 4A and Sticky K on nanophase Al2O3
Examine 4A and Sticky K on conventional Al2O3
Measure change in Contact angle on substrates with and without peptides

23. Aknowledgements Professor Sean Decatur, MHC
Professor Linda Schadler, Materials Science, RP
Wendy Barber-Armstrong
Aggie Starzyk
Catherine Pevtsova
Noelle Smith for listening

24. Nanophase Alumina Conventional Alumina ~38nm grain size
Higher surface energy
Has shown better osteoblast adhesion in past studies
>100nm grain size
Lower surface energy
Decreased osteoblast adhesion in previous studies

26. Meet the Peptides
Sticky K
Mini Helix 4A
Sticky E