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Epitaxial Growth of Peptide Nanofilaments on Inorganic Surfaces Effects of Interfacial HydrophobicityHydrophilicity.

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Angewandte
Chemie
Peptide Nanofilaments
DOI: 10.1002/ange.200503636
Epitaxial Growth of Peptide Nanofilaments on
Inorganic Surfaces: Effects of Interfacial
Hydrophobicity/Hydrophilicity**
Feng Zhang, Hai-Ning Du, Zhi-Xiang Zhang, Li-Na Ji,
Hong-Tao Li, Lin Tang, Hua-Bin Wang, Chun-Hai Fan,
Hong-Jie Xu, Yi Zhang,* Jun Hu, Hong-Yu Hu, and
Jian-Hua He*
The self-assembly of peptides and proteins has recently
attracted much attention because of its scientific importance
and widespread applications in the fields of nanomaterials,[1–5]
crystallography,[6–8] and pathology.[9–13] Particularly, onedimensional growth of peptides or proteins on inorganic
substrates has been recognized as a potential approach to the
fabrication of functional nanostructures.[14–16] In these cases
the peptide (or protein) is precisely assembled with the
assistance of inorganic templates.[7, 8] This feature allows the
arrangement of peptide molecules in a predesignated orientation on substrates by proper design of the peptide
sequences.
Previous studies showed that different substrates have
significant effects on the formation of nanofilaments.[17–20] As
it is generally accepted that the hydrophobic interaction
between the side chains is a driving force in the formation of
peptide nanofilaments, it is very important to explore the
effects of the substrate hydrophobicity/hydrophilicity on the
[*] F. Zhang, Z.-X. Zhang, L.-N. Ji, L. Tang, H.-B. Wang, C.-H. Fan,
H.-J. Xu, Y. Zhang, J. Hu, J.-H. He
Shanghai Institute of Applied Physics
Chinese Academy of Sciences
Shanghai 201800 (P.R. China)
Fax: (+ 86) 21-5955-3021
Fax: (+ 86) 21-5955-2394
E-mail: yzhang@sinap.ac.cn
hejh@sinap.ac.cn
H.-N. Du, H.-T. Li, H.-Y. Hu
Institute of Biochemistry and Cell Biology
Shanghai Institutes for Biological Sciences
Chinese Academy of Sciences
Shanghai 200031 (P.R. China)
J. Hu
Bio-X Life Sciences Research Center
College of Life Science and Biotechnology
Shanghai JiaoTong University
Shanghai 200030 (P.R. China)
[**] We thank Dr. Bin Li, Xing-Fei Zhou, Yun-Chang Guo, and Hai Li for
valuable discussions and Miss Stefanie Maerz for the helpful
revisions. This work was supported by grants from the Shanghai
Institute of Applied Physics, the National Natural Science Foundation of China (No. 10335070, 10404032, and 30400064), and the
Science and Technology Commission of Shanghai Municipality (No.
04qmx1466 and 0552nm033).
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
Angew. Chem. 2006, 118, 3693 –3695
relevant template-assisted epitaxial growth, which has not
been systematically studied so far.
Herein, we compare the self-assembly behavior of the
peptide GAV-9 on hydrophilic mica and hydrophobic HOPG
(highly ordered pyrolytic graphite) surfaces by in situ atomic
force microscopy (AFM) to investigate how interfacial
hydrophobicity/hydrophilicity influences the epitaxial
growth of peptide nanofilaments.
GAV-9, NH2-VGGAVVAGV-CONH2 (Figure 1 a), consists of hydrophobic amino acid residues and is a conserved
consensus of three neurodegenerative-disease-related proteins: a-synuclein, amyloid b protein, and prion protein,
Figure 1. Schematic representation of self-assembled GAV-9 nanofilaments and their interfacial orientations at hydrophobic and hydrophilic
surfaces. a) The “spacefill” model (left), which is represented by a
green stick (right); b) GAV-9 tapes lying horizontally on HOPG (yellow
slab); c) GAV-9 fibrils oriented upright on mica (red slab).
which are capable of forming amyloid fibrils in vivo and
in vitro.[20–22] Unlike a-synuclein, GAV-9 with a concentration
of 1.6 mm or less cannot form amyloid fibrils even when its
solution is incubated under fibrillization-accelerating conditions[21] for a long time ( 6 days), as characterized by AFM
and turbidity measurements (data not shown). Circular
dichroism experiments show that GAV-9 in fresh solutions
adopts a random-coil structure, but forms b-sheets when dried
(Supporting Information).
However, AFM studies indicate that GAV-9 forms uniform nanofilaments from random-coil monomeric solutions
through template-assisted growth on surfaces, including tapelike objects[19] on HOPG and fibrillike aggregates[23] on mica
(Figure 2). Both the tapes and fibrils grow epitaxially across
the surface and show three preferred orientations at 1208 to
each other. The Fourier transform confirms its threefold
symmetry (inset of Figure 2 b and Figure 2 e). It was also
found that orientations of the tapes and the fibrils are
consistent with the underlying atomic lattice of the substrates
(Figure 2 a and Figure 2 b, Supporting Information). These
results indicate that the self-assembly of GAV-9 is a onedimensional epitaxial growth process, similar to those previously reported.[14–17]
Solid-state ATR (attenuated total reflectance) IR spectroscopy of the fibrils on a mica surface shows an absorption
at ñ = 1628 cm 1 together with a weak band at ñ = 1690 cm 1,
indicating an antiparallel b-sheet structure[2] (Supporting
Information). Surprisingly, the fibrils are 3.0 0.1 nm in
height, which is almost equal to the length (3.1 nm) of the
GAV-9 peptide in an extended antiparallel b-sheet conformation.[24] The high quality of the fibrils with very uniform
height (Figure 2 b–d) equal to the length of a single molecule
2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3693
Zuschriften
be investigated by AFM. Furthermore, the
peak-to-peak distance of 5.0 0.2 nm
between the adjacent tapes (Figure 2 f and
Figure 2 h) indicates the presence of a gap
( 2 nm).
To investigate the dynamic details of the
ordered assembly process, in situ AFM
studies have also been carried out. When
the concentration of GAV-9 reached
1.6 mm, nucleation seeds with heights of
5–15 nm appeared on the mica surface (see
labels 1–26 in Figure 3). Fibrils then started
to grow bidirectionally from these seeds at a
growth rate of 1–20 nm s 1. The rate varied
at different locations and for different
fibrils, which may reflect changes in the
local environmental conditions. These
fibrils can extend to lengths ranging from
several microns to tens of microns, but they
Figure 2. AFM images and analysis of nanofilaments. a) The atomic lattice of the mica
never overlap with one another. On the
surface; b) GAV-9 (1.6 mm) fibrils with apparent widths of 40–70 nm on the same mica
basis of AFM images of isolated fibrils, the
surface as in a); inset: Fourier transform. the yellow arrows in a) and b) indicate the
consistency of the orientations; c) section analysis of the green line marked in b); d) section width of fibrils were found to vary from tens
to hundreds of nanometers (Figure 2 b and
analysis along a fibril marked by the blue line in b); e) GAV-9 (1.6 nm) tapes with an
apparent width of 20 nm on HOPG; inset: Fourier transform; f) layers of GAV-9 (0.3 mm)
Supporting Information). However, in situ
tapes on HOPG; yellow arrows indicate their orientations; inset: Fourier transform of the
AFM observations showed that the width of
region marked with a white box; g) section analysis of the pink line marked in e); h) section
any individual fibril remained constant
analysis of the white line marked in f).
( 12 nm, after deconvolution) after the
fibril had started to grow. As indicated by
the arrows in Figure 3 l, wider fibrils are
most likely a result of the merger of adjacent ones, not of
suggests that GAV-9 is oriented upright on mica surfaces
lateral growth of the original fibril. The scanning of the AFM
(Figure 1). Peptide fibrils growing in this “upright” style with
tip sometimes disturbed the growth of fibrils thereby changthe peptide backbone perpendicular to the substrate have not
ing the direction of growth (Figure 3, labels 2, 3, 13, 17, and
been reported previously. In this case, the electrostatic
interaction between the negatively charged
substrate and the positively charged peptide terminal is the driving force for the
ordered assembly of GAV-9. Control
experiments show that the growth of fibrils
on two positively charged surfaces is significantly inhibited (Supporting Information). The combination of the above results
strongly supports an upright orientation.
On the HOPG surface, the height and
deconvoluted width[16, 25] of GAV-9 tapes
are 0.9 0.1 nm and 3–4 nm, respectively.
They are approximately equal to the
dimensions of GAV-9 (1.0 nm in diameter,
3.1 nm in length). This indicates that the
tapes lie with their hydrophobic side chains
in contact with the hydrophobic surface of
the HOPG substrate (Figure 1). Similar
phenomena have been found in other
systems.[14–20] When the concentration of
GAV-9 increases, multiple layers of tapes
are often observed (Figure 2 f), and the
tapes sometimes adopt an orientation of
Figure 3. In situ AFM observation of GAV-9 fibrils (apparent width 40 nm) growing on
308 to each other (besides 608 or 1208). At
mica. a–l) A series of snapshots of the growing process, which took about 100 min. The
concentrations higher than 1.6 mm, the
numerical labels indicate the formation of new nucleation seeds. The height and scale bars
growth of tapes on HOPG is too fast to
shown in a) apply to all images.
3694
www.angewandte.de
2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. 2006, 118, 3693 –3695
Angewandte
Chemie
22). On HOPG, however, the process is relatively fast (at a
rate of 0.3 mm s 1 at a concentration of 0.8 mm), and interactions of the AFM tip did not seem to affect the growth
process (Supporting Information).
In summary, we have presented the in situ AFM investigation of the self-assembly of GAV-9 molecules into ordered
nanofilaments on both hydrophilic and hydrophobic surfaces.
The experimental results indicate that the growth of GAV-9
nanofilaments adopt a horizontal orientation on HOPG and
an “upright” orientation on mica. The fibrils are highly
uniform in height and may be hundreds of microns in length,
which makes them an ideal biotemplate for generating
functional nanomaterials. The substrate-directed assembly
of peptides by employing surface hydrophobicity/hydrophilicity can be envisioned as a unique approach to fabricate
specifically tailored nanostructures. Moreover, our results
point to the significance of focusing physicochemical studies
of the self-assembly of peptides on the hydrophilic/hydrophobic nature of surfaces, as the interfacial properties may
dramatically affect the self-assembly process.
Experimental Section
GAV-9 was synthesized by using the Boc solid-phase method on an
ABI 433 A peptide synthesizer (Appliedbiosystems) and cleaved
from the MBHA resin (100–200 mesh, Fluka) with hydrogen fluoride.
The peptide was purified through a TSK-40 (S) column (2.0 C 98 cm,
Tosoh). Before used, GAV-9 was dissolved in 10 mm PBS buffer
(phosphate 10 mm, NaCl 10 mm, pH 7.0).
A commercial AFM instrument (Nanoscope IIIa, Veeco) equipped with an E-scanner and a liquid cell was employed, and experiments were performed in the tapping mode. Silicon nitride cantilevers
with a nominal spring constant of 0.58 N m 1 (Veeco) were used.
Atomic resolution images of the mica and HOPG surfaces were
obtained by contact-mode AFM and scanning tunneling microscopy
(STM), respectively, prior to the in situ experiments.
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Received: October 14, 2005
Revised: February 17, 2006
Published online: April 26, 2006
.
Keywords: electrostatic interactions · hydrophobic effect ·
nanofilaments · peptides · self-assembly
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www.angewandte.de
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