ScienceWeek

SCIENCEWEEK

ScienceWeek
February 21, 2003
Vol. 7 Number 8

An Online Digest of Research in the Sciences

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Man's destiny is to know, if only because societies with
knowledge culturally dominate societies that lack it. Luddites
and anti-intellectuals do not master the differential equations
of thermodynamics or the biochemical cures of illness. They stay
in thatched huts and die young.
-- Edward O. Wilson

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Section 1

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Thematic Issue: Atomic Force Microscopy

1. Introduction.
2. On Methodology.
3. AFM in Biology: Titin and Other Proteins.
4. AFM in Biology: Paleontology.
5. AFM in Biology: Mechanical Properties of Living Cells.
6. AFM in Conformational Chemistry.
7. AFM in Supramolecular Chemistry.
8. AFM in Materials Science.

Notices and Subscription Information

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Section 2

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1. INTRODUCTION.

The new technology of scanning probe microscopy has created a
revolution in microscopy, with applications ranging from
condensed matter physics to biology. This issue of ScienceWeek
presents only a glimpse of the many and varied applications of
atomic force microscopy in the sciences.

The first scanning probe microscope, the scanning tunneling
microscope, was invented by G. Binnig and H. Rohrer in the 1980s
(they received the Nobel Prize in Physics in 1986), and the
invention has been the catalyst of a technological revolution.
Scanning probe microscopes have no lenses. Instead, a "probe" tip
is brought very close to the specimen surface, and the
interaction of the tip with the region of the specimen
immediately below it is measured. The type of interaction
measured essentially defines the type of scanning probe
microscopy. When the interaction measured is the force between
atoms at the end of the tip and atoms in the specimen, the
technique is called "atomic force microscopy". When the quantum
mechanical tunneling current is measured, the technique is called
"scanning tunneling microscopy". These two techniques, atomic
force microscopy (AFM) and scanning tunneling microscopy (STM)
have been the parents of a variety of scanning probe microscopy
techniques investigating a number of physical properties.

[Note: In general, "quantum mechanical tunneling" is a quantum
mechanical phenomenon involving an effective penetration of an
energy barrier by a particle resulting from the width of the
barrier being less than the wavelength of the particle. If the
particle is charged, the effective particle translocation
determines an electric current. In this context, "wavelength"
refers to the de Broglie wavelength of the particle, which is
given by L = h/mv, with (L) the wavelength of the moving
particle, (h) the Planck constant, (m) the mass of the particle,
and (v) the velocity of the particle.]

ATOMIC FORCE MICROSCOPY IN BIOLOGY

C. Wright-Smith and C.M. Smith (San Diego State College, US)
present a review of the use of atomic force microscopy in
biology, the authors making the following points:

1) Since its introduction in the 1980s, atomic force microscopy
(ATM) has gained acceptance in biological research, where it has
been used to study a broad range of biological questions,
including protein and DNA structure, protein folding and
unfolding, protein-protein and protein-DNA interactions, enzyme
catalysis, and protein crystal growth. Atomic force microscopy
has been used to literally dissect specific segments of DNA for
the generation of genetic probes, and to monitor the development
of new gene therapy delivery particles.

2) Atomic force microscopy is just one of a number of novel
microscopy techniques collectively known as "scanning probe
microscopy" (SPM). In principle, all SPM technologies are based
on the interaction between a submicroscopic probe and the surface
of some material. What differentiates SPM technologies is the
nature of the interaction and the means by which the interaction
is monitored.

3) Atomic force microscopy produces a topographic map of the
sample as the probe moves over the sample surface. Unlike most
other SPM technologies, atomic force microscopy is not dependent
on the electrical conductivity of the product being scanned, and
ATM can therefore be used in ambient air or in a liquid
environment, a critical feature for biological research. The
basic atomic force microscope is composed of a stylus-cantilever
probe attached to the probe stage, a laser focused on the
cantilever, a photodiode sensor (recording light reflected from
the cantilever), a digital translator recorder, and a data
processor and monitor.

4) Atomic force microscopy is unlike other SPM technologies in
that the probe makes physical (albeit gentle) contact with the
sample. The cornerstone of this technology is the probe, which is
composed of a surface-contacting stylus attached to an elastic
cantilever mounted on a probe stage. As the probe is dragged
across the sample, the stylus moves up and down in response to
surface features. This vertical movement is reflected in the
bending of the cantilever, and the movement is measured as
changes in the light intensity from a laser beam bouncing off the
cantilever and recorded by a photodiode sensor. The data from the
photodiode is translated into digital form, processed by
specialized software on a computer, and then visualized as a
topological 3-dimensional shape.

The Scientist 2001 22 January

Related Background:

ON SCANNING PROBE MICROSCOPY

A. Yazdani and C.M. Lieber (2 installations, US) present a review
of recent developments in scanning probe microscopy, the authors
making the following points:

1) The invention and development of scanning probe microscopy has
taken the ability to image matter to the atomic scale and opened
fresh perspectives on everything from semiconductors to
biomolecules, and new methods are being devised to modify and
measure the microscopic landscape in order to explore its
physical, chemical, and biological features.

2) In scanning tunneling microscopy, electrons quantum
mechanically "tunnel" between the tip and the surface of the
sample. This tunneling process is sensitive to any overlap
between the electronic wave functions of the tip and sample, and
depends exponentially on their separation. The scanning tunneling
microscope makes use of this extreme sensitivity to distance. In
practice, the tip is scanned across the surface, while a feedback
circuit continuously adjusts the height of the tip above the
sample to maintain a constant tunneling current. The recorded
trajectory of the tip creates an image that maps the electronic
wave functions at the surface, revealing the atomic landscape in
fine detail.

2) The most widely used scanning probe microscopy technique, one
which can operate in air and liquids, is atomic force microscopy.
In this technique, a tip is mounted at the end of a soft
cantilever that bends when the sample exerts a force on the tip.
By optically monitoring the cantilever motion it is possible to
detect extremely small chemical, electrostatic, or magnetic
forces which are only a fraction of those required to break a
single chemical bond or to change the direction of magnetization
of a small magnetic grain. Applications of atomic force
microscopy have included in vitro imaging of biological
processes.

3) In general, the various techniques of scanning probe
microscopy have now been applied to high-resolution spectroscopy,
the probing of nanostructures, measurements of forces in
chemistry and biology, the production of deliberate movements of
small numbers of atoms, and the use of precision lithography as a
tool for making nanometric-sized electronic devices.

4) The authors conclude: "The scanning probe microscope has
evolved from a passive imaging tool into a sophisticated probe of
the nanometer scale. These advances point to exciting
opportunities in many areas of physics and biology, where
scanning probe microscopes can complement macroscopically
averaged measurement techniques and enable more direct
investigations. More importantly, these tools should inspire new
approaches to experiments in which controlled measurements of
individual molecules, molecular assemblies, and nanostructures
are possible."

Nature 1999 401:227

ATOMIC FORCE MICROSCOPY IN SURFACE CHEMISTRY

"The atomic-force microscope (AFM) can be used to investigate
contact and hardness on the atomic scale. Analogously to the STM,
the AFM uses a feedback loop to control the distance between the
sample and a probe tip at the end of a cantilever arm. Rather
than a tunneling current, however, the AFM monitors an optical
signal as feedback to measure the level of deflection. Thus, both
attractive and repulsive interactions of the tip and sample can
be monitored. As the microscope tip approaches the surface,
attractive forces are first exerted on the tip by the surface and
can be measured to as small a value as 10^(-9) N. Upon contact
with the surface, further motion of the tip results in repulsive
forces between the tip and the sample. This procedure is capable
of producing loads that overlap the forces encountered in
macroscopic mechanical measurements. After a force of known
magnitude has been applied, that area of contact with the tip is
scanned for signs of permanent damage. For a smooth gold surface,
permanent damage has been detected only for forces as large as
about 5 X 10^(-5) N. This result suggests that the metal surface
responds to the approaching  metal tip by elastically 'bending'
in the range of 10^(-9) to 5 X 10^(-5) N. This range of forces is
the regime of elastic deformation. Only on application of a force
greater than 5 x 10^(-5) N did plastic deformation (the
irreversible breaking of metal-metal bonds) begin. Measurements
of this type permit one to determine the forces needed for
elastic and plastic deformation on the atomic scale and to
correlate the results to those obtained by macroscopic studies."

Gabor A. Somorjai: in N. Hall (ed.): The New Chemistry. Cambridge
University Press 2000, p.161.

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2. ON METHODOLOGY.

QUANTITATIVE MEASUREMENT OF SHORT-RANGE CHEMICAL BONDING FORCES.

M.A. Lantz et al (University of Basel, CH) discuss AFM in the
measurement of short-range forces, the authors making the
following points:

1) The atomic force microscope (AFM) was originally intended to
be a tool capable of measuring the forces acting between a single
pair of atoms (1) but has only recently evolved into an
instrument capable of producing atomically resolved images of
surfaces with characteristic features and defects (2-4). This
true atomic-scale contrast is generally interpreted as resulting
from the short-range chemical interaction between an atomically
sharp AFM tip and the nearest atoms on the surface of the sample.
In principle, it should therefore be possible to map the chemical
bonding potential between the foremost atom on an AFM tip and a
specific atom on the sample.

2) The measurement of short-range bonding forces with the AFM has
been difficult to achieve for several reasons. First, at room
temperature, thermal drift and piezoelectric scanner creep make
it difficult to reliably position the tip above a specific
lattice position. Second, most atomic-resolution AFM images have
been obtained using a dynamic technique in which the tip-bearing
cantilever is driven on its fundamental resonant frequency with a
typical amplitude of several nanometers. When the cantilever tip
comes close to the sample surface, the force acting on the tip
weakly perturbs the cantilever oscillation, giving rise to a
small shift f in the resonance frequency. The frequency shift is
used as a feedback parameter to control the tip-sample spacing,
and images therefore correspond to contours of constant frequency
shift. Because of the large tip excursion, the relation between
the measured frequency shift and the force acting on the tip is
not straightforward. Recently, however, progress has been made in
quantitatively understanding and inverting this relation (5). A
third difficulty arises because, in general, both short-range
forces (such as covalent bonding forces) and long-range forces
[such as van der Waals (vdW) and electrostatic forces] act on the
tip. Separating these contributions in order to isolate the
short-range chemical bonding force is a nontrivial problem.
Finally, it is difficult to determine whether the measured
chemical force involves more than just a single pair of atoms.

3) In summary: The authors report direct force measurements of
the formation of a chemical bond. The experiments were performed
using a low-temperature atomic force microscope, a silicon tip,
and a silicon (111) 7 x 7 surface. The measured site-dependent
attractive short-range force, which attains a maximum value of
2.1 nanonewtons, is in good agreement with first-principles
calculations of an incipient covalent bond in an analogous model
system. The resolution was sufficient to distinguish differences
in the interaction potential between inequivalent adatoms,
demonstrating the ability of atomic force microscopy to provide
quantitative, atomic-scale information on surface chemical
reactivity.

References (abridged):

1. G. Binnig, C. F. Quate, C. Gerber, Phys. Rev. Lett. 56, 930
(1986).

2. F. Ohnesorge and G. Binnig, Science 260, 1451 (1993).

3. F. J. Giessibl, Science 267, 68 (1995).

4. S. Kitamura and M. Iwatsuki, Jpn. J. Appl. Phys. 34, 145
(1995).

5. F. J. Giessibl, Phys. Rev. B 56, 16010 (1997).

Science 2001 291:2580.

Related Background Brief:

INVERTING DYNAMIC FORCE MICROSCOPY: FROM SIGNALS TO TIME-RESOLVED
INTERACTION FORCES. Transient forces between nanoscale objects on
surfaces govern friction, viscous flow, and plastic deformation,
occur during manipulation of matter, or mediate the local wetting
behavior of thin films. To resolve transient forces on the (sub)
microsecond time and nanometer length scale, dynamic atomic force
microscopy (AFM) offers largely unexploited potential. Full
spectral analysis of the AFM signal completes dynamic AFM.
Inverting the signal formation process, the authors measure the
time course of the force effective at the sensing tip. This
approach yields rich insight into processes at the tip and
dispenses with a priori assumptions about the interaction, as it
relies solely on measured data. Force measurements on silicon
under ambient conditions demonstrate the distinct signature of
the interaction and reveal that peak forces exceeding 200 nN are
applied to the sample in a typical imaging situation. These
forces are 2 orders of magnitude higher than those in covalent
bonds. M. Stark et al: Proc. Nat. Acad. Sci. 2002 99:8473.

Related Background Brief:

DETECTION AND LOCALIZATION OF INDIVIDUAL ANTIBODY-ANTIGEN
RECOGNITION EVENTS BY ATOMIC FORCE MICROSCOPY. The authors report
that a methodology has been developed for the study of molecular
recognition at the level of single events and for the
localization of sites on biosurfaces, in combining force
microscopy with molecular recognition by specific ligands. For
this goal, a sensor was designed by covalently linking an
antibody (anti-human serum albumin, polyclonal) via a flexible
spacer to the tip of a force microscope. This sensor permitted
detection of single antibody-antigen recognition events by force
signals of unique shape with an unbinding force of 244 +- 22 pN.
Analysis revealed that observed unbinding forces originate from
the dissociation of individual Fab fragments from a human serum
albumin molecule. The two Fab fragments of the antibody were
found to bind independently and with equal probability. The
flexible linkage provided the antibody with a 6-nm dynamical
reach for binding, rendering binding probability high, 0.5 for
encounter times of 60 ms. This permitted fast and reliable
detection of antigenic sites during lateral scans with a
positional accuracy of 1.5 nm. The authors suggest this
methodology has promise for characterizing rate constants and
kinetics of molecular recognition complexes and for molecular
mapping of biosurfaces such as membranes. P. Hinterdorfer et al:
pnas 1996 93:3477.

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3. AFM IN BIOLOGY: TITIN AND OTHER PROTEINS.

REVERSE ENGINEERING OF THE GIANT MUSCLE PROTEIN TITIN

The term "myofibril" refers to one of the long cylindrical
contractile elements that constitute the major component of the
muscle fiber, extending for the entire length of the muscle
fiber, with each myofibril composed in turn of numerous
myofilaments.

The term "sarcomere" refers to the functional unit of a myofibril
of vertebrate muscle, each sarcomere approximately 2 to 3 microns
long. In striated muscle, the sarcomeres of many parallel
myofibrils are positioned such that myosin thick filaments are
aligned in register across the myofibril and have a dense
appearance in microscope preparations. The actin thin filaments
have a lighter appearance, with a resulting alteration of light
isotropic bands called "I bands" and dark anisotropic bands
called "A bands". The I band is bisected by a dense narrow "Z
line", while the central less dense region of the A band is known
as the "H zone", which in turn is bisected by the dark "M line"
(or midline), the locus of specific proteins that link adjacent
thick filaments to each other. The Z lines are due to attachment
sites for thin filaments. The various sarcomere bands are thus
classical light-microscope features which have only in the past
few decades been analyzed in terms of various macromolecular
structures.

The term "titin" (connectin) refers to a giant protein that forms
a single-molecule elastic filament extending from the M line to
the Z line in the striated muscle sarcomere. Titin is one of the
largest polypeptides yet described. Its amino acid sequence
consists mainly of repeats of two types of approximately 100-
amino-acid motifs, known as class I and class II. There is also a
domain characteristic of protein kinases near the C terminus.
Titin is believed to play an important role in sarcomere
alignment during muscle contraction. A single molecule of titin
measures approximately 1 micron in length and has a molecular
weight of approximately 3 million daltons.

H. Li et al (Mayo Foundation, US) discuss titin, the authors
making the following points:

1) Individual titin molecules span both the A-band and I-band
regions of muscle sarcomeres. The I-band part of titin has been
identified as the region that is functionally elastic. The
authors report a study of the shortest titin isoform, the N2B
isoform found in cardiac-muscle sarcomeres. The elastic I-band
region of N2B-titin can be subdivided into four structurally
distinct regions: a proximal immunoglobulin region containing 15
tandem immunoglobulin-like (Ig) domains; a middle N2B segment
that contains a 572-residue amino-acid sequence of unknown
structure; a 186-amino-acid-long segment rich in proline (P),
glutamate (E), valine (V) and lysine (K) residues, named the PEVK
region; and a distal Ig region that contains 22 tandem Ig
modules. The authors use polyprotein engineering and single-
molecule force spectroscopy to dissect the individual mechanical
elements of the I-band of cardiac titin and reconstruct the
elasticity of cardiac muscle. Polyproteins, when mechanically
stretched by single-molecule atomic force microscopy (AFM) give
distinctive mechanical fingerprints as their modules unfold
sequentially (sawtooth patterns in the force extension curve),
and can be used to positively identify the mechanical features of
a single molecule.

2) In summary: Through the study of single molecules it has
become possible to explain the function of many of the complex
molecular assemblies found in cells(1-5). The protein titin
provides muscle with its passive elasticity. Each titin molecule
extends over half a sarcomere, and its extensibility has been
studied both in situ and at the level of single molecules. These
studies suggested that titin is not a simple entropic spring but
has a complex structure-dependent elasticity. The authors use
protein engineering and single-molecule atomic force microscopy
to examine the mechanical components that form the elastic region
of human cardiac titin. The authors demonstrate that when these
mechanical elements are combined, they explain the macroscopic
behavior of titin in intact muscle. The authors suggest their
studies demonstrate the functional reconstitution of a protein
from the sum of its parts.

References (abridged):

1. Sigworth, F. J. & Neher, E. Single Na+ channel currents
observed in cultured rat muscle cells. Nature 287, 447-449 (1980)

2. Bustamante, C., Smith, S. B., Liphardt, J. & Smith, D. Single-
molecule studies of DNA mechanics. Curr. Opin. Struct. Biol. 10,
279-285 (2000)

3. Smith, D. E. et al. The bacteriophage 29 portal motor can
package DNA against a large internal force. Nature 413, 748-752
(2001)

4. Finer, J. T., Simmons, R. M. & Spudich, J. A. Single myosin
molecule mechanics: piconewton forces and nanometer steps. Nature
368, 113-119 (1994)

5. Lu, H. & Schulten, K. Steered molecular dynamics simulations
of force-induced protein domain unfolding. Proteins Struct.
Funct. Genet. 35, 453-463 (1999)

Nature 2002 418:998

Related Background Brief:

I-BAND TITIN IN CARDIAC MUSCLE IS A THREE-ELEMENT MOLECULAR
SPRING AND IS CRITICAL FOR MAINTAINING THIN FILAMENT STRUCTURE.
In cardiac muscle, the giant protein titin exists in different
length isoforms expressed in the molecule's I-band region. Both
isoforms, termed N2-A and N2-B, comprise stretches of Ig-like
modules separated by the PEVK domain. Central I-band titin also
contains isoform-specific Ig-motifs and nonmodular sequences,
notably a longer insertion in N2-B. The authors investigated the
elastic behavior of the I-band isoforms by using single-myofibril
mechanics, immunofluorescence microscopy, and immuno-electron-
microscopy of rabbit cardiac sarcomeres stained with sequence-
assigned antibodies. The authors also overexpressed constructs
from the N2-B region in chick cardiac cells to search for
possible structural properties of this cardiac-specific segment.
The authors found that cardiac titin contains three distinct
elastic elements: poly-Ig regions, the PEVK domain, and the N2-B
sequence insertion, which extends approximately 60 nm at high
physiological stretch. Recruitment of all three elements allows
cardiac titin to extend fully reversibly at physiological
sarcomere lengths, without the need to unfold Ig domains.
Overexpressing the entire N2-B region or its NH(2) terminus in
cardiac myocytes greatly disrupted thin filament, but not thick
filament structure. The authors suggest their results strongly
indicate that the NH(2)-terminal N2-B domains are necessary to
stabilize thin filament integrity. N2-B-titin emerges as a unique
region critical for both reversible extensibility and structural
maintenance of cardiac myofibrils. W.A. Linke et al: J Cell Biol
1999 146:631.

Related Background Brief:

MECHANICAL UNFOLDING INTERMEDIATES IN TITIN MODULES. The modular
protein titin, which is responsible for the passive elasticity of
muscle, is subjected to stretching forces. Previous work on the
experimental elongation of single titin molecules has suggested
that force causes consecutive unfolding of each domain in an all-
or-none fashion. To avoid problems associated with the
heterogeneity of the modular, naturally occurring titin, the
authors engineered single proteins to have multiple copies of
single immunoglobulin domains of human cardiac titin. The authors
report the elongation of these molecules using the atomic force
microscope. The authors find an abrupt extension of each domain
by approximately 7 angstroms before the first unfolding event.
This fast initial extension before a full unfolding event
produces a reversible 'unfolding intermediate' Steered molecular
dynamics simulations show that the rupture of a pair of hydrogen
bonds near the amino terminus of the protein domain causes an
extension of about 6 angstroms, which is in good agreement with
the observations. Disruption of these hydrogen bonds by site-
directed mutagenesis eliminates the unfolding intermediate. The
unfolding intermediate extends titin domains by approximately 15%
of their slack length, and is therefore likely to be an important
previously unrecognized component of titin elasticity. P.E.
Marszalek et al: Nature 1999 402:100.

Related Background Brief:

STEERED MOLECULAR DYNAMICS SIMULATIONS OF FORCE-INDUCED PROTEIN
DOMAIN UNFOLDING. Steered molecular dynamics (SMD), a computer
simulation method for studying force-induced reactions in
biopolymers has been applied to investigate the response of
protein domains to stretching apart of their terminal ends. The
simulations mimic atomic force microscopy and optical tweezer
experiments, but proceed on much shorter time scales. The
simulations on different domains for 0.6 nanosecond each reveal
two types of protein responses: the first type, arising in
certain beta-sandwich domains, exhibits nanosecond unfolding only
after a force above 1,500 pN is applied; the second type, arising
in a wider class of protein domain structures, requires
significantly weaker forces for nanosecond unfolding. In the
first case, strong forces are needed in concert to break a set of
interstrand hydrogen bonds which protect the domains against
unfolding through stretching; in the second case, stretching
breaks backbone hydrogen bonds one by one, and does not require
strong forces for this purpose. Stretching of beta-sandwich
(immunoglobulin) domains has been investigated further revealing
a specific relationship between response to mechanical strain and
the architecture of beta-sandwich domains. H. Lu and K. Schulten:
Proteins 1999 35:453.

Related Background Brief:

THE MOLECULAR ELASTICITY OF THE EXTRACELLULAR MATRIX PROTEIN
TENASCIN. Extracellular matrix proteins are thought to provide a
rigid mechanical anchor that supports and guides migrating and
rolling cells. The authors examined the mechanical properties of
the extracellular matrix protein tenascin by using atomic-force-
microscopy techniques. The authors suggest their results indicate
that tenascin is an elastic protein. Single molecules of tenascin
could be stretched to several times their resting length. Force-
extension curves showed a saw-tooth pattern, with peaks of force
at 137pN. These peaks were approximately 25 nm apart. Similar
results have been obtained by study of titin. The authors also
found similar results by studying recombinant tenascin fragments
encompassing the 15 fibronectin type III domains of tenascin.
This indicates that the extensibility of tenascin may be due to
the stretch-induced unfolding of its fibronectin type III
domains. Refolding of tenascin after stretching, observed when
the force was reduced to near zero, showed a double-exponential
recovery with time constants of 42 domains refolded per second
and 0.5 domains per second. The former speed of refolding is more
than twice as fast as any previously reported speed of refolding
of a fibronectin type III domain. The authors suggest that the
extensibility of the modular fibronectin type III region may be
important in allowing tenascin-ligand bonds to persist over long
extensions. These properties of fibronectin type III modules may
be of widespread use in extracellular proteins containing such
domain. A.F. Oberhauser et al: Nature 1998 393:181.

Related Background Brief:

DYNAMIC STRENGTH OF MOLECULAR ADHESION BONDS. In biology,
molecular linkages at, within, and beneath cell interfaces arise
mainly from weak noncovalent interactions. These bonds will fail
under any level of pulling force if held for sufficient time.
Thus, when tested with ultrasensitive force probes, we expect
cohesive material strength and strength of adhesion at interfaces
to be time- and loading-rate-dependent properties. To examine
what can be learned from measurements of bond strength, the
authors have extended Kramers' theory for reaction kinetics in
liquids to bond dissociation under force and tested the
predictions by smart Monte Carlo (Brownian dynamics) simulations
of bond rupture. By definition, bond strength is the force that
produces the most frequent failure in repeated tests of breakage,
i.e., the peak in the distribution of rupture forces. As verified
by the simulations, theory shows that bond strength progresses
through three dynamic regimes of loading rate. First, bond
strength emerges at a critical rate of loading (> or = 0) at
which spontaneous dissociation is just frequent enough to keep
the distribution peak at zero force. In the slow-loading regime
immediately above the critical rate, strength grows as a weak
power of loading rate and reflects initial coupling of force to
the bonding potential. At higher rates, there is crossover to a
fast regime in which strength continues to increase as the
logarithm of the loading rate over many decades independent of
the type of attraction. Finally, at ultrafast loading rates
approaching the domain of molecular dynamics simulations, the
bonding potential is quickly overwhelmed by the rapidly
increasing force, so that only naked frictional drag on the
structure remains to retard separation. Hence, to expose the
energy landscape that governs bond strength, molecular adhesion
forces must be examined over an enormous span of time scales.
However, a significant gap exists between the time domain of
force measurements in the laboratory and the extremely fast scale
of molecular motions. Using results from a simulation of biotin-
avidin bonds, the authors describe how Brownian dynamics can help
bridge the gap between molecular dynamics and probe tests. E.
Evans and K. Ritchie: Biophys. J. 1997 72:1541.

Related Background:

AFM IN THE STUDY OF MEMBRANE PROTEINS: UNFOLDING PATHWAYS OF
INDIVIDUAL BACTERIORHODOPSINS.

F. Oesterhelt et al (Ludwig Maximilians-Universit„t, DE) discuss
unfolding of bacteriorhodopsins, the authors making the following
points:

1) Membrane proteins acquire their unique functions through
specific folding of their polypeptide chains stabilized by
specific interactions in the membrane. Their stability or
resistance to unfolding, which goes hand in hand with their
anchoring into the hydrophobic belt of the membrane, is usually
investigated by chemical or thermal denaturation (1,2). Such
experiments, however, provide only ensemble information about the
energetics but not about individual proteins and their anchoring
forces. As described by the fluid mosaic model (3), membrane
proteins may diffuse within the bilayer but in the normal
direction are strongly restricted to the membrane plane. It is
expected that stability of membrane proteins involves
interactions with the lipid bilayer as well as intra- and
intermolecular interactions (1). Thus, it is tempting to
determine not only the forces that anchor membrane proteins in
the membrane but also the forces that interact between their
secondary structure elements.

2) To answer this pertinent question in membrane biology, the
authors combined atomic force microscopy (AFM) (4,5) and single-
molecule force spectroscopy to image individual membrane proteins
and to measure their molecular forces. The authors chose
bacteriorhodopsin (BR), a light-driven proton pump, because it
represents one of the most extensively studied membrane proteins.
Structural analysis has revealed the photoactive retinal embedded
in seven closely packed alpha-helices, which builds a common
structural motif among a large class of related G-protein-coupled
receptors. Moreover, BR has become a paradigm for alpha-helical
membrane proteins in general and for ion transporters in
particular. Together with adjacent lipids, BR molecules assemble
into trimers, which are packed into two-dimensional hexagonal
lattices, the so-called purple membrane of Halobacterium
salinarum.

3) In summary: Atomic force microscopy and single-molecule force
spectroscopy were combined to image and manipulate purple
membrane patches from Halobacterium salinarum. Individual
bacteriorhodopsin molecules were first localized and then
extracted from the membrane; the remaining vacancies were imaged
again. Anchoring forces between 100 and 200 piconewtons for the
different helices were found. Upon extraction, the helices were
found to unfold. The force spectra revealed the individuality of
the unfolding pathways. Helices G and F as well as helices E and
D always unfolded pairwise, whereas helices B and C occasionally
unfolded one after the other. Experiments with cleaved loops
revealed the origin of the individuality: stabilization of helix
B by neighboring helices.

References (abridged):

1. T. Haltia and E. Freire, Biochim. Biophys. Acta Bioenerg.
1228, 1 (1995).

2. S. H. White and W. C. Wimley, Annu. Rev. Biophys. Biomol.
Struct. 28, 319 (1999).

3. S. J. Singer and G. L. Nicolson, Science 175, 720 (1972).

4. G. Binnig, C. F. Quate, C. Gerber, Phys. Rev. Lett. 56, 930
(1986).

5. M. Radmacher, R. W. Tillmann, M. Fritz, H. E. Gaub, Science
257, 1900 (1992).

Science 2000 288:143.

Related Background Brief:

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4. AFM IN BIOLOGY: PALEONTOLOGY

ATOMIC FORCE MICROSCOPY OF PRECAMBRIAN MICROSCOPIC FOSSILS

A. Kempe et al (Ludwig-Maximilians-Universit„t M�nchen, DE)
discuss the use of AFM in the examination of fossils, the authors
making the following points:

1) Traditionally, understanding of the history of life has been
based chiefly on studies of the morphology of fossils.
Morphology, however, can provide only limited insight about the
underlying biochemical and physiological capabilities of ancient
organisms, a deficiency particularly detrimental to understanding
the earliest, Precambrian, seven-eighths of life's history.
Unlike the more recent, shorter, and much more familiar
Phanerozoic fossil record, that of the Precambrian was dominated
by diverse prokaryotic microorganisms having only a limited range
of simple morphologies yet widely divergent metabolic
capabilities. Because prokaryotic taxa having more or less
identical morphologies can differ greatly in metabolic
capability, and because the evolutionary development of these
various capabilities had profound effects on the evolution of the
Earth's oceans, atmosphere, and surficial environment (1), there
is a fundamental need to develop new techniques that by
correlating chemistry with morphology in individual Precambrian
microscopic fossils can provide insight into their underlying
biochemical makeup.

2) Significant progress toward answering this need has been made
by using ion microprobe spectrometry to analyze the carbon
isotopic composition of single Precambrian microfossils (2,3), an
approach to understanding the chemistry of such fossils that has
recently been extended to a molecular level by laser-Raman
spectroscopic imagery of individual Precambrian fossil microbes
(4,5). In principal, information about the structural makeup of
such molecular components should be accessible by using atomic
force microscopy (AFM), a technique used routinely in material
science to elucidate the nm-scale structure of macromolecules
such as DNA. The authors use AFM to image the fine structure of
the cell walls of Precambrian microfossils, an approach to this
problem that coupled with laser-Raman spectroscopy reveals the
submicron-scale organization of their kerogenous components. This
combination of AFM and laser-Raman spectroscopy provides means
not only to elucidate the fine structure of individual
microscopic fossils but to investigate the geochemical maturation
of ancient organic matter, the mechanisms that underlie fossil
preservation by permineralization, and, potentially, to determine
whether carbonaceous microscopic fossil-like objects are true
fossils rather than pseudofossil "look-alikes."

3) In summary: Atomic force microscopy (AFM) is a technique used
routinely in material science to image substances at a submicron
(including nm) scale. The authors apply this technique to
analysis of the fine structure of organic-walled Precambrian
fossils, microscopic sphaeromorph acritarchs (cysts of planktonic
unicellular protists) permineralized in 650-million-year-old
cherts of the Chichkan Formation of southern Kazakhstan. AFM
images, backed by laser-Raman spectroscopic analysis of
individual specimens, demonstrate that the walls of these
petrified fossils are composed of stacked arrays of 200-nm-sized
angular platelets of polycyclic aromatic kerogen. Together, AFM
and laser-Raman spectroscopy provide means by which to elucidate
the submicron-scale structure of individual microscopic fossils,
investigate the geochemical maturation of ancient organic matter,
and, potentially, distinguish true fossils from pseudofossils and
probe the mechanisms of fossil preservation by silica
permineralization.

References (abridged):

1.  Schopf, J. W. (1999) Cradle of Life, The Discovery of Earth's
Earliest Fossils (Princeton Univ. Press, Princeton).

2.  House, C. H. , Schopf, J. W. , McKeegan, K. D. , Coath, C. D.
, Harrison, T. M. & Stetter, K. O. (2000) Geology 28, 707-710.

3.  Ueno, Y. , Isozaki, Y. , Yuimoto, H. & Maruyama, S. (2001)
Int. Geol. Rev. 43, 196-212.

4.  Kudryavtsev, A. B. , Schopf, J. W. , Agresti, D. G. &
Wdowiak, T. J. (2001) Proc. Natl. Acad. Sci. USA 98, 823-826.

5.  Schopf, J. W. , Kudryavtsev, A. B. , Agresti, D. G. ,
Wdowiak, T. J. & Czaja, A. D. (2002) Nature (London) 416, 73-76.

Proc. Nat. Acad. Sci. 2002 99:9117

Related Background Brief:

LASER--RAMAN IMAGERY OF EARTH'S EARLIEST FOSSILS. Unlike the
familiar Phanerozoic history of life, evolution during the
earlier and much longer Precambrian segment of geological time
centred on prokaryotic microbes. Because such microorganisms are
minute, are preserved incompletely in geological materials, and
have simple morphologies that can be mimicked by nonbiological
mineral microstructures, discriminating between true microbial
fossils and microscopic pseudofossil "lookalikes" can be
difficult. Thus, valid identification of fossil microbes, which
is essential to understanding the prokaryote-dominated,
Precambrian 85% of life's history, can require more than
traditional palaeontology that is focused on morphology. By
combining optically discernible morphology with analyses of
chemical composition, laser--Raman spectroscopic imagery of
individual microscopic fossils provides a means by which to
address this need. The authors apply this technique to
exceptionally ancient fossil microbe-like objects, including the
oldest such specimens reported from the geological record, and
demonstrate that the results obtained substantiate the biological
origin of the earliest cellular fossils known. J.W. Schopf et al:
Nature 2002 416:73.

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5. AFM IN BIOLOGY: MECHANICAL PROPERTIES OF LIVING CELLS

COMBINING CONSTITUTIVE MATERIALS MODELING WITH ATOMIC FORCE
MICROSCOPY TO UNDERSTAND THE MECHANICAL PROPERTIES OF LIVING
CELLS.

M. McElfresh et al (Lawrence Livermore National Laboratory, US)
discuss the mechanical properties of living cells, the authors
making the following points:

1) The surface of the living cell is a highly complex
heterogeneous structure containing a variety of lipid, protein,
and carbohydrate components. The organization of the cell's
exterior "sensing elements" and other specialized regions of the
membrane is tailored to reflect the function of the cell and
serves vital roles in cell-cell interactions, cell signaling, and
cell-surface interactions. The changes that occur in these
important interaction phenotypes during the development of cancer
and other diseases may be understood in much more detail, thereby
allowing the relationships between specific phenotypes to cell
and tissue normo- and pathophysiology, prognosis, and therapy to
be discerned (1).

2) Recent studies have shown that the components that comprise
the membrane are segregated into domains that are dynamic and
change in response to external and internal stimuli (2-5). This
segregation appears to be controlled by a variety of factors,
including the composition of the lipids, interactions with the
cytoskeleton or extracellular matrix, and physical or structural
barriers to diffusion. Although these barriers usually limit the
random movement of receptors used in signaling and recognition
and maintain them in a particular environment, proteins and
carbohydrates are often relocalized and recruited into a
particular region of the cell surface to facilitate cell
function. In some cases, such as the sperm cell, dramatic changes
in the composition of the membrane and the location and
distribution of its proteins (receptors) occur throughout its
development. In other cases, more subtle changes often occur
later in the life of the cell and lead to cancer or other
diseases, such as multiple sclerosis.

3) Atomic force microscopy (AFM) has developed rapidly during the
past decade, providing nanometer-scale resolution in the imaging
of biological materials ranging in size from single molecules to
intact cells. Although the best data have been obtained from
studies of macromolecules (proteins, nucleic acids, and their
complexes), AFM images of mouse and bull sperm have been obtained
that rival the resolution of electron microscopy (EM). Unlike EM,
however, AFM imaging can be performed in fluid on living cells.
More recent developments in AFM now allow the detection of
molecular recognition events between single molecules using
ligands attached to AFM tips for the recognition of receptors
bound on rigid surfaces. By monitoring the cantilever deflection
during approach-retraction cycles (i.e., force-volume/force-
distance curves) at a constant (lateral) position on the sample,
unbinding forces (i.e., the maximum force at the moment of
receptor-ligand detachment) have been determined for various
ligand-receptor pairs, including biotin-avidin, DNA bases,
antibody-antigen, and cell-recognition proteins. This development
has made it possible to use a single receptor molecule bound to
the tip of an AFM cantilever to map the locations of ligands
bound on solid surfaces. The goal of our project is to enable
this "recognition mapping" method to be used in the study of the
surfaces of living cells.

4) In summary: The authors report a study of the properties of
living cells and cell membranes by using atomic force microscopy.
During atomic force microscopy (AFM) measurement, there is a
strong mechanical coupling between the AFM tip and the cell. The
purpose of this study is to present a model of the overall
mechanical response of the cell that allows us to separate out
the mechanical response of the cell from the local surface
interactions we wish to quantify. These local interactions
include recognition (or binding) events between molecules bound
to an AFM tip (e.g., an antibody) and molecules or receptors on
the cell surface (e.g., the respective antigen). The reported
computational model differs from traditional Hertzian contact
models by explicitly taking into account the mechanics of the
biomembrane and cytoskeleton. The model also accounts for the
mechanical response of the living cell during arbitrary
deformation. The indentation of a bovine sperm cell is used to
test the validity of this model, and further experiments are
proposed to fully parameterize the model.

References (abridged):

1. Lekka, M. , Lekki, J. , Marszalek, M. , Golonka, P. ,
Stachura, Z. , Cleff, B. & Hrynkiewicz, A. Z. (1999) Appl. Surf.
Sci. 141, 345-349.

2. Primakoff, P. & Myles, D. G. (1983) Dev. Biol. 98, 417-428.

3. Friend, D. S. (1989) Ann. NY Acad. Sci. 567, 208-221.

4. Edidin, M. (1997) Curr. Opin. Struct. Biol. 7, 528-532.

5. Retvield, A. & Simons, K. (1998) Biochim. Biophys. Acta 1376,
467-479.

Proc. Nat. Acad. Sci. 2002 99:6493

Related Background:

SINGLE PILUS MOTOR FORCES EXCEED 100 pN

B. Maier et al (Columbia University, US) discuss bacterial pilus
motor forces, the authors making the following points:

1) Type IV pili are polymeric filaments found on many Gram-
negative bacteria. They play a crucial role in human
pathogenesis, because they mediate adhesion to host mammalian
cells evoking downstream cellular responses (1), biofilm
formation (2), twitching motility (3,4), and DNA uptake (5).
Recently, type IV pili have been shown to generate considerable
force by retraction (3). For some pilus-dependent functions, the
amount of force generated by pilus retraction is critical, e.g.,
in host-cell responses and movement of the bacteria through
viscous mucous layers. Thus, it is of major interest to determine
the level of force generated by a single pilus retraction and the
factors that regulate force development.

2) Molecular motors normally move objects by repetitive, ATP-
powered motions on polymer filaments. Type IV pilus polymers are
several micrometers in length and 6 nm in diameter with 1,300
PilE (pilin) subunits/æm. It has been shown that PilT, an ATPase
associated with various cellular activities (AAA), is required
for pilus retraction (3) and force generation. However, it is
unknown whether the pilus retraction process, which most likely
involves depolymerization into a membrane, is functionally
similar to other motors or fundamentally different because of
membrane depolymerization. PilT may act as a molecular motor, as
a chaperone in a ratchet-like process, or as a decapping protein.

3) In summary: Force production by type IV pilus retraction is
critical for infectivity of Neisseria gonorrhoeae and DNA
transfer. The authors investigated the roles of pilus number and
the retraction motor, PilT, in force generation in vivo at the
single-molecule level, and found that individual retraction
events are generated by a single pilus fiber, and only one PilT
complex powers retraction. Retraction velocity is constant at low
forces but decreases at forces greater than 40 pN, giving a
remarkably high average stall force of 110 ñ 30 pN. Further
insights into the molecular mechanism of force generation are
gained from the effect of ATP-depletion, which reduces the rate
of retraction but not the stall force. Energetic considerations
suggest that more than one ATP is involved in the removal of a
single pilin subunit from a pilus. The authors suggest these
results are most consistent with a model in which the ATPase PilT
forms an oligomer that disassembles the pilus by a cooperative
conformational change.

References (abridged):

1. Lee, S. L. , Bonnah, R. A. , Hugashi, D. L. , Atkinson, J. P.
, Milgram, S. L. & So, M. (2002) J. Cell Biol. 156, 951-957.

2. Singh, P. K. , Parsek, M. R. , Greenberg, E. P. & Welsh, M. J.
(2002) Nature 417, 552-555.

3. Merz, A. J. , So, M. & Sheetz, M. P. (2000) Nature 407, 98-
101.

4. Skerker, J. M. & Berg, H. C. (2001) Proc. Natl. Acad. Sci. USA
98, 6901-6904.

5. Wolfgang, M. , Lauer, P. , Park, H.-S. , Brossay, L. , H‚bert,
J. & Koomey, M. (1998) Mol. Microbiol. 29, 321-330.

Proc. Nat. Acad. Sci. 2002 99:16012

Related Background Brief:

PILUS RETRACTION POWERS BACTERIAL TWITCHING MOTILITY. Twitching
and social gliding motility allow many gram negative bacteria to
crawl along surfaces, and are implicated in a wide range of
biological functions. Type IV pili (Tfp) are required for
twitching and social gliding, but the mechanism by which these
filaments promote motility has remained enigmatic. The authors
use laser tweezers to demonstrate that Tfp forcefully retract.
Neisseria gonorrhoeae cells that produce Tfp actively crawl on a
glass surface and form adherent microcolonies. When laser
tweezers are used to place and hold cells near a microcolony,
retractile forces pull the cells toward the microcolony. In
quantitative experiments, the Tfp of immobilized bacteria bind to
latex beads and retract, pulling beads from the tweezers at
forces that can exceed 80 pN. Episodes of retraction terminate
with release or breakage of the Tfp tether. Both motility and
retraction mediated by Tfp occur at about 1 micron/sec and
require protein synthesis and function of the PilT protein. The
authors suggest their experiments establish that Tfp filaments
retract, generate substantial force and directly mediate cell
movement. J.J. Merz et al: Nature 2000 407:98.

Related Background Brief:

DIRECT OBSERVATION OF EXTENSION AND RETRACTION OF TYPE IV PILI.
Type IV pili are thin filaments that extend from the poles of a
diverse group of bacteria, enabling them to move at speeds of a
few tenths of a micrometer per second. They are required for
twitching motility, e.g., in Pseudomonas aeruginosa and Neisseria
gonorrhoeae, and for social gliding motility in Myxococcus
xanthus. The authors report direct observation of extension and
retraction of type IV pili in P. aeruginosa. Cells without
flagellar filaments were labeled with an amino-specific Cy3
fluorescent dye and were visualized on a quartz slide by total
internal reflection microscopy. When pili were attached to a cell
and their distal ends were free, they extended or retracted at
rates of about 0.5 microns/sec (29øC). They also flexed by
Brownian motion, exhibiting a persistence length of about 5
microns. Frequently, the distal tip of a filament adsorbed to the
substratum and the filament was pulled taut. From the absence of
lateral deflections of such filaments, we estimate tensions of at
least 10 pN. Occasionally, cell bodies came free and were pulled
forward by pilus retraction. Thus, type IV pili are linear
actuators that extend, attach at their distal tips, exert
substantial force, and retract. J.M. Skerker and H.C. Berg: pnas
2001 98:6901.

Related Background Brief:

STRUCTURE OF THE FIBRE-FORMING PROTEIN PILIN AT 2.6 A RESOLUTION.
The crystallographic structure of Neisseria gonorrhoeae pilin,
which assembles into the multifunctional pilus adhesion and
virulence factor, reveals an alpha-beta roll fold with a striking
85 A alpha-helical spine and an O-linked disaccharide. Key
residues stabilize interactions that allow sequence
hypervariability, responsible for pilin's celebrated antigenic
variation, within disulphide region beta-strands and connections.
Pilin surface shape, hydrophobicity and sequence variation
constrain pilus assembly to the packing of flat subunit faces
against alpha 1 helices. Helical fiber assembly is postulated to
form a core of coiled alpha 1 helices banded by beta-sheet,
leaving carbohydrate and hypervariable sequence regions exposed
to solvent. H.E. Parge et al: Nature 1995 378:32.

Related Background:

ON THE MOTILITY OF BACTERIA ON SOLID SURFACES

A.J. Merz and K.T. Forest (Dartmouth Medical School, US) discuss
bacterial motility, the authors making the following points:

1) Considerations of bacterial motility usually refer to cells
swimming and tumbling through fluid media, propelled by rotary
flagella. Over the last thirty years, studies of flagellar
motility have yielded insights into molecular motor function,
signal transduction and type III bacterial protein secretion. But
bacterial life is not limited to the aqueous phase, and bacterial
motility is not limited to swimming: many bacteria crawl, glide
or twitch their way over solid substrates [1 5] . Bacterial
surface locomotion is involved in many aspects of microbiology
including morphogenesis, biofilm formation and microbe--host
interactions. Studies of surface motility engines can also
improve our understanding of protein export and of proteinaceous
channels that conduct macromolecules.

2) We can recognize two modes by which Gram-negative bacteria
move over surfaces. Adventurous gliding results from compressive
forces generated by the hydration, expansion and rearward
extrusion of polyelectrolyte slime. Twitching or "social gliding"
motility is due to tensile forces generated through the
attachment and retraction of type IV pilus fibers. Many questions
remain about the molecular machines that power twitching and
adventurous gliding, but they share at least one common feature:
both rely on the flux of large volumes of macromolecules through
proteinaceous pores in the bacterial outer membrane.

3) Several mechanisms have been proposed to account for gliding
motility including treadmill-like motors on the cell surface
[1,3,6]  and secretion of surfactants that draw the cell forward
[3,4] . Recent experiments support another idea: that the gliding
of filamentous bacteria -- linked chains of dozens to hundreds of
cells -- is powered by compressive forces arising from the
rearward secretion of slime, a polyelectrolyte gel composed of
complex carbohydrates [3].

4) In summary: It has been known for decades that bacteria
locomote over surfaces, but the mechanisms that power motility
have been unclear. Recent experiments have begun to explain two
modes of surface motility. Twitching or social gliding motility
is powered by the retraction of type IV pili. Adventurous gliding
motility is powered by the rearward secretion of carbohydrate
slime. In both cases, cell movement depends on the translocation
of enormous volumes of macromolecules through outer membrane pore
complexes. The authors describe molecular models for surface
motility and discuss how these models can inform studies of
macromolecule secretion across bacterial membranes.

References (abridged):

1. Hoiczyk E. (2000) Gliding motility in cyanobacteria:
observations and possible explanations. Arch. Microbiol., 174:11-
17

2. Kaiser D. (2000) Bacterial motility: how do pili pull? Curr.
Biol., 10:R777-780

3. McBride M.J. (2001) Bacterial gliding motility: multiple
mechanisms for cell movement over surfaces. Annu. Rev.
Microbiol., 55:49-75

4. Spormann A.M. (1999) Gliding motility in bacteria: insights
from studies of Myxococcus xanthus Microbiol. Mol. Biol. Rev.,
63:621-641

5. Wall D. and Kaiser D. (1999) Type IV pili and cell motility.
Mol. Microbiol., 32:1-10.

Current Biology 2002 12:R297

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6. AFM IN CONFORMATIONAL CHEMISTRY.

CHAIR-BOAT TRANSITIONS IN SINGLE POLYSACCHARIDE MOLECULES
OBSERVED WITH FORCE-RAMP AFM

P.E. Marszalek et al (Mayo Foundation, US) discuss chair-boat
transitions in polysaccharides, the authors making the following
points:

1) Single molecule force spectroscopy has become an important
tool to examine the conformations of proteins and polysaccharide
molecules under a stretching force (1-5). Recent experiments have
demonstrated that a force field can trigger conformational
changes in these molecules that cannot be observed by traditional
NMR or x-ray crystallographic techniques. Force spectroscopy
involves measuring the force required to extend a molecule by a
certain amount (2-5). The data are compiled into a force-
extension relationship that gives a characteristic fingerprint
for the conformational transitions of the molecule under study.
For example, force-extension relationships of polysaccharides
such as amylose and dextran display a characteristic plateau (2,
4) that marks forced transitions of the glucose monomers from
their chair conformation to the boat-like conformation (4).
Similarly, two distinct plateaus in the force-extension curve of
pectin report a transition of the galactose rings from the chair
conformation to the boat conformation and a subsequent flip of
the boat to the inverted chair conformation (5). These
observations provide an interesting perspective on the behavior
of polysaccharides under mechanical tension and suggest that
fingerprints of forced conformational transitions can be used as
a means for identifying single polysaccharides molecules in
solution.

2) In summary: Under a stretching force, the sugar ring of
polysaccharide molecules switches from the chair to the boat-like
or inverted chair conformation. This conformational change can be
observed by stretching single polysaccharide molecules with an
atomic force microscope. In those early experiments, the
molecules were stretched at a constant rate while the resulting
force changed over wide ranges. However, because the rings
undergo force-dependent transitions, an experimental arrangement
where the force is the free variable introduces an undesirable
level of complexity in the results. The authors demonstrate the
use force-ramp atomic force microscopy to capture the
conformational changes in single polysaccharide molecules. Force-
ramp atomic force microscopy readily captures the ring
transitions under conditions where the entropic elasticity of the
molecule is separated from its conformational transitions,
enabling a quantitative analysis of the data with a simple two-
state model. This analysis directly provides the physico-chemical
characteristics of the ring transitions such as the width of the
energy barrier, the relative energy of the conformers, and their
enthalpic elasticity. The authors suggest their experiments
enhance the ability of single-molecule force spectroscopy to make
high-resolution measurements of the conformations of single
polysaccharide molecules under a stretching force, making an
important addition to polysaccharide spectroscopy.

References (abridged):

1.  Fisher, T. E. , Marszalek, P. E. & Fernandez, J. M. (2000)
Nat. Struct. Biol. 7, 719-724.

2.  Rief, M. , Oesterhelt, F. , Heymann, B. & Gaub, H. E. (1997)
Science 275, 1295-1297.

3.  Oberhauser, A. F. , Marszalek, P. E. , Erickson, H. P. &
Fernandez, J. M. (1998) Nature (London) 393, 181-185.

4.  Marszalek, P. E. , Oberhauser, A. F. , Pang, Y.-P. &
Fernandez, J. M. (1998) Nature (London) 396, 661-664.

5.  Marszalek, P. E. , Pang, Y. P. , Li, H. , Yazal, J. E. ,
Oberhauser, A. F. & Fernandez, J. M. (1999) Proc. Natl. Acad.
Sci. USA 96, 7894-7898.

Proc. Nat. Acad. Sci. 2002 99:4278

Related Background:

SINGLE MOLECULE FORCE SPECTROSCOPY ON POLYSACCHARIDES BY ATOMIC
FORCE MICROSCOPY

M. Rief et al (Ludwig-Maximilians-Universitat, DE) discuss force
spectroscopy of polysaccharides, the authors making the following
points:

1) A recent series of single molecule experiments has provided
detailed insight into intermolecular and intramolecular forces,
revealing relevant information on molecular mechanisms (1-4). In
previous experiments, the authors and others chemically linked
molecular pairs such as biotin and avidin (3,5), or conjugated
DNA strands, between the tip of an atomic force microscope (AFM)
cantilever and support structures. Molecule-specific bond forces
between binding pairs were measured upon separation and compared
with known thermodynamic parameters (4). The authors have now
used this approach to probe elastic properties of single polymer
strands.

2) Experimental geometry: Dextrans (average molecular weight
500,000) linked to a gold surface through epoxy-alkanethiols were
activated with one carboxymethyl group per glucose unit on
average and reacted with streptavidin such that several molecules
were chemically bound to each dextran filament. The mean distance
between the grafting points of two different polymer strands was
about 200 angstroms, and the hydrated "polymer brush" extended
1000 to 2000 angstroms into the solution. Because in
physiological buffer dextran behaves like an ideal polymer, the
coil overlap is expected to be low. In these experiments
streptavidin served as a molecular handle for the manipulation of
the polymer to be investigated. An AFM cantilever with biotin
bound to the AFM tip, following the protocol given in (3), was
used to pull on individual dextran filaments through the biotin-
streptavidin bond.

3) In summary: Recent developments in piconewton instrumentation
allow the manipulation of single molecules and measurements of
intermolecular as well as intramolecular forces. Dextran
filaments linked to a gold surface were probed with the atomic
force microscope tip by vertical stretching. At low forces the
deformation of dextran was found to be dominated by entropic
forces and can be described by the Langevin function with a 6
angstrom Kuhn length. At elevated forces the strand elongation
was governed by a twist of bond angles. At higher forces the
dextran filaments underwent a distinct conformational change. The
polymer stiffened and the segment elasticity was dominated by the
bending of bond angles. The conformational change was found to be
reversible and was corroborated by molecular dynamics
calculations.

References (abridged):

1. S. B. Smith, Y. Cui, C. Bustamante, Science 271, 795 (1996).

2. P. Cluzel et al., ibid., p. 792; T. T. Perkins, D. E. Smith,
R. G. Larson, S. Chu, ibid. 268, 83 (1995); M. Radmacher, M.
Fritz, H. G. Hansma, P. K. Hansma, ibid. 265, 1577 (1994); J.
K„s, H. Strey, E. Sackmann, Nature 368, 226 (1994); J. T. Finer,
R. M. Simmons, J. A. Spudich, ibid., p. 113; K. Svoboda, C. F.
Schmidt, B. J. Schnapp, S. M. Block, ibid. 365, 721 (1993); U.
Dammer, et al., Biophys. J. 70, 2437 (1996); P. Hinterdorfer, W.
Baumgartner, H. J. Gruber, K. Schilcher, H. Schindler, Proc.
Natl. Acad. Sci. U.S.A. 93, 3477 (1996).

3. E.-L. Florin, V. T. Moy, H. E. Gaub, Science 264, 415 (1994).

4. V. T. Moy, E. L. Florin, H. E. Gaub, ibid. 266, 257 (1994).

5. G. U. Lee, D. A. Kidwell, R. J. Colton, Langmuir 10, 354
(1994).

Science 1997 275:1295

Related Background Brief:

POLYSACCHARIDE ELASTICITY GOVERNED BY CHAIR-BOAT TRANSITIONS OF
THE GLUCOPYRANOSE RING. Many common, biologically important
polysaccharides contain pyranose rings made of five carbon atoms
and one oxygen atom. They occur in a variety of cellular
structures, where they are often subjected to considerable
tensile stress. The polysaccharides are thought to respond to
this stress by elastic deformation, but the underlying molecular
rearrangements allowing such a response remain poorly understood.
It is typically assumed, however, that the pyranose ring
structure is inelastic and locked into a chair-like conformation.
The authors describe single-molecule force measurements on
individual polysaccharides that identify the pyranose rings as
the structural unit controlling the molecule's elasticity. In
particular, the authors find that the enthalpic component of the
polymer elasticity of amylose, dextran and pullulan is eliminated
once their pyranose rings are cleaved. The authors interpret
these observations as indicating that the elasticity of the three
polysaccharides results from a force-induced elongation of the
ring structure and a final transition from a chair-like to a
boat-like conformation. The authors expect that the force-induced
deformation of pyranose rings they report here plays an important
role in accommodating mechanical stresses and modulating ligand
binding in biological systems. P.E. Marszalek et al: Nature 1998
396:661.

Related Background:

ATOMIC LEVERS CONTROL PYRANOSE RING CONFORMATIONS.

P.E. Marszalek et al (Mayo Foundation, US) discuss pyranose ring
conformations, the authors making the following points:

1) Atomic force microscope (AFM) manipulations of single
polysaccharide molecules have recently expanded conformational
chemistry (1) to include force-driven transitions between the
chair and boat conformers of the pyranose ring structure (2). The
application of a force to a single molecule will deform it
elastically and also induce conformational transitions. Although
it is easy to understand the origin of an elastic deformation,
the mechanics of the conformational transition is less clear.

2) Pyranose-based sugars have two distinct chair conformations,
4C1 and 1C4 (3), separated by an energy barrier of 11 kcal/mol
(4). In addition to the chair conformers, pyranoses have
intermediate conformers corresponding to the boat conformation,
whose energy is 5-8 kcal/mol above the energy of the 4C1 chair
(5). Thermally driven transitions do occur between these
conformers. However, in the absence of an applied force, the most
stable conformation of a pyranose is that of the 4C1 chair (4-9).
Application of a force of 200 pN to polymers of glucopyranose
such as amylose drives a conformational change in the pyranose
ring that is evident as a sudden elongation of the molecule,
marking a prominent enthalpic component of the elasticity of the
molecule (2). This enthalpic component results from an increase
in the distance between glycosidic oxygen atoms caused by a
force-induced transition between the chair and boat conformations
of the pyranose ring (2).

3) In summary: Atomic force microscope manipulations of single
polysaccharide molecules have recently expanded conformational
chemistry to include force-driven transitions between the chair
and boat conformers of the pyranose ring structure. The authors
now expand these observations to include chair inversion, a
common phenomenon in the conformational chemistry of six-membered
ring molecules. The authors demonstrate that by stretching single
pectin molecules, they could change the pyranose ring
conformation from a chair to a boat and then to an inverted chair
in a clearly resolved two-step conversion. The two-step extension
of the distance between the glycosidic oxygen atoms O1 and O4
determined by atomic force microscope manipulations is
corroborated by ab initio calculations of the increase in length
of the residue vector O1O4 on chair inversion. The authors
postulate that this conformational change results from the torque
generated by the glycosidic bonds when a force is applied to the
pectin molecule. Hence, the glycosidic bonds act as mechanical
levers, driving the conformational transitions of the pyranose
ring. When the glycosidic bonds are equatorial, the torque is
zero, causing no conformational change. However, when the
glycosidic bond is axial, torque is generated, causing a rotation
around C-C bonds and a conformational change. This hypothesis
readily predicts the number of transitions observed in pyranose
monomers with 1a-4a linkages (two), 1a-4e (one), and 1e-4e
(none). The authors suggest their results demonstrate single-
molecule mechanochemistry with the capability of resolving
complex conformational transitions.

References (abridged):

1. Barton, D. H. R. (1970) Science 169, 539-544.

2. Marszalek, P. E., Oberhauser, A. F., Pang, Y.-P. & Fernandez,
J. M. (1998) Nature (London) 396, 661-664.

3. Barrows, S.E., Dulles, F. J., Cramer, C. J., French, A. D. &
Truhlar, D. G. (1995) Carbohydr. Res. 276, 219-251.

4. Joshi, N. V. & Rao, V. S. R. (1979) Biopolymers 18, 2993-3004.

5. Dowd, M. K., French, A. D. & Reilly, P. J. (1994) Carbohydr.
Res. 264, 1-19.

Proc. Nat. Acad. Sci. 1999 96:7894

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7. AFM IN SUPRAMOLECULAR CHEMISTRY

UNRAVELING THE NANOSTRUCTURE OF SUPRAMOLECULAR ASSEMBLIES OF
HYDROGEN-BONDED ROSETTES ON GRAPHITE: AN ATOMIC FORCE MICROSCOPY
STUDY

H. Sch”nherr et al (University of Twente, NL) discuss the
application of AFM to supramolecular chemistry, the authors
making the following points:

1) The self-assembly of small molecular building blocks into
supramolecular aggregates by using non-covalent interactions is
anticipated to open the path toward the realization of molecular
devices and well-defined nanometer-scale structures and objects
(1). Expanding on the profound knowledge in the fields of
supramolecular chemistry (2-5) and intermolecular (surface)
forces, recent progress in nanostructuring has enabled several
groups to arrange supramolecular aggregates in two dimensions on
surfaces by clever design of the interactions and by using
scanning probe microscopy approaches at variable temperatures.
Other promising approaches include molecular beam epitaxy. As
shown very recently, supramolecular assemblies in three
dimensions serve as potentially valuable templates for the
formation of, e.g., single crystal silver nanowires.

2) The authors report on their recent progress in the formation
and structural analysis of nanometer-scale aggregates by using
self-assembled rosette structures based on hydrogen-bonding.
Their previous work on molecular boxes derived from the
corresponding double rosettes showed that nano-rod structures can
be obtained under certain conditions on graphite surfaces.
Because the synthetic pathways for selective functionalization of
these molecules have been developed fully in recent years, it is
possible to attach functional units, such as receptors and
reporters, at virtually any preselected location in the
molecules. If the lateral assembly of higher order structures,
such as nano-rods or crystals, can be controlled similarly well
in two dimensions, it will be possible to position and pattern
functional nanostructures by spontaneous self-assembly processes.
These nanostructures possess, hence, considerable potential as
templates, receptor arrays, versatile molecular print-boards,
etc.

3) In summary: The self-organization of multicomponent tetra-
rosette assemblies into ordered nanostructures on graphite
surfaces has been studied by atomic force microscopy (AFM). Real-
space information on the level of individual molecules allowed
the authors to analyze the underlying structure in unprecedented
detail. In highly ordered nano-rod domains, tetra-rosettes
arrange in the form of parallel rows with a spacing of 4.6 ñ 0.1
nm. High resolution AFM revealed the internal packing of the
tetra-rosette assemblies in these rows, which can be described by
an oblique lattice. The authors suggest these results, together
with recent improvements in synthetic approaches, contribute to
the development of a general strategy to develop H-bonding-based
nanostructures with molecular precision.

References (abridged):

1.  Lehn, J.-M. (1995) Supramolecular Chemistry: Concepts and
Perspectives (VCH, New York).

2.  Lehn, J.-M. (1988) Angew. Chem. Int. Ed. Engl. 27, 89-112.

3.  Cram, D. J. (1988) Angew. Chem. Int. Ed. Engl. 27, 1009-1020.

4.  Pedersen, C. J. (1988) Angew. Chem. Int. Ed. Engl. 27, 1021-
1027.

5.  Reinhoudt, D. N., ed. (1999) Supramolecular Materials and
Technologies (Wiley, New York).

Proc. Nat. Acad. Sci. 2002 99:5024.

Related Background Brief:

SELECTIVE ASSEMBLY ON A SURFACE OF SUPRAMOLECULAR AGGREGATES WITH
CONTROLLED SIZE AND SHAPE. The realization of molecule-based
miniature devices with advanced functions requires the
development of new and efficient approaches for combining
molecular building blocks into desired functional structures,
ideally with these structures supported on suitable substrates.
Supramolecular aggregation occurs spontaneously and can lead to
controlled structures if selective and directional non-covalent
interactions are exploited. But such selective supramolecular
assembly has yielded almost exclusively crystals or dissolved
structures; the self-assembly of absorbed molecules into larger
structures, in contrast, has not yet been directed by controlling
selective intermolecular interactions. The authors report the
formation of surface-supported supramolecular structures whose
size and aggregation pattern are rationally controlled by tuning
the non-covalent interactions between individual absorbed
molecules. Using low-temperature scanning tunneling microscopy,
the authors demonstrate that substituted porphyrin molecules
adsorbed on a gold surface form monomers, trimers, tetramers or
extended wire-like structures. The authors find that each
structure corresponds in a predictable fashion to the geometric
and chemical nature of the porphyrin substituents that mediate
the interactions between individual adsorbed molecules. The
authors suggest their findings indicate that careful placement of
functional groups that are able to participate in directed non-
covalent interactions will allow the rational design and
construction of a wide range of supramolecular architectures
absorbed to surfaces. T. Yokoyama et al: Nature 2001 413:585.

Related Background Brief:

CONTROLLED ROOM-TEMPERATURE POSITIONING OF INDIVIDUAL MOLECULES:
MOLECULAR FLEXURE AND MOTION. Two-dimensional positioning of
intact individual molecules was achieved at room temperature by a
controlled lateral "pushing" action of the tip of a scanning
tunneling microscope. To facilitate this process, four bulky
hydrocarbon groups were attached to a rigid molecule. These
groups maintained sufficiently strong interactions with the
surface to prevent thermally activated diffusional motion, but
nevertheless allowed controllable translation. Simulations
demonstrated the crucial role of flexure during the positioning
process. The authors suggest these results outline the key role
of molecular mechanics in the engineering of predefined
properties on a molecular scale. T.A. Jung et al: Science 1996
271:181.

Related Background Brief:

NANOSCALE SCIENCE OF SINGLE MOLECULES USING LOCAL PROBES.
Experiments on individual molecules using scanning probe
microscopies have demonstrated an exciting diversity of physical,
chemical, mechanical, and electronic phenomena. They have
permitted deeper insight into the quantum electronics of
molecular systems and have provided unique information on their
conformational and mechanical properties. Concomitant
developments in experimentation and theory have allowed a diverse
range of molecules to be studied, varying in complexity from
simple diatomics to biomolecular systems. At the level of an
individual molecule, the interplays of mechanical and electronic
behavior and chemical properties manifest themselves in an
unusually clear manner. In revealing the crucial role of thermal,
stochastic, and quantum-tunneling processes, they suggest that
dynamics is inescapable and may play a decisive role in the
evolution of nanotechnology. J.K. Gimzewski and J. Joachim:
Science 1999 283:1683.

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8. AFM IN MATERIALS SCIENCE

"There are over 34,000 described species of Araneae [spiders].
Each species uses silk, and some ecribellate orb weavers
(Araneoidea) have a varied tool kit of task-specific silks with
divergent mechanical properties. Araneoid major ampullate silk,
the primary dragline, is extremely tough. Minor ampullate silk,
used in web construction, has high tensile strength. An orb web's
capture spiral, in part composed of flagelliform silk, is
stretchy and can triple in length before breaking. Each of these
fibers is composed of one or more proteins encoded by the spider
silk fibroin gene family. Previously sequenced araneoid fibroins
are dominated by iterations of four simple amino acid motifs:
polyalanine (An) (n, iterations of amino acid or motif),
alternating glycine and alanine (GA), GGX (where X represents a
small subset of amino acids), and GPG(X)n (P, proline). However,
orb weavers represent only a fraction of spider diversity. The
silks of non-araneoid spiders have not been characterized at the
DNA sequence level... Spiders draw fibers from dissolved fibroin
proteins that are stored in specialized sets of abdominal
glands."

J. Gatesy et al: Science 2001 291:2603.

SEGMENTED NANOFIBERS OF SPIDER DRAGLINE SILK: ATOMIC FORCE
MICROSCOPY AND SINGLE-MOLECULE FORCE SPECTROSCOPY.

E. Oroudjev et al (University of California Berkeley, US) discuss
spider silk, the authors making the following points:

1) The last decade has seen a significant increase in the
scientific literature on spider dragline silk. This interest is
due to the impressive mechanical properties of spider dragline
silk, at a time when biomaterials and biomimetics are both
exciting interest in the rapidly growing field of materials
research. The viscoelastic fibers of spider dragline silk combine
both a high tensile strength that is comparable to steel and is
only slightly inferior to Kevlar (2/3 of its tensile strength),
and a high elasticity (30% elongation before failure) that is
comparable to rubber (1-4). This unique combination makes spider
dragline silk mechanically superior to almost any other natural
or man-made material. It is apparent that the mechanical
properties of the dragline silk protein's intramolecular
structure as well as the intermolecular organization of these
proteins in the fiber are critical for spider silk performance
(2,5). The authors report the partial mechanical and structural
characterization of a recombinant dragline silk protein. The
authors suggest this recombinant silk protein provides a valuable
test system for establishing the relationships between protein
structure and mechanical properties in spider silk.

2) Spider dragline silk can be pictured as a composite material
consisting of a semiamorphous matrix filled with tiny (<10 nm)
nano-crystalline-like particles. The amino acid sequence for
spider dragline silk proteins is comprised of poly(A)
[poly(alanine)], for some silks substituted by poly(GA)
[poly(glycyalanine)], and glycine-rich sequences (2). Despite
intensive structural studies on spider dragline silk proteins,
their exact structural organization remains to be solved. The 4-
to 10-residue-long poly(A) and poly(GA) motifs are thought to be
involved in the formation of beta-sheet nano-crystalline-like
particles. Glycine-rich sequences are thought to fold into some
non-alpha-helical helical structure for GGX or into beta-turns
for GPGGX, thus forming the semiamorphous matrix (2). On the
other hand, a few reports suggest that at least part of these
glycine-rich motifs can also fold into beta-sheets and/or form an
interphase between crystalline-like objects and a semiamorphous
matrix. NMR and x-ray diffraction experiments show that the
crystalline-like particles are well oriented along the silk fiber
with polypeptide chains parallel and alanine residues
perpendicular to the fiber axis. These findings suggest that
protein molecules are overall well oriented in the silk fiber.
This high degree of molecular orientation, together with
structural organization of dragline silk proteins, is a
prerequisite for the unique mechanical properties of the whole
silk fiber.

3) In summary: Despite its remarkable materials properties, the
structure of spider dragline silk has remained unsolved. Results
from two probe microscopy techniques provide new insights into
the structure of spider dragline silk. A soluble synthetic
protein from dragline silk spontaneously forms nanofibers, as
observed by atomic force microscopy. These nanofibers have a
segmented substructure. The segment length and amino acid
sequence are consistent with a slab-like shape for individual
silk protein molecules. The height and width of nanofiber
segments suggest a stacking pattern of slab-like molecules in
each nanofiber segment. This stacking pattern produces nano-
crystals in an amorphous matrix, as observed previously by NMR
and x-ray diffraction of spider dragline silk. The possible
importance of nanofiber formation to native silk production is
discussed by the authors. Force spectra for single molecules of
the silk protein demonstrate that this protein unfolds through a
number of rupture events, indicating a modular substructure
within single silk protein molecules. A minimal unfolding module
size is estimated to be approximately 14 nm, which corresponds to
the extended length of a single repeated module, 38 amino acids
long. The structure of this spider silk protein is distinctly
different from the structures of other proteins that have been
analyzed by single-molecule force spectroscopy, and the force
spectra show correspondingly novel features.

References (abridged):

1. Hinman, M. B. , Jones, J. A. & Lewis, R. V. (2000) Trends
Biotechnol. 18, 374-379.

2. Hayashi, C. Y. , Shipley, N. H. & Lewis, R. V. (1999) Int. J.
Biol. Macromol. 24, 271-275.

3. Tirrell, D. A. (1996) Science 271, 39-40.

4. Cunniff, P. M. , Fossey, S. A. , Auerbach, M. A. , Song, J. W.
, Kaplan, D. L. , Adams, W. W. , Eby, R. K. , Mahoney, D. &
Vezie, D. L. (1994) Polym. Adv. Technol. 5, 401-410.

5. Vollrath, F. & Knight, D. P. (2001) Nature (London) 410, 541-
548.

Proc. Nat. Acad. Sci. 2002 99:6460

Related Background Brief:

LIQUID CRYSTALLINE SPINNING OF SPIDER SILK. Spider silk has
outstanding mechanical properties despite being spun at close to
ambient temperatures and pressures using water as the solvent.
The spider achieves this feat of benign fiber processing by
judiciously controlling the folding and crystallization of the
main protein constituents, and by adding auxiliary compounds, to
create a composite material of defined hierarchical structure.
Because the "spinning dope" (the material from which silk is
spun) is liquid crystalline, spiders can draw it during extrusion
into a hardened fiber using minimal forces. This process involves
an unusual internal drawdown within the spider's spinneret that
is not seen in industrial fibre processing, followed by a
conventional external drawdown after the dope has left the
spinneret. Successful copying of the spider's internal processing
and precise control over protein folding, combined with knowledge
of the gene sequences of its spinning dopes, could permit
industrial production of silk-based fibers with unique properties
under benign conditions. F. Vollrath and D.P. Knight: Nature 2001
410:541.

Related Background Brief:

FIBER MORPHOLOGY OF SPIDER SILK: THE EFFECTS OF TENSILE
DEFORMATION. The fiber morphology of the dragline silk of Nephila
clavipes has been investigated by the detailed analysis of wide-
angle X-ray diffraction (WAXD) patterns. WAXD gives the crystal
lattice dimensions, the orientation distribution? the crystalline
fraction, and an estimate of the crystal size. It is found that
the crystals are very small and well oriented. The mean (minimum)
crystal dimensions are 2 x 5 x 7 nm, and the angle, phi, between
the molecular chains in the crystals and the fiber axis has a
full width at half-maximum (fwhm) of 15.7 degrees and an
orientation function f=0.981. The X-ray crystallinity is in the
range 10-15%, and the amorphous diffraction is divided 60:40
between an isotropic ring and an oriented halo with fwhm 30
degrees. This means one-third of the material is in the oriented
amorphous state, with a chain orientation of fwhm 43 degrees and
f=0.87. When the fiber is extended up to 10%,, the orientation of
the crystals increases as predicted for affine deformation at
constant volume. There is no observable change in crystallinity
and apparently a small reduction in the lateral crystal size on
deformation. D.T. Grubb and L.W. Jelinski: Macromolecules 1997
30:2860.

Related Background Brief:

SUPERCONTRACTION AND BACKBONE DYNAMICS IN SPIDER SILK: C-13 AND
H-2 NMR STUDIES. The high-performance mechanical properties of
certain spider silks can be radically altered by the addition of
water. For example, unconstrained silk fibers from the major
ampullate gland of the golden orb-weaving spider, Nephila
clavipes, contract to about half of their original length when
immersed in water. The authors use solid-state C-13 and H-2 NMR
to study N. clavipes silk fibers, so as to address the molecular
origins of supercontraction in the wet silk. Using C-13 NMR, the
authors study backbone dynamics and demonstrate that, when in
contact with water, a substantial fraction of the glycine,
glutamine, tyrosine, serine, and leucine residues in the protein
backbone show dramatic increases in the rate of large-amplitude
reorientation. 2H NMR of silk samples that incorporate leucine
deuterated at one terminal methyl group provides a probe for
dynamics at specific side chains along the fiber. Only a subset
of these leucine residues is strongly affected by water. The
authors suggest that the highly conserved YGGLGS(N)QGAGR blocks
found in the silk protein play a major role in the
supercontraction process. Amino acid sequences are proposed to
produce artificial spider silk with similar mechanical
properties, but without the undesired phenomenon of
supercontraction. A possible use of the "supercontracting
sequence" is also suggested. Z.T Yang et al: .J. Am. Chem. Soc.
2000 122:9019.

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