Subnanometer atomic force microscopy of
peptide–mineral interactions links clustering and
competition to acceleration and catastrophe
R. W. Friddlea,b,1, M. L. Weavera,c,1, S. R. Qiua, A. Wierzbickid, W. H. Caseyc, and J. J. De Yoreob,2
a
Physical and Life Sciences Directorate, Lawrence Livermore National Laboratory, Livermore, CA 94551; bMolecular Foundry, Lawrence Berkeley National
Laboratory, Berkeley, CA 94720; cDepartments of Chemistry and Geology, University of California, Davis, CA 95616; and dDepartment of Chemistry,
University of South Alabama, Mobile, AL 36688
Edited by Joanna Aizenberg, Harvard University, and accepted by the Editorial Board November 3, 2009 (received for review July 23, 2009)
In vitro observations have revealed major effects on the structure,
growth, and composition of biomineral phases, including stabili-
zation of amorphous precursors, acceleration and inhibition of
kinetics, and alteration of impurity signatures. However, decipher-
ing the mechanistic sources of these effects has been problematic
due to a lack of tools to resolve molecular structures on mineral
surfaces during growth. Here we report atomic force microscopy
investigations using a system designed to maximize resolution
while minimizing contact force. By imaging the growth of calcium-
oxalate monohydrate under the influence of aspartic-rich peptides
at single-molecule resolution, we reveal how the unique interac-
tions of polypeptides with mineral surfaces lead to acceleration,
inhibition, and switching of growth between two distinct states.
Interaction with the positively charged face of calcium-oxalate
monohydrate leads to formation of a peptide film, but the slow
adsorption kinetics and gradual relaxation to a well-bound state
result in time-dependent effects. These include a positive feedback
between peptide adsorption and step inhibition described by a
mathematical catastrophe that results in growth hysteresis, charac-
terized by rapid switching from fast to near-zero growth rates for
very small reductions in supersaturation. Interactions with the ne-
gatively charged face result in formation of peptide clusters that
impede step advancement. The result is a competition between ac-
celerated solute attachment and inhibition due to blocking of the
steps by the clusters. The findings have implications for control of
pathological mineralization and suggest artificial strategies for di-
recting crystallization.
biomineralization ∣ calcium oxalate monohydrate ∣ crystal growth ∣
peptide adsorption ∣ molecular resolution
T he interaction of proteins with inorganic constituents is a de-
fining feature of biomineral systems. They direct crystalliza-
tion of precursors (1), inhibit pathological mineralization (2, 3),
and initiate mineral resorption (4). In vitro observations reveal
major effects on structure, growth, and composition including
stabilization of amorphous phases (5), acceleration (6) and inhi-
bition of kinetics (2), and alteration of impurity signatures (7).
Defining the microscopic mechanism behind these effects has
been difficult due to a lack of tools to resolve either proteins
or the molecular structure of mineral surfaces during growth.
In principle, atomic force microscopy (AFM) has the resolving
power to investigate interactions between proteins and crystal
surfaces at the molecular scale. However, in practice this scenario
presents the difficult challenge of imaging soft biomolecules on a
hard, dynamic surface and requires a delicate balance between
lateral resolution, temporal resolution, and sample disruption.
To capture the morphological evolution of atomic steps and their
interaction with macromolecules, one must scan rapidly. Thus
cantilever stiffness must be low to minimize the force applied as
it passes over topographic features such as steps and weakly ad-
sorbed macromolecules. At the same time, high-resolution imag-
ing requires sharp probes. Silicon probes, which offer excellent
www.pnas.org/cgi/doi/10.1073/pnas.0908205107
sharpness, are too stiff to scan at high rates in contact mode
without displacing adhered molecules or fracturing the brittle
tip. The common solution is to use cantilever probes fabricated
from silicon nitride, which are much softer; but the malleability of
silicon nitride renders these probes dull and limits their resolving
power. To overcome these limitations, we employed probes com-
prised of sharpened Si tips on flexible, low-stress Si3 N4 cantile-
vers (Applied Nanostructures). To augment the relatively weak
laser signal reflected from the thin Si3 N4 cantilever beam
(600 nm thick, uncoated), we utilized a 3-mW diode laser instead
of the ∼1-mW lasers usually employed.
We used this system to investigate peptide interactions with
calcium-oxalate monohydrate (COM, CaC2 O4 · H2 O), the domi-
nant mineral phase in human kidney stones (8) (see Methods for
a description of materials and imaging conditions). Aside from
clinical relevance, COM is an excellent system for investigating
peptide interactions, because it presents two strongly contrast-
PHYSICS
ing faces. One—ð−101Þ—is calcium-rich and electrostatically
positive, whereas another—(010)—is oxalate-rich and strongly
negative (9).
The importance of both specific and nonspecific interactions
between COM surfaces and polypeptides or polyionic molecules
has been demonstrated previously. Polypeptides altered step
kinetics in a face- and step-specific manner, and their pre-
sence in solution reduced adhesion forces between carboxylate-
functionalized AFM tips and COM surfaces (10, 11). In vitro stu-
dies of clinical relevance found that COM growth was strongly
inhibited by acidic urinary molecules, particularly citrate and
osteopontin (OPN) (12, 13). [In this regard, COM is reminiscent
of calcium carbonate in that proteins commonly found in associa-
tion with carbonate biominerals are highly acidic and rich in
carboxylic side chains (14).] However, the consequences of intro-
ducing polyanions to COM differ significantly between small
molecules and polypeptides. For example, the effects of citrate
on COM growth were well explained by classical growth models
of step pinning (15), and electrostatic models of citrate binding
provided a clear rationale for the face- and step-specific effects
(12): Due to repulsive and attractive interactions of the carboxylic
groups with oxalate and calcium ions, respectively, citrate had
little effect on the negative face while binding strongly to the
positive face. In contrast, OPN, which contains peptide segments
Author contributions: S.R.Q., W.H.C., and J.J.D.Y. designed research; R.W.F., M.L.W., and
A.W. performed research; R.W.F., M.L.W., S.R.Q., and J.J.D.Y. analyzed data; and R.W.F.,
M.L.W., and J.J.D.Y. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission. J.A. is a guest editor invited by the Editorial Board.
Freely available online through the PNAS open access option.
1
R.W.F. and M.L.W. contributed equally to this work.
2
To whom correspondence should be addressed. E-mail: jjdeyoreo@lbl.gov.
This article contains supporting information online at www.pnas.org/cgi/content/full/
0908205107/DCSupplemental.
PNAS ∣ January 5, 2010 ∣ vol. 107 ∣ no. 1 ∣ 11–15
consisting of more than 50% aspartic acid (Asp) residues, exhib-
ited face-specific effects that appear to contradict this simple pic-
ture. On the positive face, they formed surface-aggregates having
little effect on growth but strongly inhibited growth of the nega-
tive face (12). Clearly, the simple binding rationale applicable to
small molecules such as citrate is not conserved in their larger,
polypeptide counterparts bearing similar chemical functionality.
The underlying source of these differences is undoubtedly the
unique adsorption behavior of polypeptides at surfaces (16). Poly-
peptide binding to an oppositely charged surface requires cross-
ing a series of kinetic barriers, such as displacement of shielding
counterions, configurational relaxation of the polymer, and coor-
dination with oppositely charged sites on the crystal to reach
the final collapsed state in which the polypeptide residues form
specific bonds to lattice sites (16). In contrast, at like-charged
surfaces, polypeptide solutions containing di- and trivalent coun-
terions exhibit fluctuating polarizations that can diminish the
double-layer repulsion between like-charged objects, allowing
the van der Waals attraction to dominate (16, 17). This effect
can lead to polypeptide clustering (dictated by the balance of
enthalpic gain and entropic cost of aggregation) as well as non-
specific binding to the surface.

Results
Atomic Resolution of COM Crystal Surfaces During Growth. To attain
high-resolution control images of the surface, we performed in
situ AFM on the ð−101Þ and (010) faces of COM at supersatura-
tion ∼0.8, in the absence of peptide. (Supersaturation is defined
 
aðCa2þ Þ aðC O2− Þ
Δμ 1
by σ ≡ kB T ¼ 2 ln K sp
2 4
, where Δμ represents the differ-
ence in chemical potential per COM molecule between solution
and crystal, kB is Boltzmann’s constant, T is the absolute tem-
perature, the ai ’s are the activities of the respective components,
and K sp is the solubility constant. See Methods for details.) As
Fig. 1 shows, even at room temperature, we obtained true single-
molecule resolution of faces, atomic steps, kinks, and even what
appear to be single attachment events during growth. In Figs. 1A
and B, the AFM images of the steps on the (010) face are com-
pared to the interpretations on the basis of the crystal structure.
Image enhancement by Fourier filtering revealed submolecular
details such as protruding ends of vertically aligned oxalate
groups (high “peaks”) and recessed, flat-lying oxalate groups
(low “holes”) (Fig. 1C). (See also Fig. S1.) Comparison of Fig. 1C
with Fig. 1D shows the greater vertical relief of the (010) face as
compared to the ð−101Þ face. The overlying molecular models
are based on classical molecular dynamics (MD) simulations
as described below.
Peptide Adsorption and Step Interactions on COM Surfaces. To inves-
tigate the consequences of polypeptide adsorption dynamics in a
well-controlled system, we synthesized two 27-residue peptides
consisting of ðDDDXÞ6 DDD (X ¼ S or G) designed to act as
surrogates for the Asp-rich region of OPN, which contains 19
Asp residues occurring primarily in groups of two to three. Not
surprisingly, in vacuo simulations of binding supported the simple
electrostatic picture: The negative oxygen atoms of the oxalate
ions that protrude from the (010) terrace were predicted to repel
the negative carboxyl side groups along the peptide (Fig. 1C),
whereas the positive calcium-rich ð−101Þ terrace provided a
favorable binding environment (Fig. 1D). Peptide binding to
the positive face was predicted to be nearly three times greater
than to the negative face (see Methods). Thus, one might naively
expect the impact of these peptides to mimic that of citrate. How-
ever, our in situ observations presented a strikingly different
picture.
As Figs. 2B and 3B show, at low resolution, the morphologies
of the two faces were similar. But high-resolution imaging
Fig. 1. (A–B) AFM height images of steps on the (010) face showing indivi-
dual kinks and single molecules at the step edge. Also given are schematics
showing the relationship between AFM images and underlying COM struc-
ture. Color scheme: Red—O, green—Ca, and gray—C. (C) AFM deflection im-
age of the (010) face showing step, kinks, and electrostatic map of
ðDDDSÞ6 DDD for configuration of highest binding energy. (D) AFM deflection
image of the ð−101Þ face and electrostatic map for configuration of highest
binding energy for ðDDDSÞ6 DDD as predicted by the in vacuo MD simulations.
Red is most negative; blue is most positive. In an intuitive and simple electro-
static picture of binding, the negative (yellow) oxygen atoms of the oxalate
ions that protrude from the (010) terrace are expected to repel the negative
(yellow-to-red) carboxyl side groups along the peptide. In contrast, on the
ð−101Þ face, the MD simulations predict that the positive (blue) calcium-rich
terrace should provide a favorable binding environment for the carboxyl-rich
peptide. Unit cell parameters (9) are a ¼ 9.976 Å, b ¼ 14.588 Å, c ¼ 6.291 Å,
and β ¼ 107.05°. Electrostatic potentials in C and D were rendered by
using MOE 2005.06 (Chemical Computing Group). Molecular structures in
Figs. 1 and 3 were rendered by using WebLab ViewerPro 3.7 (Molecular
Simulations).
revealed starkly contrasting peptide adsorption behavior. As ex-
pected, peptides bound to the ð−101Þ face so that, even at peptide
concentrations <10 nM, the face was covered by peptides
(Fig. 3C). In contrast, the (010) atomic structure was still well
resolved in the presence of peptide, showing that individual pep-
tides did not bind to that face (Fig. 2C). Instead, despite the like
charge of the peptides and the face, the peptides formed surface-
adsorbed clusters about 10 nm in size (Figs. 2B and C). Cluster
number density increased with peptide concentration (Fig. S2),
but cluster size remained nearly constant.
By following the step dynamics at molecular resolution, we
found that these contrasting adsorption behaviors led to distinct,
face-specific controls on growth (Fig. 4). On the ð−101Þ face, as
steps moved through the adsorbed peptides, they became se-
verely roughened due to a high density of pinning sites. But
on the (010) face, the steps still consisted of straight segments
with visible kinks. When a step encountered a peptide cluster,
it formed a hinge point where it was temporarily stopped. Even-
tually, the steps overcame these obstacles and recovered to their
straight morphology, leaving the peptide clusters unperturbed by
the passing step. This process occurred repeatedly without any
significant change to cluster location or shape (movies of these
growth processes are provided in SI Text).

12 ∣ www.pnas.org/cgi/doi/10.1073/pnas.0908205107 Friddle et al.

Fig. 2. The (010) face. (A) A typical dislocation hillock under pure growth
conditions. (B) Morphology during growth in 5 nM ðDDDSÞ6 DDD, at low ionic
strength (I ¼ 7 × 10−4 M), showing peptide clusters and pinned steps.
(C) High-resolution image of peptide clusters on the terraces, one in front
of the approaching steps and one within with the upper step. The crystal
lattice is well resolved, and the adsorbed peptides are confined to the clusters
(I ¼ 0.15 M). (D) Step speed versus supersaturation σ at various concentra-
tions of ðDDDSÞ6 DDD showing dual functionality—inhibition at low supersa-
turation (or high peptide level) and acceleration at high supersaturation (or
low peptide level). Standard errors in step speed measurements range from
10% at the lowest values of supersaturation to <0.5% at the highest values.
In all cases, the standard errors are smaller than the symbols. Solid curves are
fits to the model combining step pinning by peptide clusters with reduction
in the activation barrier by an amount proportional to the peptide concen-
tration (see Supplementary Information for details.) (Figs. S1 and S2 show
dependencies of cluster density and degree of inhibition/acceleration on
peptide concentration.)
Crystal Growth Kinetics in the Presence of Peptide. Quantification
of the atomic step speed v revealed unreported consequences
of these distinct adsorption behaviors on growth kinetics as well
as insights into previously unexplained effects. On the ð−101Þ
face, the steps exhibited two distinct states: Upon decreasing
supersaturation σ from high values, v suddenly jumped from that
of the pure system into a “dead zone”—i.e., a region of finite
supersaturation in which no growth occurs (Fig. 3E, red, yellow,
and green curves). But upon increasing σ from near-equilibrium
values, v followed a smooth trajectory with no abrupt changes
(Fig. 3E, blue curve). In contrast, on the (010) face, dual func-
tionality was exhibited: At low supersaturation or high peptide
concentration, v was reduced, whereas at high σ or low peptide
concentration, steps accelerated (Figs. 2D and Fig. S3). This dual
effect is similar to what was observed for calcite, both with these
same peptides as well as other Asp-rich peptides (6). Hence this
behavior is apparently not an anomalous phenomenon.
Discussion
Asymmetric Growth Kinetics from Slow Peptide Adsorption Kinetics.
We propose that the phenomenon of bistable growth observed on
the ð−101Þ face is a natural consequence of polypeptide adsorp-
tion kinetics at faces to which they strongly bind through specific
interactions. The series of barriers that slow the adsorption of
polypeptides (16) results in their passage through multiple con-
figurations before reaching a lowest-energy collapsed state (18).
Friddle et al.
Fig. 3. The ð−101Þ face. (A) Steps originating at a dislocation hillock under
pure growth conditions. (B) Morphology during growth in 10 nM
ðDDDSÞ6 DDD solution, at low ionic strength (7 × 10−4 M), showing extensive
step pinning. (C) High-resolution image collected in pure solution showing
lattice structure overlaid with a ball-and-stick model. The color scheme is
the same as in Fig. 1A and B. (D) High-resolution image showing that, unlike
the (010) face, the ð−101Þ face becomes covered by a uniform film of peptide
preventing visualization of the underlying lattice [ðDDDSÞ6 DDD, I ¼ 0.15 M].
PHYSICS
(E) Step speed versus supersaturation for various concentrations of
ðDDDSÞ6 DDD. Symbols are experimental values taken after introducing
the indicated peptide concentration into otherwise pure solution. Red
squares and blue circles were collected while reducing and increasing super-
saturation, respectively. Standard errors are as described in Fig. 2. Solid curves
give the theoretical dependence that takes into account the slow adsorption
kinetics of the peptides for decreasing (downward arrow) and increasing
(upward arrow) supersaturation. (See Supplementary Information for de-
tails.) The model is in excellent agreement with the data and predicts two
distinct states of growth with a discontinuous drop in step speed to zero
as supersaturation is reduced and a slow steady rise as supersaturation is
increased.
Under supersaturated conditions, step growth competes with this
slow peptide binding: When an approaching step arrives at a pep-
tide binding site, if the peptide is well-bound, it can block solute
access to kinks; but if it is weakly adhered, as the peptide bonds
to the crystal fluctuate, step propagation can proceed past the
site. Thus, whether a peptide impedes step motion depends on
the characteristic time scale τ available to relax into the well-
bound state.
The time window for peptide binding is the terrace exposure
time T, which is given by L∕v, where L is the distance between
steps. Analysis of the step speed data on the ð−101Þ face shows
that the characteristic time τ for ðDDDSÞ6 DDD adsorption is
∼40 s and, at the higher values of σ used in these experiments,
T ≪ τ (19). However, because L and v decrease and increase with
supersaturation, respectively, upon reduction of σ, T rapidly in-
creases. When sufficiently low σ is reached, the coverage of well-
bound peptides is then large enough to appreciably slow the steps.
As they slow, T increases further, resulting in yet higher peptide
coverage, which again reduces v, raising the peptide coverage yet
again, and so on. Because of this positive feedback, step speed
exhibits the characteristic feature of a mathematical “catas-
trophe” (20); i.e., there is a point at which v spontaneously jumps
PNAS ∣ January 5, 2010 ∣ vol. 107 ∣ no. 1 ∣ 13

Fig. 4. (A–F) Series of sequential images showing interaction of step on (010)
face with peptide clusters in a solution containing 10 nM DDDS (I ¼ 0.15 M).
An individual step approaches two adjacent clusters (A and B), is transiently
pinned (C and D), and then recovers (E and F). Frame interval, 4.2 seconds.
(G–L) Time sequence showing inhibited growth of step on the ð−101Þ face
under I ¼ 0.15 M. A single large peptide aggregate near the center of the
image provides a point of reference. The continuous film of peptides on this
face transiently pins subsegments of the oncoming step at many sites along
the step front. Frame interval, 8.4 seconds. Note the sudden extension of the
step towards the reference cluster in H, followed by inhibition of the same
step region in I. This behavior of slight acceleration as steps approach clusters
was often observed on both faces.
from a finite value to zero and does not exhibit intermediate
values. In contrast, when the crystal is first exposed to peptides
at a value of σ below the jump, step speed is near zero (T ≫ τ)
and peptide adsorption proceeds unperturbed until equilibrium
coverage of well-bound peptides is reached. Even as σ increases,
these peptides present a large barrier to desorption that steps
cannot overcome. As a consequence, the dynamic relation be-
tween time scales is lost and v follows a trajectory well-predicted
by the classic pinning model (13, 15) in the absence of time depen-
dence (Fig. 3E).
Thus the slow, multistate binding of peptides to the ð−101Þ
face breaks the symmetry in the step kinetics: Upon decrease
of σ from high levels, weakly bound peptides are displaced by
rapidly advancing steps, but upon increase from equilibrium,
the peptides present nearly permanent adsorbates, the symmetry
is broken, and the step exhibits two distinct states—uninhibited
growth or arrested growth. Because the surface peptide coverage
will increase with solution peptide concentration, the supersa-
turation at which the jump between states occurs should also
increase, as is observed experimentally. The red and blue curves
14 ∣ www.pnas.org/cgi/doi/10.1073/pnas.0908205107
in Fig. 3E give the predictions of this model for a two-state
adsorption mechanism. (See SI Text for details of the model.)
Antagonistic Effects of Enhanced Desolvation and Step Blocking. We
propose that the dual modulation of growth on the (010) face
derives from the unique behavior of polyelectrolytes adhering
to like-charged surfaces as discussed above (16, 17). The clusters,
which result from nonspecific adhesion mediated by van der
Waals attraction, are weakly bound and present only a transient
barrier to solute addition to steps. Their effects should become
significant only at high coverage where they impede step motion.
On the other hand, simulations predict that Asp residues on pep-
tides in solution can assist cation desolvation at mineral surfaces
(21), which is generally recognized to be the rate-limiting step in
mineral growth (22, 23). This phenomenon should lead to step
acceleration. Thus we suggest that Asp-rich peptides near the
(010) face impose two opposing controls on step advancement:
Peptides in solution near the step lower the barrier to solute
incorporation into the step, whereas adsorbed clusters cause
transient blocking of step advancement. At low peptide concen-
trations, when cluster density is low, enhancement by peptides
in solution near the step dominates, whereas at high peptide con-
centration, when the cluster density is high, step blocking by the
clusters overcomes the enhancement. The curves in Fig. 2D were
calculated by including a decrease in the desolvation barrier of a
magnitude proportional to the concentration of peptides in solu-
tion along with pinning by peptide clusters according to the classic
step-pinning model (13, 15). (See SI Text for details.)
Conclusion
The results from this simple system demonstrate that the high-
resolution in situ imaging obtainable with hybrid AFM probes
can provide unique insights into the consequences of interactions
characteristic of macromolecules at inorganic surfaces. More-
over, those consequences create a versatile palette of controls
over mineral growth: Depending on the strength of the interac-
tion with the faces and steps, the adsorption dynamics and
the tendency towards cluster formation, macromolecules act as
"switches, throttles, or brakes" on crystal formation.
Methods
Molecular Modeling. Energy minimizations were performed on the peptide/
COM system by using the Compass force field as implemented in MS Model-
ing version 4.0 software (Accelrys) with the dielectric constant equal to 20 to
attenuate the Coulomb force acting on the peptide at the COM surface.
During minimizations, the COM lattice was held fixed. Binding energies were
obtained by subtracting the optimized energy of the adsorbate–surface sys-
tem from the energies of the surface and adsorbate when separated beyond
the interaction distance. Flat and stepped surfaces of COM were built by
using CERIUS version 4.2 surface builder (Accelrys) from the unit cell derived
from the crystallography data published by Deganello and Piro (9). Because
the reported unit cell by Deganello and Piro did not include hydrogen atoms,
hydrogen atom positions were determined computationally by using a com-
mercial density functional theory software package (CASTEP; Accelrys) with
optimization at the Perdew-Burke-Ernzerhof exchange-correlation func-
tional of the generalized gradient approximation level of theory with 3D per-
iodic boundary conditions applied to the COM unit cell; the geometric
dimensions of the unit cell were fixed at its crystallographically determined
values, and all atoms were frozen except the hydrogens, which were free
to move.
AFM Imaging. All in situ images were collected at 25 °C in contact mode
(Digital Instruments E scanner; Nanoscope IIIA) on surfaces of COM crystals
anchored inside the enclosed fluid cell. The AFM head was equipped with
a 3-mW laser diode, and the probes consisted of hybrid silicon tips on silicon
nitride cantilevers (HYDRA rectangular lever, k ¼ 35 pN∕nm, and tip radius
<8 nm; Applied Nanostructures, Inc, www.appnano.com). Solution flow
was temporarily interrupted during high-resolution image collection. Details
of the temperature control and flow systems, image collection, and data
analysis are provided elsewhere (24, 25).
Friddle et al.
Peptide Synthesis. Peptides were synthesized according to standard proce-
dures (26) by sequential addition of Fmoc amino acids. After synthesis, the
peptides were purified and molecular weights verified by mass spectrometry
as described previously (27).
Materials
The COM crystals used for these experiments were grown in vitro with
a gel method (28). Aqueous solutions of calcium oxalate with calcium-to-
oxalate ratio ¼ 1 were prepared by using reagent-grade K2 C2 O4 and CaCl2 ·
2H2 O dissolved in distilled deionized (18 MΩ >) water. The ionic strength was
fixed by using reagent-grade KCl.
Inductively coupled plasma–atomic emission spectroscopy was used to
confirm the purity of all reactants. Solution pH was adjusted to 7.0
0.05
before each experiment by using a calibrated glass electrode and potas-
sium hydroxide. The calcium and oxalate concentrations were varied from
0.1 to 0.35 mM, respectively, leading to a range of supersaturation
a a

ðCa2þ Þ ðC2 O2− Þ
σ ¼ 12 ln K sp
4
between 0 and 1.2. Here we ignore the activity of water,
which is assumed to be close to unity and unchanged during the experiments.
Similarly, the activity of the pure calcium-oxalate monohydrate solid is unity
and is independent of peptide level. The relationship between the calcium
1. Politi Y, et al. (2008) Transformation mechanism of amorphous calcium carbonate into
calcite in the sea urchin larval spicule. Proc Natl Acad Sci USA, 105:17362–17366.
2. Shiraga H, et al. (1992) Inhibition of calcium-oxalate crystal-growth invitro by uropon-
tin—Another member of the aspartic acid-rich protein superfamily. Proc Natl Acad Sci
USA, 89:426–430.
3. Graether SP (2000) Beta-helix structure and ice-binding properties of a hyperactive
antifreeze protein from an insect. Nature, 406:325–328.
4. Lange PF, Wartosch L, Jentsch TJ, Fuhrmann JC (2006) ClC-7 requires Ostm1 as a beta-
subunit to support bone resorption and lysosomal function. Nature, 440:220–223.
5. Gower LB, Odom DJ (2000) Deposition of calcium carbonate films by a polymer-
induced liquid-precursor (PILP) process. J Cryst Growth, 210:719–734.
6. Elhadj S, De Yoreo JJ, Hoyer JR, Dove PM (2006) Role of molecular charge and
hydrophilicity in regulating the kinetics of crystal growth. Proc Natl Acad Sci USA, 103:
19237–19242.
7. Stephenson AE, et al. (2008) Peptides enhance magnesium signature in calcite: Insights
into origins of vital effects. Science, 322:724–727.
8. Hoyer JR, Otvos L, Urge L (1995) Role of osteopontin in urinary stone formation. Ann
NY Acad Sci, 760:257–265.
9. Deganello S, Piro OE (1981) The crystal structure of calcium oxalate monohydrate
(whewellite). N Jb Miner Mh, 2:81–88 (translated from German).
10. Sheng XX, Jung TS, Wesson JA, Ward MD (2005) Adhesion at calcium oxalate crystal
surfaces and the effect of urinary constituents. Proc Natl Acad Sci USA, 102:267–272.
11. Jung T, et al. (2004) Probing crystallization of calcium oxalate monohydrate and the
role of macromolecule additives with in situ atomic force microscopy. Langmuir, 20:
8587–8596.
12. Qiu SR, et al. (2004) Molecular modulation of calcium oxalate crystallization by
osteopontin and citrate. Proc Natl Acad Sci USA, 101:1811–1815.
13. Weaver ML, et al. (2007) Inhibition of calcium oxalate monohydrate growth by citrate
and the effect of the background electrolyte. J Cryst Growth, 306:135–145.
14. Mann S (2001) Biomineralization: Principles and Concepts in Bioinorganic Materials
Chemistry (Oxford Univ Press, New York).
Friddle et al.
and oxalate activities and the respective solution concentrations can be
found in ref. 13.
Ionic activities were estimated through speciation calculations in which
the Davies equation was used to approximate activity coefficient corrections.
Equilibrium activities were taken to be those at which the speed of atomic
steps on the COM crystal surface was measured to be zero. In a previous
study, for calcium-oxalate solutions at ionic strength 0.05 M, we determined
the value of K sp to be 1.56 × 10−9 M2 , which was close to the reported value
of 1.66 × 10−9 M2 (29).
ACKNOWLEDGMENTS. The authors are grateful to Dr. John R. Hoyer of the
University of Delaware, who performed the peptide synthesis and character-
ization, to Dr. E. Alan Salter for his assistance in molecular modeling and
preparation of figures, and to Prof. Pupa Gilbert for valuable discussion.
Development of subnanometer AFM was supported by Office of Science,
Office of Basic Energy Sciences of the US Department of Energy under
Contract DE-AC52-07NA27344. Theoretical analysis was supported by Office
of Science, Office of Basic Energy Sciences of the US Department of Energy
under Contract DE-AC02-05CH1123. AFM investigations of COM growth
were supported by Grant DK61673 from the National Institutes of Health.
15. Cabrera N, Vermilyea DA (1958) Growth of crystals from solution. Growth and Perfec-
tion of Crystals; Proceedings, eds Doremus RH (Wiley, New York), pp 393–410.
16. Harding JH, et al. (2008) Computational techniques at the organic-inorganic interface
in biomineralization. Chem Rev, 108:4823–4854.
17. Pastre D, et al. (2003) Adsorption of DNA to mica mediated by divalent counterions: A
theoretical and experimental study. Biophys J, 85:2507–2518.
18. Bachmann M, Janke W (2005) Conformational transitions of nongrafted polymers
near an absorbing substrate. Phys Rev Lett, 95:058102.
19. De Yoreo JJ, Wierzbicki A, Dove PM (2007) New insights into mechanisms of biomo-
lecular control on growth of inorganic crystals. CrystEngComm, 9:1144–1152.
20. Arnol’d VI (1986) Catastrophe Theory (Springer, New York).
21. Piana S, Jones F, Gale JD (2007) Aspartic acid as a crystal growth catalyst. CrystEng-
Comm, 9:1187–1191.
22. Piana S, Jones F, Gale JD (2006) Assisted desolvation as a key kinetic step for crystal
growth. J Am Chem Soc, 128:13568–13574.
PHYSICS
23. Petsev DN, Chen K, Gliko O, Vekilov PG (2003) Diffusion-limited kinetics of the
solution-solid phase transition of molecular substances. Proc Natl Acad Sci USA,
100:792–796.
24. DeYoreo JJ, Orme CA, Land TA (2001) Using atomic force microscopy to investigate
solution crystal growth. Advances in Crystal Growth Research, eds Sato K, Nakajima
K, Furukawa Y (Elsevier Science, Amsterdam), pp 361–380.
25. Qiu SR, et al. (2005) Modulation of calcium oxalate monohydrate crystallization by
citrate through selective binding to atomic steps. J Am Chem Soc, 127:9036–9044.
26. Fields GB, Noble RL (1990) Solid-phase peptide-synthesis utilizing 9-fluorenylmethox-
ycarbonyl amino-acids. Int J Peptide Protein Res, 35:161–214.
27. Hoyer JR, Asplin JR, Otvos L (2001) Phosphorylated osteopontin peptides suppress crys-
tallization by inhibiting the growth of calcium oxalate crystals. Kidney Int, 60:77–82.
28. Cody AM, Horner HT, Cody RD (1982) SEM study of the fine surface features of
synthetic calcium oxalate monohydrate crystals. Scan Electron Microsc, 1:185–197.
29. Tomazic BB, Nancollas GH (1979) A study of the phase transformation of calcium
oxalate trihydrate monohydrate. Invest Urol, 16:329–335.
PNAS ∣ January 5, 2010 ∣ vol. 107 ∣ no. 1 ∣ 15