Macromolecules for The Demented
and methods for their study
Help from Keunok Yu, Jirun Sun, Bethany Lyles, George Newkome and
LSU’s Alz-Hammer’s Research Team
Krispy Kreme Donut Day, September 2003
Supported by National Institutes of Health-AG, NSF-DMR and NSF-IGERT
•
•
•
•
How Alzheimer’s happens
Attempts to prevent or reverse it
Characterization challenges
Alzheimer’s model systems with materials implications
Amyloid Diseases
• Several diseases are caused by the misfolding of
proteins into self-associating structures (fibrils)
w/ predominantly b-sheet secondary structure
• Alzheimer’s Disease: Amyloid b-protein (Abin
neurons/brain
• Type II Diabetes: amylin in Islets of Langerhans
• Mad Cow Disease (BSE): PrpSc in brain --
transmittable by protein aggregates
• Huntington’s Disease, triplet repeat expansion: (Gln)n
Age:
Positron emission tomography
20
80 Normal 80 Alzheimer’s
Postmortem Coronal Sections
Normal
Alzheimer’s
PET images courtesy of the Alzheimer's Disease Education and Referral Center/National Institute on Aging; Postmortem images
courtesy of Edward C. Klatt, Florida State University College of Medicine
APP = Amyloid Precursor Protein
http://www.bmb.leeds.ac.uk/staff/nmh/amy.html
APP = the larger, lighter pink one
•Transmembrane protein
•Normal function not known
•Educated guesses
May help stem cells develop identity
Or help relocate cells to final location
May “mature” cells into structural type
May protect brain cells from injury
Synaptic action
Copper homeostasis
•Anyway, you need it.
•Normal “clipping” of APP by a “secretase” enzyme (in red, and also assumed
to be a transmembrane protein) is shown.
•There are several secretases, also associated proteins, and they seem to
mutate easily: there is a genetic link.
•It is not exactly clear why things go awry with advanced age.
Clipping APP the right & wrong ways
NH2 terminus
Correct
Unlike the α-secretase enzyme, which
cuts APP into shorter protein
fragments in the cell's cytoplasm,
the γ-secretase cleaves its target
within the hydrophobic membrane
of the cell. The transmembrane
proteins called presenilins permit
access to the γ site on APP.
Mutations in the genes coding for
presenilins are frequent causes for
autosomal dominant Alzheimer's
disease.
Incorrect
Feature article by Vernon M. Ingram
American Scientist on-Line
Vol. 91, #4 July-August 2003
http://www.americanscientist.org/template/IssueTOC/issue/394
Exporting the dangerous fragments
Figure 6. The Aβ1-42
protein fragment is
exported from the cell
immediately after being
cut from the parent APP
molecule. Once it
reaches the extracellular
space, the peptide
refolds to form a sticky
shape that clumps
together as an insoluble
aggregate.
Feature article by Vernon M. Ingram
American Scientist on-Line
Vol. 91, #4 July-August 2003
http://www.americanscientist.org/template/IssueTOC/issue/394
Amyloid hypothesis: fibrils or protofibrils cause
cell death, possibly as the body’s own defenses
tries to clear such “foreign” matter.
Peter Lansbury Group
http://focus.hms.harvard.edu/1998/June4_1998/neuro.html
Competing hypothesis: channel formation disrupts Ca+2 metabolism
Introduction of Varying Salts to Increase
β-amyloid Aggregation, Ab 10-35
NaCl
NaNO3
10 mm x 10 mm scanning probe microscope images (on mica)
of 300 mM Ab10–35 incubated for 8 days at room temperature
in 15 mM phosphate buffer containing 50 mM salt.
NaF
Alzheimer research group, Team goal:
Mediate or alter the Aggregation of Ab
R = regular
H = hydrophobic
Exemplify with Double-stick Tape
Peptide-based Mediation Requires a Specific Sequence
 Hydrophobic KLVFF region is responsible for βamyloid aggregation
 Incorporation of such region for β-sheet breaking or
capping
H-(Lys)-Val-Leu-Phe-Phe-(Lys)6-NH2
A peptide construct incorporating the KLVFF region developed
by Professor Regina Murphy at the University of Wisconsin-Madison
Peptide-based Inhibitors of Ab Fibrillogenesis
Ab16-20 core
Ab16-22 core
"Disrupter"
H3N
H3N
H
H3N
O
H
N
H
O
N
H
H
O
H
N
H
O
N
H
H
O
O
H
N
6
H
H-Lys-Leu-Val-Phe-Phe-(Lys)6-NH2
"Disrupter"
CH3 O
H
NH2
N
H3N
O
NH3
N
H
H
H
O
CH3
N
H
CO2
O
N
H
N
O
Meredith
Ab core analog
H2N
O
H2N
H
N
H
NH2
HN
H
O
O
N
H
H
N
H
H
O
O
N
H
NH3
H
HN
H2N
NH2
H CH3
H
N
H
NH2
O
H-Lys-(Me)Leu-Val-(Me)Phe-Phe-(Me)Ala-Glu-NH2
Murphy
"Disrupter"?
CH3 O
H
H-DLeu-DPhe-DLeu-DArg-DArg-NH2
Norstedt
LSU Peptide-based Mediators
K
L
V
F
F
AMY-1 x = 1, y = 6
AMY-2 x = 6, y = 1
AMY-3 x = 1, y = 1
MCP 1
K
L
V
F
F
MCP 2
O
H2N
Dbzg
O
OH
H2N
OH
Dibg
Mediators Developed by Professor Robert Hammer & Professor Mark McLaughlin
Synthesis & Vetting of Peptide
with aaAA-Blocker
KH
H2N
O
H
N
O
V H
N
iBu iBu H
O
H
N
O
N
Bn Bn H
F H
O
H
N
Pr Pr
O
(NH
K H
NH
)
O 6
2
H-Lys-Dibg-Val-Dbzg-Phe-Dpg-(Lys)6-NH2
HPLC of crude peptide
MALDI-MS of purified peptide
Calc’d for (M + Na) = 1731.3
Determining Mediator Efficacy
Using Transmission Electron Microscopy
Control
50 mM Ab1-40
4.5 months
10 mM
50 mM Ab1-40
50 mM AMY-1
4.5 months
10 mM
10 mM
Control
50 mM Ab1-40
5 mM AMY-1
4.5 months
1:1 Ab:Inhibitor
10:1 Ab:Inhibitor
Even a sub-stoichiometric amount of AMY-1 inhibitor is effective
TEM image after 4.5 months at Room Temperature
50 mM phosphate buffer/ 150 mM NaCl pH 7.4
But such limited success is very after-the-fact.
Can we use diffusion-based and other methods
to determine the early stages of aggregation?
Can we follow it in real-time in vitro?
Two Possible choices:
• Dynamic light scattering
• Fluorescence photobleaching recovery
A series of dynamic light scattering runs can identify a
peptide that has an effect on large fibrils.
50000
Addition of 5mL of 500mM
Murphy Peptide dissolved
in water
40000
b -Amyloid1-40 incubated
R h,app / Å
30000
in 100% DMSO followed
by dilution in PBS pH 7.4
20000
10000
Sonicate in water bath
for 10 mins with probe sonicator
0
0
10000
20000
30000
40000
Time/s
50000
60000
70000
That’s OK for simple screening, but
there are problems with DLS
1) the size is only an apparent value, because of
the single angle used for measurement;
2) the presence of small protofibrils, and the
effect of inhibitors on them, is difficult to
ascertain, especially in the presence of larger
fibrils that dominate the scattering;
3) reversibility is not easily studied; and,
4) experimentally tedious for early stages of
aggregation.
Modulation FPR Device
Lanni & Ware, Rev. Sci. Instrum. 1982
SCOPE
5-10% bleach depth
IF
PA
c
X
TA/PVD *
PMT
*
D
S
*
DM
OBJ
M
RR
*
M
ARGON ION LASER
AOM
* = computer link
Labeling β-Amyloid fragment 1-43
H2N-DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIAL-COOH
When fluorescein is attached, we call it L-Ab
Fluorescein has
about 7% the
mass of Ab.
Sticht, H.; Bayer, P.; Willbold, D.; Dames, S.; Hilbich, C.; Beyreuther, K.;
Frank, R.; Rösch, P.Eur. J. Biochem. 1995, 233, 293-298.
Diffusion Results – Great Reproducibility
But Dye Shrinks it…and may stabilize against aggregation.
6.0
5.5
pH 2.7
pH 6.9
pH 11
Sodium Fluorescein Dye
5.0
4.5
-6
D/10 cm s
2 -1
4.0
3.5
3.0
2.5
2.0
Theoretical/Experimental D0 for monomeric β-amyloid1-40
1.5
1.0
0.5
0.0
0
5
10
15
20
25
30
35
Days
100 μM Mixture β-amyloid1-40 in phosphate buffer – pH 2.7, 6.9 and 11
Theory/Experimental result for monomeric Ab1-40 from: Massi, F.; Peng, J.W.; Lee, J.P.; Straub, J.E. Stimulation Study of the Structure and Dynamics of the
Alzheimer’s Amyloid Peptide Congener in Solution. Biophysical Journal 2001, 80, 31-44.
Back to DLS: L-Ab does not prevent formation of
large fibrils when mixed with unlabeled material
and fibrils increase in size with added salt.
100 mM b-Amyloid1-40 in PBS, pH 7.4
7.0
6.5
6.0
5.5
-9
D/10 cm s
2 -1
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
Mixed labeled & unlabled
0.5
0.0
0
25
50
75
100
125
150
Ionic strength/ mM
175
200
Epifluorescence also shows L-Ab is
actually incorporated into macrofibers.
Bottom Line: we think L-Ab is OK to study.
Two FPR Contrast Decay Modes are Often
Observed: Fast = small; Slow = large.
Contrast / Arbitrary
1
pH 2.7
pH 6.9
pH 11
0.1
0.01
1E-3
1
10
100
t/s
1000
Doing More Experiments Faster with Less
Precious Amyloid: Dialysis FPR
Pump
Exchange Fluid
Cover slip
Sample
PTFE spacer
Dialysis membrane
O-ring
Evolution of protofibrils from labeled monomer after dialysis
against a weak citrate buffer at pH 5.0. After one hour,
large aggregates appear and represent ~ 18% of the signal.
FPR of 50mM labeled b-amyloid1-40 in 10 mM KOH
dialysis againt 10mM Citrate buffer pH 5.0
2.0
-6
Diffusion Coefficient/ 10 cm s
2 -1
2.5
1.5
Amplitude 81.5% ; 82%
1.0
0.5
Amplitude 18.5% ; 18%
0.0
0
20
40
60
80
Time/min
100
120
140
Finding a convenient buffer for controlled self assembly. This run
is at pH 4 Acetate Buffer. Adding calcium hastens aggregation.
One Pot Dialysis FPR of 50mM 5CFb-Amyloid1-40 in 10mM KOH
Slow
Fast
2 Exponential Analysis
1E-6
100mM Acetate buffer pH4
0.1N HCl pH1
1E-7
Percent Amplitude
Diffusion Coefficient/ cm s
2 -1
50mM PB PH 7.2
0
5
10
15
20
Scan #
1E-8
5mM CaCl2
10mM CaCl2
15mM CaCl2
25mM CaCl2
1E-9
0 2 4
6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
2
Time/10 mins
Fast
Slow
Amplitudes
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5
0
25
30
35
Reversing Amyloid Aggregation…by pH
FPR Study: Reversibility of b-Amyloid Aggregation
100mM 5-CF-b-Amyloid1-40+ b-Amyloid1-40 pH 11
dialysis against 50mM PB pH 7.4
-6
D/10 cm s
2 -1
1E-6
1E-7
dialysis against 50mM PB pH 2.7
1E-8
0
200
400
600
800
1000
1200
1400
1600
1800
Time/min
Diffusion from in situ FPR of 5-carboxyfluorescein-Ab1-40 (25% mixed with
unlabeled 75% Ab1-40) starting at pH 11, then alternately dialyzed between 50
mM phosphate (pH 2.7) and 50 mM phosphate (pH 7.4).
What has any of this got to do with
Nanomaterials???
Ab = $400,000/gram
Need cheaper model systems.
They also have materials applications.
Bolaform amphiphiles have a
dumb-bell shape.
hydrophilic
hydrophilic
hydrophobic
Arborol example: [9]-10-[9]
9 watery hydroxyl
groups
OH
HO
HO
H
N
HO
HO
HO
HO
HO
HO
HO
N
H
O
O
10 oily
methylene groups
O
N
H
O
O
OH
H
N
OH
OH
O
N
H
H
N
OH
OH
OH
OH
OH
Toolbox
Small angle X-ray scattering: analysis of the
structure (inverse space)
q
Basic idea:
Detector
I
Sample
Synchrotron
I
Scattering
envelope
X-ray
q
Intensity
Melting an 8% Arborol Gel
0.012
pr25
0.01
pr26
pr27
0.008
pr28
0.006
pr29
pr30
0.004
pr32
0.002
pr33
pr34
0
-0.002
pr35
0
0.5
1
1.5
2
q/nm
Eleven runs
Start time (30oC): 3:35 am
End time (90oC): 5:27 am
2.5
pr36
Stacked dumbbell model
1
Based on molecular
modeling, SAXS,
FF-SEM, DSC,
AFM, POM…and
common sense.
qI(q)
0.1
0.01
1E-3
1E-4
0.0
0.2
0.4
0.6
0.8
1.0
2
-2
1.2
1.4
1.6
q / nm
z
x
I(q) = S(q)P(qr)
1
S(q)  2
M
M
M

j1( j i ) i 1
sin(qrij )
N+N1
3N+(N-1)
(back) N+
2N+
5
3
3N+5 5
(back) N+
2N+
3
1
3N+3 3
(back)N+
3N+1 1 Origin(0
(back) 4N+2
,0,0)
2N+
1
4N+
1 2N+
2
2
qrij
 3

P(qr )   3   sin x  x cos x 
x

N1
2N+(N1)
y 5
3N+2
(back) N+
2N+
4
2
3N+4
(back) N+
2N+
6
6
3N+6 4
(back)N+
3
N
6
4N (back)
2
4
N
b
a
2
Nradius, r
Synthesis of Inhibitor [9]-6
O
Br
bromohexane
+
O
OEt
O
NaC
OEt
OEt
O
DMF/Benzene
OEt
OEt
OEt
O
O
triethyl carbonate
OH
OH
3 H2 N
OH
H
N
OH
OH
OH
O
O
N
H
OH
DMSO
O
[9]-6
H
N
OH
OH
OH
OH
OH
Self-assembly of [9]-12-[9]
Starting point is “extruded” fibers
1300
[9]-10-[9]only
[9]-10-[9] plus [9]-6
1200
1100
Rh/(Å)
1000
900
800
Rh from linear fit of gamma vs q2
of DLS data at five angles:
40, 50, 60, 70 and 90.
700
600
500
400
0
1
2
Number of Days
3
4
Scientific Conclusions
• Promising inhibitors have been designed and constructed.
Probably even more expensive than Ab itself.
• DLS can screen promising ones.
• Dialysis FPR can observe Ab deconstruction in real time. So
far, only by pH, but dialysis experiments with precious
inhibitor are coming.
• Model systems to practice with can teach us better
methods…and have some materials science applications.
• Many things not shown: e.g., Ab slows diffusion of the lipids
that make cell membranes. Is this important?
Broader Conclusions
• Membrane proteins (or fragments) are hard to
study.
• Don’t expect a cure soon and you won’t be
disappointed.
• Take your statins once the doctor tells you to start,
then hope for the best.
• Science in the service of practical problems is
increasingly multidisciplinary.
• Scientists spend a lot more time scratching their
heads and wondering what’s going on than it must
seem from textbooks.
Discussion Points
•
•
•
•
We have spent $1.4 M for this research (so far).
Perhaps 10 papers will appear eventually.
About six Ph.D. students will be trained.
Could 100,000 teams like ours (that’s 2000
in every state of the United States!) cure
Alzheimer’s, Mad Cow, Huntington’s and
other related afflictions?
Lecture Checkup
(do NOT write your name)
• What is the single most important thing you learned from
this lecture?
• What is the ONE thing you wish you understood better
about this lecture?
• Action item: what will you do to understand better?