Amazing A-beta All Stars
…what serious money and great
talent tell us about b-amyloid
P. Thiyagarajan, et al.—Argonne National
Laboratory & University of Chicago
Applied Crystallography Article
J. Appl. Cryst., 33, 535-539 (2000)
This article describes the fibril
structure of a sub-peptide of Ab,
sometimes with the attach-ment
of a blocking polyethylene glycol.
The idea is to figure out whether
and how an attachment to the
Ab will affect the fibril formation.
PEG
H2NAb 10-35
SANS = Small Angle Neutron Scattering
Argonne National Laboratory (near Chicago)
Radially-averaged intensity profile
Detector with time of flight capabilities,
can measure over a wide range of
scattering vectors simultaneously
Sample
0.5 Å to 15 Å neutrons in each pulse
Pulsed neutron source
SANS
Radially-averaged intensity profile
Destructive interference between
neutrons scattered from different
parts of the Amyloid fibrils causes the
intensity to decline with angle. The
specific pattern of that decline can be
interpreted in terms of fibril thickness
and mass per unit length.
I(q)
q
Interferences due to fibrillar shape can be “taken out” of the data by
multiplying intensity by scattering vector magnitude, q. The
remaining decline in intensity is due to finite fibril thickness.
Rod-Modified Guinier Plot—Slope
Gives Fibril Cross-sectional Radius
Rg,c
Findings
Fibril thickness depends strongly on pH.
Fibril-fibril associations are less at low pH (even
though, from our experience, such conditions
lead to rapid self-assembly of Ab).
Fibrils are thicker when PEG is present (by 23 –
30 Å)
PEG is located along the periphery of the fibril.
Interdisciplinary Considerations
Think what goes into such an experiment
• Someone to build/program SANS (several $M)
• Someone to make peptide
• Someone to attach PEG
• Someone to collect & analyze data
• Someone with the intellectual lead to direct project,
set standards, monitor competitors & write the results.
• Nine (9) authors in all!
Robert Tycko
National Institutes of Health, Maryland
Current Topics Article (i.e., review)
Biochemistry, 42(11), 3151-3159 (2003)
If I wrote one article like this one in my whole
life, that would represent a great career.
General purpose of the article: review
structural methods, especially solid state
NMR, for determining b-amyloid structure.
Amyloid does not crystallize, so
crystallography won’t suffice.
The information required exceeds
what small angle X-ray and Small
angle neutron scattering can provide.
No choice but to haul out a big NMR
(and serious labeling, simulation, etc.).
13C
labeling off-center will produce
different dipole-dipole coupling for
Free peptides
Various derivatives
(circles) and simulated for
different distances (lines)
“These data
indicate an inregister, parallel
alignment….”
Tycko Figure 2
13C-13C
exchange using a 9.4T NMR
indicates which specifically labeled residues are closely coupled.
Multiple-quantum NMR absorptions
require proximal nuclei.
“An n-quantum 13C NMR signal is
observable only if at least n 13C nuclei are
sufficiently close in space to be linked by a
network of couplings with coupling
constants d ~ 1/tMQ”
Again, simulations are performed to show
that parallel alignment of the peptides
occurs.
Figure 3
Proposed Model of Amyloid
Each molecule has 2 beta
strands, red and blue
Hydrophobic
Polar
Positively charged
Figure 6
EM provides less detail, but it is so
much easier!
Helmuth MÖhwald & Coworkers
Max Planck Institute for Colloids and Interfaces
Golm, Germany
This group is devoted to surface studies.
One mechanism of nano-organization and self
assembly that we have not discussed is organization
of nanostructures at surfaces.
So…here is a good example concerning how Ab
organizes near the air-water interface.
Air is hydrophobic (despite the humidity in Baton
Rouge or Bangkok!) so this is a model for interaction
with other hydrophobic surfaces in the body—cell
walls, some protein surfaces, etc. That kind of
interaction is a little more complex—a colleague
studies it back at LSU.
Langmuir Trough
0.0015
Reflection Infrared
Absorption
Wilhelmy plate surface tension measurement
Movable barrier
Surface Reflection Absorption Infrared
“The central message
of this work is that a
stable b sheet-enriched
state of the amyloid is
formed at the airwater interface, in
contrast to the initial
bulk solution
containing high ahelix/random coil….”
Stability
It was also found that the reflection IR spectra were
stable to several compression/dilation cycles.
In the plane of the air-water interface there exist
antiparallel b-sheet structures.
Certain alignments of the self-assembled structure
could be ruled out, but the essential information is
that the surface induces b-sheet formation.
For structural details, the Tycko work is better.
Alignment
Certain alignments of the
self-assembled structure
could be ruled out, but the
essential information is that
the surface induces b-sheet
formation. For structural
details, the Tycko work is
better.
Kim & Lee
Fluorescence Nanoscopy Laboratory
Department of Chemistry Ewha Womens University
Seoul, Korea
Nanomedicine: can a Buckeyball cure
Alzheimer’s?
My colleagues laughed at this paper.
Afterall, we are seriously about making peptide
inhibitors at LSU, and everyone knows (or
thinks they know) that the answer will be a
peptide.
But let’s have a look.
Buckey inhibitor?
Fluorescence data shows that the selected Fullerene
suppresses Ab aggregation. Compare to Melatonin, a
circadean hormone.
IC50 values
(inhibitory concentration for 50% reduction)
Fullerene works about as good as anything else!
Point of Discussion
Scattering and NMR provide indirect evidence
for the structure.
That is the most detailed info we have, but….
In the future, what will be the role of NMR,
scattering and other techniques?
If we are to build Erick Drexler’s nano-robots,
won’t they have nano-vision?
Tim Lodge story.
What’s
Next?
Advancements in Cryo-TEM may give soft matter microscopy
almost the power that solid state enjoys (i.e., sub-nanometer)
Jimenez et al.
EMBO Journal
18(4), 815-821 (1999)
“890 cut-out repeats* were iteratively aligned by a
multivariate statistical analysis.”
Then the images are manipulated in 3D
*Image sections
Microscopy at 25 Å resolution
Conclusion
Disease-inspired inventions and innovations to study
nanometer-scale objects will continue to be
important as non-biological nanoscience develops.
With apologies to Richard Feynman, there is always
room at the interfaces !
Some interesting interfaces
•
biological-materials
•
fundamental-applied
•
theory-imaging
•
nano-micro