Protein Assemblies in Health and in Diseases: Biological AFM Havisha Garimella Intern/Mentor Mount Hebron High School Dr. Albert Jin

Preview of Presentation Personal Interest Internship Experience Research Question and Thesis Paper Project Product Purpose of Product Future Aspirations Conclusion Questions

Personal Interest Interested in medical field Wanted to study biomedical engineering after Independent Research G/T Surrounded by many relatives in medical field

Internship Experience Learned imaging equipment and techniques Did data interpretation Helped me to work independently

Atomic Force Microscopy High resolution imaging machine o Imaging (in biological fluids) o Fore-distance relations o Analyses (mathematical, etc.) Common AFM results o Structural information o Viscoelastic properties

Atomic Force Microscopy

Amyloid β Protein Large deposits found in brain of Alzheimer's patients Sequential cleavage of the amyloid precursor protein Monomers form fibers Fibers create tangles called plaques Pathogenicity unknown Causes various other neurodegenerative diseases

Research Question and Thesis Will using the AFM to identify the path of Amyloid β help us learn about the fiber formations and how to inhibit the protein assembly? By identifying the build up of the Amyloid β multimers, we will work to establish the mechanism of assembly for this protein fiber relevant to Alzheimer's disease, and learn how to inhibit it.

Paper Discussed : o Atomic Force Microscopy (AFM) o Alzheimer's Disease o Amyloid Beta Protein o Data Collection (images) o Results o Conclusion

Project Objective is to observe and record conformational changes at all stages of the polymerization of amyloid beta and identify specific protein oligomers in vitro that correlate to the loss of axon function in Alzheimer’s disease using atomic force microscopy

…Project Set up AFM: 1. Prepared mica layer on top of silicon disc for Multimode AFM. Peeled layer off using Scotch tape for a fully flat surface. 2. Aligned laser onto cantilever and engaged tip onto surface. 3. Set to tapping mode for readout. 4. Executed high-resolution scans. 5. Analyzed data with Nanoscope software and ImageJ.

…Project Fiber formation was tracked with 1mg/ml of Abeta(1-40aa) in 40 mM Tris-HC, pH 8.0, with 100 mM NaCl at 25 deg. C, using both AFM Amyloid β is taken from a vial of lyophilized Abeta and added to the salt solution, at which point the assembly process begins.

…Project At various time points (i.e. 40 mins, 2hrs)5ul of the assembling fibers is looked at When preparing the samples to look at under the AFM, three different preparations of the mica surface (after peeling a fresh layer) were prepared. Observed that APS (Aminopropyl silatrane) surface worked best; probably because it is the smoothest (causes uniform surface)

…Project 40 minute on APS mica. Can see the fibers beginning to form

…Project 2 hr on APS mica. Can see that there are more fibers and they have grown longer.

…Project 70 hr (end point) on APS mica. Can see that the fibers are much longer and associating with one another possibly forming plaques.

Structure of Amyloid β Protein  40 min. Fibers beginning to form 2 hr : 70 hr (end point). The fibers are much longer and associating with one another possibly forming plaques.

Structure of Amyloid β Protein  No growth without NaCl More salt, more fiber growth :

Structure of Amyloid β Protein After 80 min; monomers build up next to each other :

Structure of Amyloid β Protein

Product Display Board for science fair

Purpose of Product Science fair Easily conveys data and results

Future Aspirations Analyze images Modeling Integrate data from dynamic light scattering

Conclusion Studying this protein is a necessity because experts are certain that amyloid β causes other neurodegenerative diseases as well. However, the pathogenicity and exact assembly of the amyloid β protein is unknown. The amyloid β protein requires biophysical characterization via atomic force microscopy (AFM).

Conclusion Protein elongates Protein initially aggregates a lot, but later aggregates disperse Protein oligomers stack up next to each other Thickness decreases once fibers form and aggregates disperse Thickness increases again after fibers tangle

Questions?

Works Cited cts_and_figures.asp hat_is_alzheimers.asp#tangles Geisse N.A. (2009). AFM and combined optical techniques. MaterialsToday. 12(7,8):40-45.