1. Observations of Peptide Behavior on the Nanoscale:
Peptide Absorption on Nanophase Al2O3
AND
Nanoscale Structure of H1 Prion Fibrils
Elizabeth Ballou, ‘04
Summer Research 2003
Sean Decature, Advisor

2. “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, more dense, and have other unique properties compared to their conventional grain sized counterparts.
Uses in orthopedics and dentistry include strengthening the interface between natural bone and prosthetics or implants.

5. 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

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

7. 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).

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

9. 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

10. 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

11. Circular Dichroism reveals secondary structure of aqueous proteins

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

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

14. 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.

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

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

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

18. 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

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

20. 4A ATR-IR Data

21. Sticky K ATR-IR Data

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. Nanoscale Formation of H1 Prion Fibrils
Goal: To examine how H1 Prion peptides behave as aggregates on the nanoscale

24. Prion Diseases
Prion related diseases include Mad Cow Disease, Creutzfeldt-Jakob Disease, and Alzhimers.
Deterioration of organs results from a build-up of protein aggregates called Amyloid Fibrils.
Cross-section of brain with stained prion plaque

25. Mutation of the Prion Protein
PrPc: Native form
Primarily -helical
Four main helices: H1, H2, H3, H4
PrPsc:Abnormal isoform
Primarily -sheet
H1, H2, H3, H4 now form sheet

26. Pathway of ß-Sheet Formation
ß-strand

27. ß-Sheets have a Right-handed Twist
ß-Sheet with twist
(as seen along axis)

28. PrPsc Forms Fibrils and Placques
B-sheets fold together to make coiled-coil structures called Amyloid Fibrils.

29. Ordered Structures Exhibit Birefringence
Birefringence: The quality of transmitting light unequally in different directions
In biological materials, this indicates a certain degree of ordering to the structure, such as might be seen in a crystal
Amyloid fibrils exhibit birefringence because of their regular ß-sheet structure

30. Methodology Synthesize H1 peptide Incubate in 20 mM Hepes buffer
Stain sample with congo red dye
View birefringence through polarizer
Record IR of sample

31. Birefringence can be detected using polarized light
This slide shows H1 stained with Congo Red dye.
H hours without polarizer
When viewed through polarized light, birefringent particles can be seen.
H hours under polarized light

32. Congo Red
H1 fibrils
H fibrils after incubating for 12 hours

33. H1 FTIR Analysis
Figure 3: H1 after incubation in Hepes buffer for 5 days

34. Summary and Further Experiments
H1 Fibrils form aggregates with regular, ß-sheet structure
Continue to observe H1 precipitate over time
Use ATR-IR to examine Amyloid fibrils

35. Aknowledgements Professor Sean Decatur, MHC
Professor Linda Schadler, Materials Science, RPI
Professor Marian Rice, Biology, MHC
Wendy Barber-Armstrong
Catherine Pevtsova
Aggie Starzyk