REVIEW
NATURE BIOTECHNOLOGY VOLUME 21 NUMBER 10 OCTOBER 2003 1171
FOCUS ON NANOBIOTECHNOLOGY
Molecular self-assembly is a powerful approach for fabricating
novel supramolecular architectures. It is ubiquitous in the natural
world (see Box 1): lipid molecules form oil drops in water; four
hemoglobin polypeptides form a functional tetrameric hemoglo-
bin protein; ribosomal proteins and RNA coalesce into functional
ribosomes.
Molecular self-assembly is mediated by weak, noncovalent
bonds—notably hydrogen bonds, ionic bonds (electrostatic interac-
tions), hydrophobic interactions, van der Waals interactions, and
water-mediated hydrogen bonds. Although these bonds are rela-
tively insignificant in isolation, when combined together as a whole,
they govern the structural conformation of all biological macromol-
ecules and influence their interaction with other molecules. The
water-mediated hydrogen bond is especially important for living
systems, as all biological materials interact with water.
All biomolecules, including peptides and proteins, interact and
self-organize to form well-defined structures that are associated
with functionality
1
.By observing the processes by which supramol-
ecular architectures are assembled in nature
1–3
,we can begin to
exploit self-assembly for the synthesis of entirely novel synthetic
materials. Peptides and proteins are versatile building blocks for
fabricating materials. Nature has already used them as scaffolds to
produce a dizzying array of materials, including collagen, keratin,
pearl, shell, coral and calcite microlenses, and optical waveguides.
In this review, I focus on the fabrication of diverse molecular struc-
tures through self-assembly of peptides, proteins, and lipids. As the use
of genetically engineered polypeptides for specifically binding selected
inorganic compounds to assemble functional nanostructures has
recently been reviewed
4
, it will not be discussed in detail here. For
other aspects of nanomaterials synthesis, namely chemistry-driven
approaches to materials synthesis (e.g.,self assembly of organic ligands
and metal ions into three-dimensional hollow cages or metalloden-
drimers) and in vitro systems for artificial protein synthesis, the reader
is referred elsewhere
5–8
.
Fabrication of nanofibers
Work in my laboratory has focused on fabricating several self-
assembling peptides and proteins for a variety of studies and biomate-
rials (Fig. 1). Examples include ionic self-complementary peptides
9–11
,
which form β -sheet structures in aqueous solution with two distinct
surfaces—one hydrophilic, the other hydrophobic (rather like the pegs
and holes in Lego bricks). The hydrophobic residues shield themselves
from water and self-assemble in water in a manner similar to that seen
in the case of protein folding in vivo.The unique structural feature of
these ‘molecular Lego’ peptides is that they form complementary ionic
bonds with regular repeats on the hydrophilic surface (Fig. 1a). The
complementary ionic sides have been classified into several moduli
(modulus I, modulus II, modulus III, modulus IV, etc., and mixtures
thereof). This classification scheme is based on the hydrophilic sur-
faces of the molecules, which have alternating positively and negatively
charged amino acids alternating by one residue, two residues, three
residues and so on. For example, charge arrangements for modulus I,
modulus II, modulus III and modulus IV are – + – + – + –+, – – + + –
– + +, – – – + + + and – – – – + + + +,respectively. The charge orienta-
Fabrication of novel biomaterials through
molecular self-assembly
Shuguang Zhang
Two complementary strategies can be used in the fabrication of molecular biomaterials. In the ‘top-down’ approach, biomaterials
are generated by stripping down a complex entity into its component parts (for example, paring a virus particle down to its capsid
to form a viral cage). This contrasts with the ‘bottom-up’ approach, in which materials are assembled molecule by molecule (and
in some cases even atom by atom) to produce novel supramolecular architectures. The latter approach is likely to become an
integral part of nanomaterials manufacture and requires a deep understanding of individual molecular building blocks and their
structures, assembly properties and dynamic behaviors. Two key elements in molecular fabrication are chemical complementarity
and structural compatibility, both of which confer the weak and noncovalent interactions that bind building blocks together during
self-assembly. Using natural processes as a guide, substantial advances have been achieved at the interface of nanomaterials and
biology, including the fabrication of nanofiber materials for three-dimensional cell culture and tissue engineering, the assembly
of peptide or protein nanotubes and helical ribbons, the creation of living microlenses, the synthesis of metal nanowires on DNA
templates, the fabrication of peptide, protein and lipid scaffolds, the assembly of electronic materials by bacterial phage selection,
and the use of radiofrequency to regulate molecular behaviors.
Center for Biomedical Engineering NE47-379 and Center for Bits & Atoms,
Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge,
Massachusetts 02139-4307, USA. Correspondence should be addressed to S.Z.
(shuguang@mit.edu)
Published online 30 September 2003; doi:10.1038/nbt874
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REVIEW
tion can also be designed in the reverse orientation, which can yield
entirely different molecules. These well-defined sequences allow the
peptides to undergo ordered self-assembly, in a process resembling
some situations found in well-studied polymer assemblies.
A broad range of peptides and proteins have been shown to produce
very stable nanofiber structures, also called amyloid fibers
12–22
.
(In physiological settings, the formation of such amyloid fibrils is
known to have a role in several diseases, including bovine spongi-
form encephalopathy, Alzheimer disease, type II diabetes and
Creutzfeld–Jakob disease; e.g.,see ref. 23.) These nanofibers are very
well ordered and possess remarkable regularity and, in some cases, hel-
ical periodicity. The mechanism whereby they undergo self-assembly
is now being elucidated
24–27
.These nanofibers are similar in scale to
extracellular matrices that are crucial in allowing a variety of cells to
adhere together to form functional tissues. Furthermore, these
nanofibers, if their formations can be precisely controlled, could serve
as scaffold to organize nanocrystals for use in the electronic industry
(see below).
Fabricating bionanotubes
Nature has selected, evolved and produced a
host of amphiphilic molecules that contain
distinct hydrophobic and hydrophilic seg-
ments. These amphiphilic molecules readily
partition in water to form various semien-
closed environments. One of the best exam-
ples is phospholipids—the predominant
constituents of the plasma membrane that
encapsulate and protect the cellular contents
from the environment, an absolute prerequi-
site for almost all living systems.
Phospholipids readily undergo self-assembly
in aqueous solution to form distinct struc-
tures that include micelles, vesicles and
tubules. This is largely a result of the
hydrophobic forces that drive the nonpolar
region of each molecule away from water
and toward one another. The dimensions
and shape of the supramolecular lipid struc-
tures depend upon various factors, such as
the geometry and curvature of the polar
head and the shape and length of the nonpo-
lar tails
28
.
Schnur and colleagues
29,30
pioneered lipid
tubule self-assembly to build materials and
ushered in a new era of fabricating novel
materials using simple building blocks
31,32
.
They have not only extensively studied all
aspects of the materials from single molecular
chemistry and chirality to all size scales, but
have also developed unexpected applications,
such as an anti-fouling coating for ships com-
prising self-assembled lipid tubules
31,32
.
These experiments stimulated my research
group to ask what might be the simplest
amphiphilic biopolymers in the prebiotic
environment. Accordingly, we designed many
simple amphiphilic peptides that consist
exclusively of natural amino acids. One such
class of molecules is surfactant-like peptides
(Fig. 1b)
33–35
.Although individually they
have completely different compositions and
sequences, these surfactant-like peptides share a common feature: the
hydrophilic heads have one or two charged amino acids and the
hydrophobic tails have four or more consecutive hydrophobic amino
acids (see figures in refs. 33–35). For example, the peptide V
6
D
(VVVVVVD) has six hydrophobic valine residues beginning from the
N-terminus, followed by a negatively charged aspartic acid residue—
thus having two negative charges, one from the side chain and the
other from the C terminus
33
.In contrast, G
8
DD (GGGGGGGGDD)
has eight glycines followed by two aspartic acids, with three negative
charges
34
.Similarly, A
6
K (AAAAAAK) or KA
6
(KAAAAAA) has six
alanines as the hydrophobic tail and a positively charged lysine as the
hydrophilic head
35
.
Perutz, in his last set of papers
20,36,37
, unequivocally demonstrated
the formation of nanotubes from polyglutamines. He showed that
the length of the polyglutamines—the number of consecutive gluta-
mines on the polypeptide chain—plays a key role in the formation of
β -helix structure. These experimental results were confirmed using
computer modeling and simulations (ref. 38; A. Windle, personal
1172 VOLUME 21 NUMBER 10 OCTOBER 2003 NATURE BIOTECHNOLOGY
Figure 1 Fabrication of various peptide materials. (a) The ionic self-complementary peptide has
16 amino acids, ∼ 5 nm in size, with an alternating polar and nonpolar pattern. The peptides form
stable β -strand and β -sheet structures; thus, the side chains partition into two sides, one polar and the
other nonpolar
48–50
. They undergo self-assembly to form nanofibers with the nonpolar residues inside
(green), and + (blue) and – (red) charged residues form complementary ionic interactions, like a
checkerboard. These nanofibers form interwoven matrices that further form a scaffold hydrogel with
very high water content, >99.5%. This is similar to agarose gel and other hydrogels. (Images courtesy
of H. Yokoi.) (b) A type of surfactant-like peptide, ∼ 2 nm in size, that has a distinct head charged
group, either positively charged or negatively charged, and a nonpolar tail consisting of six hydrophobic
amino acids. The peptides can self-assemble into nanotubes and nanovesicles with a diameter of
∼ 30–50 nm. These nanotubes go on to form an inter-connected network
33–35
similar to what has been
observed in carbon nanotubes. (Image courtesy of S. Santoso.) (c) Surface nanocoating peptide. This
type of peptide has three distinct segments: a functional segment, which interacts with other proteins
and cells; a linker segment that not only can be either flexible or stiff, but also sets the distance from
the surface; and an anchor for covalent attachment to the surface
47
. These peptides can be used as ink
for an inkjet printer to print directly on a surface, instantly creating any arbitrary pattern, as shown
here. Neural cells from rat hippocampal tissue form defined patterns. (Images courtesy of S. Fuller
and N. Sanjana.) (d) Molecular switch peptide, a type of peptide with strong dipoles that can undergo
drastic conformation changes, between α -helix and β -strand or β -sheet, under external stimuli
87
. It is
conceivable that metal nanocrystals could be attached to these dipolar peptides to fabricate them into
tiny switches.
a
b
c
d
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