Download Biological AFM Setup

Protein Assemblies in Health and in Diseases: Biological AFM and
Related Studies
Havisha Garimella1,2
Preceptors: Dr. Albert J. Jin1, Dr. Paul Smith1
1Laboratory
Abstract
Interpretation
Results
Alzheimer's disease affects more than 5 million people; it cannot yet be cured[1].
Amyloid β protein is known to be associated with Alzheimer’s, as large deposits
of it were found in the brain of patients with Alzheimer's. Thousands of monomers
of the amyloid β build up exponentially in the brain, forming fibers. The problem
lies in the fact that the pathogenicity of the amyloid β, or how it exactly causes
Alzheimer’s, is unknown. There is a belief, though not as well known, that the true
culprit is the small soluble oligomers that makeup the polymers, with protein
conformational changes from random coil to alpha-helix, and to beta-sheets. The
atomic force microscopy (AFM), which provides high resolution 3D visualization at
the nanoscale [2], is used in the study. The machine is used to identify and record
protein oligomers’ conformational changes and shape in vitro, at all stages of the
polymerization of the amyloid β, that correlate to the loss of axon function in
Alzheimer’s. The structural biology and mechanics of protein assembly of the
amyloid β protein can be outlined through the AFM, which will give us insight into
its pathogenicity. The study of the amyloidosis of amyloid β is central to the
pathology of Alzheimer’s. It is necessary to understand on a macromolecular level
what triggers the complex folding mechanisms and shifts the equilibrium from
functional to pathological isoforms of proteins. By doing so we can map out the
pathway of amyloid β and inhibit the protein assembly.
Healthy Brain
of Cellular Imaging and Macromolecular Biophysics, National Institute of Biomedical Imaging and Bioengineering, NIH, Bethesda, MD, USA;
2Mount Hebron High School, Maryland, USA.
AFM Characterization
The reason why the APS modified surface worked, as opposed to the Ca2+ modified surface, is because the
APS surface is much smoother. Ca has a tendency to crystallize and therefore does not produce as uniform
of a surface.
Data and Analysis
No growth without NaCl
5 nm
2 hr on APS mica. Can see that
there are more fibers and they
have grown longer
Conclusion
0 nm
Advanced
Alzheimer’s
40 min on APS mica. Can see the
fibers beginning to form
More salt enables proteins to overcome charge repulsion and so they come
together. There is a significant change in height from the 40 minutes to the 2
hours. This could be because initially, when the fiber is forming, there are a lot of
nonstructural aggregates, i.e. salt, forming around it, so it causes the height to be
larger than normal. After the monomers build up and fibers form, the aggregates
disperse, giving a more accurate measurement of the height of the protein itself.
There are so many short pieces of fibers in the solution because when the
aggregate disperses, some of the protein comes with it. Because there is less salt
aggregate, the fibers are not as thick as it was in the initial stages; however, the
thickness does increase when fibers get tangled (7.4 nm). Also, there is a
significant increase in length. It is believed that the oligomers do no stack on top
of each other, but instead build up next to each other because the length gets
larger, but not the height.
100 nm
70 hr on APS mica. Can see that the fibers
are much longer and associating with one
another possibly forming plaques.
Left. Healthy brain compared to brain affected by
Alzheimer's[3].
Above. (Shown above.) Diagram of AFM setup [2].
Materials & Methods
Biological AFM Setup
The MultiMode AFM was used. Setup was
as follows:
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.
Above. MultiMode AFM (Bruker, CA).
Parameters for AFM Characterization
1mg/mL of Aβ (1-40aa) in 40 mM Tris-HC, pH 8.0, with 100 mM NaCl at 25o C
was imaged
• Protein taken from lyophilized Aβ and added to the salt solution, at which
point the assembly process begins.
• At various time points 5 µL of the assembling fibers solution is taken out and
diluted 100-fold into no salt 40mM, Tris-HC, pH 8.0, to stop the assembly.
Doing so allows us to look at the various time points of fiber formation under
the AFM, without worrying that the fibers are still assembling.
• Samples imaged on mica surface of; aminopropyl silatrane (APS). The
modification gives the surface + charge (see diagram). This was because it
was thought that the fibers were negatively charged because the calculated
isoelectric point of the Aβ was 6.
Studying this protein is a necessity because experts are certain that
amyloid β causes Alzheimer’s and other neurodegenerative diseases as
well. The key part of this study is to first characterize the protein assembly
of amyloid β. There is very little information regarding the structure and
kinetics of the Aβ, thus before one can understand the protein’s
relationship with Alzheimer’s, it is necessary to outline the fiber formations
of the protein. From the data and analysis, it is concluded that the Aβ
protein elongates. It initially aggregates a lot with the salt and biological
media, but later as the fibers form, the nonstructural aggregates disperse.
Also, the protein oligomers stack up next to each other. Moreover, another
vital discovery is that more salt enables this protein to grow. Significant
progress has been made as there are very few studies published on the
structural analysis of this protein; however, through this experiment, we
were able to provide biophysical characterization of it. This is very
important because by understanding the structure, we will be able to map
out the pathway, learn what specific oligomers correlate to axon loss, and
inhibit the protein assembly. The initial steps of the project have been
completed, which is to grow the fibers and record and study
conformational changes at all stages of the polymerization of amyloid β.
Now the future work required is to integrate dynamic light scattering,
mathematical modeling, optical Imaging, raman and fluorescence
spectroscopy, and theoretical analysis to identify its pathogenicity, to learn
how to prevent the protein growth, and to simply enhance our information
regarding its macromolecular pathway. The study of the amyloidosis of
amyloid β is central to the pathology of Alzheimer’s.