disassembly and assembly of peptide fibrils via local...
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ChemMatrix
Deprotection = 20%Piperidine/DMFCoupling = COMU, DIEA
NHOO
O
O
NH2
Cleavage = TFA:TIPS:DMB 92.5 : 2.5 : 5
2hrs
Fmoc SPPSNH
HN
O
O
N
O
NNH
HN
O
O
O OH
NH
OHHN
O
O
NNH O
O
NHN
O
NH
NHH2N
O
NNH O O
NH
OHHN
O
HOO
HN
O
O OH
NH
OHHN
O
NH O
H2NO
N
NH
Spacer Q11
4 aa 11 aa
Spacer
Q11
4 aa
11 aa
NH
HN N
H
HN N
H
HN N
H
HN N
H
HN
O
O
O
O
O
O
NH2OOHO
NH2O
OH2N
O
OH2N
O
O
NH2
O
NH2O
OH2N
OH2N
NH2=
HN NH
HNO
O
O
O
OHOH
=
Q11 Self-assembly domain
MUC1Q11 was synthesized on solid support. Purity of the peptide was verified by HPLC and Mass Spectroscopy.
Disassembly and assembly of peptide fibrils via local mechanical perturbation
Arpad Karsai1, Teri Jo Lanoue1, Fadi Khoury1, Hamed Malekan1, Weifeng Lin1, Victoria Tran1, Daniel Cox2, Xi Chen1, Gang-yu Liu1* 1* Department of Chemistry, University of California, Davis, CA 95616, USA. 2 Department of Physics, University of California, Davis, CA 95616, USA.
Acknowledgments The project was funded by University of California, Research Investments in the Sciences and Engineering (RISE) program, NIH and W. M. Keck Foundation.
MUC1, 20 residues long human glycoprotein segment
Lyophilized peptide was dissolved in 1/6xPBS buffer (pH 7.4) with 200 µM or 400 µM final concentration and was incubated for 1 – 5 days at room temperature. Fibril formation were studied with Atomic Force Microscope (AFM) imaging. Samples were deposited on freshly cleaved mica surface and were scanned in aqueous condition using tapping mode imaging using Olympus Biolever A. Mechanical perturbation was carried out by: 1. Contact mode AFM imaging using set point
equivalent with 1.0nN Force vertical force. 2. AFM force measurements imposing 2.0 - 4.0nN
vertical force to the sample. 3. Low set point high free amplitude (ratio ≤ 0.6) AC
mode scanning.
MUC1Q11 is a 35 residues peptide known to form fibrils, which exhibit potential for cancer vaccine development. Using time-dependent and high resolution atomic force microscopy (AFM) imaging, this presentation reveals that MUC1Q11 forms fibrils on mica, following a nucleation process along the crystal axis of mica(0001) surfaces. Under 10 to 20 μM concentration and room temperature, individual fibrils as long as 1 micron could form within 2 hrs, covering 15% of mica surfaces. This natural growth process could be significantly altered using an AFM probe to cut the newly formed fibrils into short fragments, because these newly exposed sites became new nuclei to initiate growth of fibrils, leading to pine-needle like branches, and much higher coverages e.g. 45% in 30 min. This poster reveal the impact of local mechanical perturbation and the protein assembles formed accordingly.
• Using an atomic force microscopy, we have applied a local force to break MUC1Q11 fibrils to smaller fragments where the cutting sites became new nuclei to initiate growth.
• The growth of the new fibrils follow the direction of the short fragments along the primary axis of the surface. The location, surface coverage or density of the new fibrils are dictated by the cutting density and time of growth.
• We have demonstrated high degree of control over the assembly and surface coverage, e.g. we were able to attain much higher coverages than the naturally formed fibrils on surfaces. Pine-needle like assembly is formed following our cut.
• Using local mechanics to impact fibril protein assembly at nanometer level provide a new platform to engineer new hierarchical structures of proteins, and therefore offer great promise to understand and control self-assembly of protein fibrils, and to produce new biomaterials, as well as to improve protein aggregate based immunotherapy.
Introduction
Methods
MUC1Q11 comprise two distinct units linked via a spacer:
Before cutting the fibrils 0 min 5 min 12 min 18 min 24 min A B C D E F
400nm 100nm
Fibrils grow from cutting points resulting multiple new elongating fibrils.
A B C Before cutting the fibrils 5 min 24 min
D Zoom-out image, 30 min
400nm 400nm
Mechanical cutting is the reason of the growth of new fibrils as it can be seen on the time-laps images and zoom out image.
New fibrils exhibit individual growth behaviors
0
5
10
15
20
25
30
35
40
45
50
0 20 40 60 80 100 120
Sur
face
cov
erag
e of
fibr
ils (%
)
Time (min)
A B C
Fibrils exhibit variable individual growth behaviors
Mechanical cutting increase the density of fibrils
200nm
0 min 20 min
200nm
A B B E
Before cutting the fibrils 20 min, n=8 40 min, n=32 0 min 5 min, n=2
400nm
C D
Perpendicular cutting results knew growing ends from cutting points
A B C D
400nm
Before cutting Zoom-out image, 50 min 20 min, n=8 40 min, n=32
Low set point AC mode imaging cause continuous cuts on fibrils and new fibrils growing from cutting points
800nm 800nm
After 117 minutes After 60 minutes
n refers to the number of mechanical cutting (trace + retrace = 2)
0
50
100
150
200
250
0 5 10 15 20 25
0
50
100
150
200
250
0 5 10 15 20 25
0
50
100
150
200
250
0 5 10 15 20 25
4
3 2
1
4
3 2
1
4
3 2
1 200 nm
Before cutting 5 min 15 min 25 min
0
50
100
150
200
250
0 5 10 15 20 25
Fibril #2
Fibril #3
Time (min)
Leng
th (n
m)
Time (min)
Time (min) Time (min)
Fibril #1
Fibril #4
Linear growth with pause
Linear growth with stop Growth with decreasing growth rate
Shows growth on both end
Shows growth on both end, stopped by other adjacent fibrils
Linear growth with pause and sudden
burst
Fibril stops with no obvious reason
A B C D
Leng
th (n
m)
Leng
th (n
m)
Leng
th (n
m)
15 min 35 min 20 min 25 min 30 min
1
2a
5
3 2b
6 4
400nm
1
2a
5
3 2b
6 4
1
2a
5
3 2b
6 4
1
2a
5
3 2b
6 4
1
2a
5
3 2b
6 4
A B C D E
Leng
th (n
m)
050
100150200250300350400
0 20 400
50100150200250300350400
0 20 400
50100150200250300350400
0 20 40
050
100150200250300350400
0 20 400
50100150200250300350400
0 20 400
50100150200250300350400
0 20 400
50100150200250300350400
0 20 40
Fibril #1 Fibril #2a
Fibril #2b Fibril #3 Fibril #4 Fibril #5 Fibril #6
Time (min)
Mostly linear growth Growth with decreasing growth rate Growth with sudden bursts Linear growth
with stop
Leng
th (n
m)
Leng
th (n
m)
Leng
th (n
m)
Linear growth speed vary between 7.9nm/min and 12.9nm/min.
Time (min) Time (min) Time (min) Time (min) Time (min) Time (min)
Conclusions
Liner growth speed vary between 4.12 nm/min and 8.47 nm/min.
Arrows indicate sudden growth