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

“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

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

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.

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

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

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. 291-301 (2001).

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

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

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

Circular Dichroism reveals secondary structure of aqueous proteins http://www.newark.rutgers.edu/chemistry/grad/chem585/lecture1.html

Peptides with Predictable Structures 4A capped: Ac-AAAAKAAAAKAAAAKAAAAY-NH2 In a helix, charged lysine side chains will encircle the structure. http://marqusee9.berkeley.edu/kael/helical.htm

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

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. http://marqusee9.berkeley.edu/kael/helical.htm

CD Data: Sticky K forms random coils in solution

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

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

IR Peaks are caused by atomic vibrations Amide I (1600-1700 cm-1): Due to C=O stretch Amide II (1510-1590 cm-1): Due to N-H deformation Both regions are sensitive to secondary structure

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

4A ATR-IR Data

Sticky K ATR-IR Data

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

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

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

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

Pathway of ß-Sheet Formation ß-strand

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

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

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

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

Birefringence can be detected using polarized light This slide shows H1 stained with Congo Red dye. H11819 16 hours without polarizer When viewed through polarized light, birefringent particles can be seen. H11819 16 hours under polarized light

Congo Red H1 fibrils H1 2021 fibrils after incubating for 12 hours

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

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

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