Crystal Growth Inhibitors for the Prevention of Cystine Kidney

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                                                                                                                       inhibitors—L-cystine dimethylester (L-CDME)
                                                                                                                       and L-cystine methylester (L-CME)—to specific
                                                                                                                       crystal surfaces, as revealed by in situ real-time
Crystal Growth Inhibitors for the                                                                                      atomic force microscopy (AFM) (7, 8) and par-
                                                                                                                       allel bulk crystallization studies (9).
Prevention of L-Cystine Kidney                                                                                             L-cystine stones are aggregates of individual
                                                                                                                       crystals with hexagonal habits (Fig. 2). L-cystine

Stones Through Molecular Design                                                                                        can be crystallized in vitro at physiological pH
                                                                                                                       (6 ≤ pH ≤ 8) by means of slow evaporation
                                                                                                                       (10), acidification of basic L-cystine solutions
Jeffrey D. Rimer,1*† Zhihua An,1* Zina Zhu,1* Michael H. Lee,1 David S. Goldfarb,2                                     to neutral pH (11), or gradual cooling of solu-
Jeffrey A. Wesson,3 Michael D. Ward1‡                                                                                  tions supersaturated with L-cystine (12). Under
                                                                                                                       these conditions, L-cystine crystallizes as hex-
                                                                                                                       agonal plates (Fig. 2B) with large (001) basal
Crystallization of L-cystine is a critical step in the pathogenesis of cystine kidney stones. Treatments
                                                                                                                       surfaces that can achieve widths of 400 mm
for this disease are somewhat effective but often lead to adverse side effects. Real-time in situ
                                                                                                                       and are bounded by six equivalent {100} faces.
atomic force microscopy (AFM) reveals that L-cystine dimethylester (L-CDME) and L-cystine
                                                                                                                       The typical thickness of these crystals ranges




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methylester (L-CME) dramatically reduce the growth velocity of the six symmetry-equivalent
                                                                                                                       from 10 to 30 mm. The crystal structure (hexag-
{100} steps because of specific binding at the crystal surface, which frustrates the attachment
                                                                                                                       onal P6122 space group, a = b = 0.5422 nm, c =
of L-cystine molecules. L-CDME and L-CME produce L-cystine crystals with different habits that reveal
                                                                                                                       5.6275 nm) reveals L-cystine molecules orga-
distinct binding modes at the crystal surfaces. The AFM observations are mirrored by reduced
                                                                                                                       nized as a helix about the 61 screw axis so that
crystal yield and crystal size in the presence of L-CDME and L-CME, collectively suggesting a new
                                                                                                                       six cystine molecules span the ~5.6-nm unit cell
pathway to the prevention of L-cystine stones by rational design of crystal growth inhibitors.
                                                                                                                       length of the c axis (13). The L-cystine mole-
                                                                                                                       cules exhibit intermolecular NH3+…–O(C=O) hy-
          idney stones comprising L-cystine (Fig.               can suppress but may not completely prevent            drogen bonding along the 61 screw axis (Fig.

K         1) affect at least 20,000 individuals in
          the United States. Although this num-
ber is substantially smaller than the number of
                                                                stone formation. Severely afflicted individuals
                                                                often rely on additional treatment with L-cystine–
                                                                binding thiol drugs such as D-penicillamine
                                                                                                                       2C, I), intermolecular S…S interactions between
                                                                                                                       the helices at intervals of c/2 along each of the
                                                                                                                       six equivalent {100} directions (Fig. 2C, II), and
individuals afflicted by calcium oxalate mono-                  [HSC(CH3)2CH(NH2)COOH] and a-mercaptopro-              NH3+…–O(C=O) hydrogen bonding (Fig. 2D,
hydrate (COM) stones (approximately 10% of                      pionylglycine [HSCH(CH3)C(O)NHCH2COOH],                III and IV) between adjacent helices in the (001)
the U.S. population), L-cystine stones are larger,              which react with L-cystine to generate more sol-       plane. The hexagonal plate habit reflects the
recur more frequently, and are more likely to                   uble asymmetric disulfides (2). These drugs, how-      multiple strong intermolecular interactions in
cause chronic kidney disease (1). The formation                 ever, have an unpleasant odor and can cause            the (001) plane. The basal surfaces of L-cystine
of L-cystine stones is a consequence of exces-                  adverse side effects, such as nausea, fever, fa-       grown at neutral pH are decorated with {100}
sive levels of L-cystine in the urine because of                tigue, skin allergies, and hypersensitivity (5). For   steps that are observable through either optical
defective reabsorption of filtered cystine (2). This            patients with very high L-cystine concentrations,      (Fig. 2B) or scanning electron microscopy (SEM)
condition is the result of an autosomal reces-                  L-cystine–binding thiol drugs may not reduce L-        (fig. S2).
sive disorder caused by mutations in one of the                 cystine levels sufficiently at dosages (up to 2000         Crystal growth near equilibrium is common-
two genes, either SLC3A1 on chromosome 2                        mg per day) regarded as below the threshold            ly described by the terrace-ledge-kink model
(type A cystinuria) or SLC7A9 on chromosome                     for toxicity, and patient adherance to these pre-      (14), in which steps created by dislocations ad-
19 (type B cystinuria), which code for compo-                   scribed medications and high fluid intake can          vance across crystal terraces by the addition of
nents of the major proximal renal tubule cys-                   be problematic (5). We report an alternative ap-       solute molecules to kink sites along the ledge
tine and dibasic amino acid transporter (3). This               proach to the prevention of L-cystine kidney           (a ledge is the intersection of a step and ter-
condition is exacerbated by the low solubility                  stones that is based on crystal growth inhibition      race). Steps originating from screw dislocations
of L-cystine (4), which favors facile formation of              achieved through the binding of tailored growth        typically exhibit a spiral growth pattern, with the
crystals that aggregate into stones, often with
centimeter dimensions (Fig. 2A).
                                                                                                                                      Fig. 1. Molecular structures of L-
    Current treatments for L-cystine stone preven-
                                                                                                                                      cystine and the inhibitors L-CDME
tion, including dilution through high fluid intake                                                                                    and L-CME.
(5) and increasing urine pH through ingestion
of alkalinizing potassium or sodium salts (5, 6),

1
  Department of Chemistry and the Molecular Design Institute,
New York University (NYU), 100 Washington Square East,
New York, NY 10003–6688, USA. 2Nephrology Section, New
York Harbor Veterans Affairs Medical Center, and School of
Medicine, NYU, New York, NY 10010, USA. 3Nephrology Divi-
sion, Department of Veterans Affairs Medical Center and the
Medical College of Wisconsin, 5000 West National Avenue,
Milwaukee, WI 53295, USA.
*These authors contributed equally to this work.
†Present address: Department of Chemical and Biomolecular
Engineering, University of Houston, S222 Engineering Build-
ing, 4800 Calhoun Avenue, Houston, TX 77204–4004, USA.
‡To whom correspondence should be addressed. E-mail:
mdw3@nyu.edu


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RESEARCH ARTICLES
      first turn occurring once every step has reached      solute, as demonstrated for amino acid and adipic     step pinning through adsorption of L-CDME
      its critical length (15). Real-time in situ AFM of    acid crystals (20–22). The effect of molecular        at the {100} steps (Fig. 3, E and F). This effect
      the L-cystine (001) face during growth in             inhibitors on the crystal growth of b-hematin—        was reversible because the steps once again be-
      aqueous solutions containing L-cystine revealed       a synthetic malaria pigment—and their poten-          come linear after addition of aqueous solu-
      steps emanating from screw dislocations, gen-         tial role as antimalarial drugs has been reported     tions containing only L-cystine (fig. S5). The step
      erating hexagonal hillocks in a spiral growth         (23, 24). Polyvalent macromolecules capable of        velocity decreased monotonically with increas-
      pattern. Occasionally, multiple dislocations were     binding to multiple crystal sites, such as pep-       ing L-CDME concentration, becoming negligi-
      observed (Fig. 3, A and B, and movie S1),             tides and proteins, also can influence ice crystal-   ble above 30 mg/liter (Fig. 4, A and C). Similarly
      merging to generate a range of step heights from      lization (25) and the formation of biominerals,       to the step roughening, the inhibitor effect was
      1 nm to 60 nm, with the larger steps observed         including calcium carbonate (26, 27) and cal-         reversible, with the rates returning to the original
      distant from the dislocation cores, where step        cium oxalates (28–30).                                value once the growth medium was replaced with
      bunching would be expected (fig. S3). In con-             AFM revealed that addition of L-CDME, a           aqueous solutions containing only L-cystine. The
      trast, hillocks generated by single isolated dis-     structural mimic of L-cystine in which the car-       reduction of the step velocity was equivalent
      locations were bounded by six well-defined            boxylate groups are replaced by methylester           along all six directions in the (001) plane, as ex-
      major {100} steps, each with a ~6-nm height cor-      groups, resulted in roughening of the otherwise       pected for the hexagonal symmetry of the crystal.
      responding to the unit cell length along c, sepa-     highly linear {100} step edges and rounding of        These observations are consistent with attachment
      rating (001) terraces. Each hillock terrace was       the hillock corners, which is consistent with         of L-CDME to the {100} step planes through a
      decorated with six minor {100} steps at 60° in-




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      tervals, each with a ~1-nm height corresponding       Fig. 2. (A) Human stones with millimeter-
      to a single L-cystine molecule, creating the ap-      scale dimensions [courtesy of M. Lewis,
      pearance of a pinwheel. These minor steps most        International Cystinuria Foundation]. (B) A
      likely reflect a splitting of the dislocation into    hexagonal L-cystine crystal prepared in
      six equivalent dislocations described by a Burgers    vitro. The faint lines on the top surface of
      vector having a magnitude of c/6. Consecutive         the crystal, parallel to the edges, are the
      images during crystal growth revealed a clock-        {100} steps. (C) Two adjacent helices of
      wise rotation of the pinwheel at the dislocation      L-cystine molecules, viewed on the (100)
      core (a left-handed screw) accompanied by con-        plane, each winding about a 61 screw axis
      tinuous generation of new hillocks. Attachment        that coincides with the c axis. Six L-cystine
      of L-cystine molecules to both the minor and          molecules, denoted C1 to C6, span the
      major steps on the surrounding terraces results       5.6-nm c axis. Key intermolecular inter-
      in outward advancement of the steps with re-          actions include amine-carboxylate hydro-
      spect to the dislocation core (Fig. 3C and movie      gen bonds along the helix (I, dN…O =
      S2). The spiral growth pattern also is observed       2.87 Å) and S…S interactions (II, dS…S =
      for D-cystine, the unnatural enantiomer, but with     3.47 Å) between helices at intervals of c/2,
      counterclockwise (a right-handed screw) rota-         depicted here for C1 and C4 along the
                                                            [010] direction (identical S…S interactions
      tion of the pinwheel (Fig. 3D and movie S3). A
                                                            occur at symmetry-related sites along the
      preference for screw dislocations of opposite
                                                            other five equivalent directions). (D) In-
      handedness for enantiomeric crystals has been         termolecular amine-carboxylate hydrogen
      predicted (16).                                       bonds in the (001) plane (III, dN…O =
          Quantitative determination of crystal growth      2.79 Å and IV, dN…O = 2.81 Å). Atom
      rates at the near-molecular level were obtained       color code is gray, carbon; red, oxygen;
      by using AFM to measure the {100} step ve-            blue, nitrogen; yellow, sulfur; and white,
      locity (Fig. 4, A and B, and fig. S4). In order to    hydrogen. (E) Schematic illustration of a
      achieve growth rates within a reasonable mea-         hexagonal L-cystine crystal, with Miller in-
      surement timeframe, the L-cystine concentration       dices. The six planes flanking (001) belong
      was adjusted to 480 mg/liter (2 mM), which is         to the {100} family.
      five times larger than L-cystine solubility at room
      temperature (17). The {100} step velocity (Vo),
      determined through measurement of the step ad-        Fig. 3. (A and B) Real-
      vance in successive images, was 11 nm/s, which        time in situ AFM images
      is equivalent to 50 molecules/nm2 s. The step         of a L-cystine crystal, ac-
      velocity was equivalent along all six directions,     quired 12 min apart. A
      as expected for the hexagonal symmetry.               pair of hexagonal hillocks
          Crystallization outcomes such as habit, chi-      generated by two closely
                                                            spaced dislocations serve
      rality, and polymorphism can be influenced by
                                                            as landmarks. (C and D)
      tailored growth inhibitors that reduce crystalli-
                                                            AFM images of a single
      zation rates through binding at specific step         dislocation center of (C)
      sites (18, 19). These inhibitors, which have been     L-cystine and (D) D-cystine
      described as “imposters,” (15) consist of a binder    crystal during growth. (E
      moiety that emulates a critical structural element    and F) AFM image of a
      of the solute that attaches to a specific crystal     hexagonal growth hil-
      site and a perturber moiety that obstructs the ap-    lock on the (001) face of
      proach of additional solute molecules to neighbor-    L-cystine (E) before and
      ing sites, pinning step motion. Growth inhibitors     (F) after addition of L-CDME (5 mg/liter; 0.02 mM), revealing roughening of the {100} steps due to step
      may be monomers that closely resemble the             pinning. Images were acquired in aqueous solutions containing 2 mM L-cystine.


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                                                                                                                                     RESEARCH ARTICLES
combination of intermolecular (cystine)S…S(L-          presence of L-CDME and L-CME may influ-                teresting insight into the binding modes of these
CDME), (cystine)C(=O)O–…+H3N(L-CDME),                  ence the aggregation of crystals and their at-         inhibitors at the L-cystine crystal surface.
and (cystine)N-H…O=C(L-CDME) interac-                  tachment to renal cells, which are also critical           The P6122 space group symmetry generates
tions in a manner that mimics the attachment           steps in stone formation (31). L-CDME also             inequivalent projections of the L-cystine mole-
of L-cystine solute molecules at the {100} steps,      promoted the formation of small amounts of             cules on each flank of a hexagonal hillock. Each
with the ester methyl groups of bound L-CDME           minute crystals of tetragonal L-cystine (Fig. 5B).     projection winds around the hillock by trans-
molecules blocking the attachment of L-cystine         The formation of the tetragonal polymorph, which       lations of Tc/6 on adjacent faces. In the 5.6-nm
solute molecules at neighboring crystal sites.         ordinarily crystallizes only under more basic          span of the hillock, diad axes create two distinct
The steric bulk of the ester methyl group is not       conditions (pH > 8) (32), can be attributed to         pairs of symmetry-related projections in which
sufficient to prevent binding of L-CDME to the         the suppressed growth of the hexagonal form            the L-cystine molecules are oriented in oppo-
{100} steps.                                           by L-CDME.                                             site directions along c—for example, C1/C3 and
     The AFM measurements demonstrate that                 The unsymmetrical L-CME, a cystine mimic           C4/C6 on the (010) face (Fig. 6). The two re-
L-CDME slows growth along the naturally fast           with only one ester methyl group, also inhibited       maining sites, located on the diad special posi-
growth directions within the (001) plane. This         L-cystine crystallization. AFM measurements re-        tions, create two additional distinct projections
microscopic behavior was mirrored in bulk crys-        vealed that the {100} step velocity declined with      along directions containing the aforementioned
tallization by a gradual change in the crystal habit   increasing L-CME concentration, but to a lesser        intermolecular S…S contacts. Molecular models
from (001) plates to small hexagonal needles ori-      extent than L-CDME (Fig. 4, B and C). L-CME            reveal that binding of L-CDME at (001)∩{100}
ented along the [001] direction as the L-CDME          also reduced the size of L-cystine crystals sub-       ledge sites [illustrated in Fig. 6 by the intersec-




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concentration was increased (Fig. 5A and fig. S6).     stantially, and it reduced the crystal yield, but to   tion of (001) and (010) planes] is precluded by
At L-CDME concentrations as low as 5 mg/liter          a lesser extent than did L-CDME (Fig. 4D).             the steric obstruction introduced by one of the
(0.02 mM; equivalent to 1% of the L-cystine con-       Step roughening was observed in the presence           ester methyl groups (Fig. 6). Consequently, attach-
centration), the area of the (001) face was re-        of L-CME, but at higher concentrations as com-         ment of L-CDME must occur at L-cystine sites
duced by a factor of 1000, whereas the length of       pared with that of L-CDME (fig. S5). These ob-         above the ledge sites. It is reasonable to sug-
the needles was comparable with the thickness          servations are consistent with weaker inhibition       gest that L-CDME would bind preferentially
of the hexagonal plates grown in the absence of        of L-CME as compared with that of L-CDME.              at the diad sites because of the additional S…S
L-CDME (~30 mm), resulting in crystals that            The L-cystine crystals grown in the presence of        interactions—for example, the C2 and C5 sites
were 1000 times smaller. Moreover, the total           L-CME exhibited a tapered hexagonal habit, with        on the (010) face. Binding of L-CDME would
crystallization yields decreased with increas-         six faces intersecting the basal (001) plane at an
ing L-CDME concentration, approaching com-             angle of 85°, which is attributable to the emer-
plete inhibition above 2 mg/liter (Fig. 4D),           gence of six equivalent {101} surfaces (Fig. 5, C
which is consistent with the AFM observations.         and D, and fig. S7). The different crystal habits
The dramatic change in crystal habit in the            generated by L-CDME and L-CME provide in-




                                                                                                              Fig. 5. (A) Minute L-cystine crystals grown in the
                                                                                                              presence of L-CDME (5 mg/liter; 0.02 mM) exhibit
                                                                                                              a hexagonal needle-like habit with prominent
                                                                                                              {100} faces and high c/a aspect ratios, approach-
                                                                                                              ing 30 for many crystals. (B) Small quantities of
                                                                                                              the tetragonal P41 polymorph are formed in the
                                                                                                              presence of L-CDME (5 mg/liter). (C) L-CME (10
                                                                                                              mg/liter; 0.04 mM) produces tapered hexagonal
                                                                                                              needles with six {101} faces. Some crystals exhibit
                                                                                                              the tapered habit at both ends of the crystal, as
                                                                                                              expected for the crystal symmetry (fig. S7). The
Fig. 4. (A and B) The position of the 5.6-nm-high {100} steps, as measured from the center of the spiral      observation of only one half of the tapered crystal
dislocations during growth in aqueous solutions containing 2 mM L-cystine with various concentrations of      suggests that growth often begins on a surface.
L-CDME or L-CME. The step velocities in the absence of inhibitor, determined from the slopes of the lines,    (D) The tapered needles formed in the presence of
are Vo = 11.4 T 0.3 and 11.3 T 0.1 nm/s, respectively. The SD is based on the average of the velocities       10 mg/liter L-CME occasionally grow from a nidus
determined for three independent steps. (C) Comparison of the effectiveness of L-CDME and L-CME on the        that may be an amorphous L-cystine particle or a
inhibition of the {100} step velocity, expressed as V/Vo. (D) The mass yield of L-cystine crystals obtained   microscopic unidentified foreign object. Crystal
after crystallization for 72 hours in the presence of various concentrations of L-CDME or L-CME. The error    growth was performed in aqueous solutions con-
bars represent 1 SD based on three measurements for each inhibitor concentration.                             taining 3 mM L-cystine (700 mg/liter).


                                        www.sciencemag.org           SCIENCE        VOL 330       15 OCTOBER 2010                                                   339
RESEARCH ARTICLES
      prevent steps from advancing further above            at each site of a diad-related pair (blue-blue or          In contrast, L-CME can bind at (001)∩{100}
      the inhibitor site, leading to step bunching that     green-green on each hillock face in Fig. 6) dif-       ledge sites in its up orientation because the car-
      would terminate in {100} planes and produc-           fer only with respect to their “up” or “down” ori-     boxylic acid terminus is indistinguishable from
      ing hexagonal needles with small cross sections.      entation (denoted by the white arrows). The C2         the ends of L-cystine (the ester methyl group
      L-CDME binding to other sites cannot be ex-           symmetry of L-CDME would result in equiv-              would prevent binding at the ledge in the down
      cluded; the projections of L-cystine molecules        alent binding to either site of a diad pair.           orientation). The terrace of the ledge site ef-
                                                                                                                   fectively breaks the twofold up-down sym-
                                                                                                                   metry of the diad-related pairs. Consequently,
                                                                                                                   each flank of the hexagonal hillock contains
                                                                                                                   six crystallographically, and therefore chemical-
                                                                                                                   ly, inequivalent (001)∩{100} ledge sites along
                                                                                                                   the 5.6-nm span of the c axis (fig. S8). The
                                                                                                                   tapered crystals grown in the presence of L-CME
                                                                                                                   can be explained only by highly selective bind-
                                                                                                                   ing of L-CME at one of these sites. Crystal step
                                                                                                                   motion would then be pinned at these sites,
                                                                                                                   repeating at an interval of c, creating a vicinal
                                                                                                                   face with a tangent plane intersecting the (001)




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                                                                                                                   plane at an angle of 85°, which is identical to
                                                                                                                   the angle of the tapered faces on the macro-
                                                                                                                   scopic crystals and assignable to {101} planes.
                                                                                                                   The different mode of binding in the presence
                                                                                                                   of L-CDME and L-CME, particularly the dis-
                                                                                                                   crimination of L-CME for one of the six pos-
                                                                                                                   sible ledge sites, is a remarkable illustration of
                                                                                                                   molecular recognition between tailored inhib-
                                                                                                                   itors and crystal surface sites. Moreover, the step
                                                                                                                   velocity measurements, step roughening, crystal
                                                                                                                   habit effects, and crystal yields represent a rare
                                                                                                                   example of a correspondence between inhib-
                                                                                                                   itor effects at the microscopic level and macro-
                                                                                                                   scopic crystallization behavior.
                                                                                                                       L-CDME is extraordinarily effective com-
                                                                                                                   pared with inhibitors examined for other kid-
                                                                                                                   ney stone–forming materials. Molecular additives
                                                                                                                   have been found to be substantially less effec-
                                                                                                                   tive as crystal growth inhibitors of COM as
                                                                                                                   compared with that of their polymeric forms;
                                                                                                                   for example, aspartic acid is more than 1000
                                                                                                                   times less active for COM inhibition than
                                                                                                                   poly(aspartic acid), in which the carboxylate
                                                                                                                   side chains are thought to bind to calcium sites
                                                                                                                   on the crystal surfaces. The effective inhibition
                                                                                                                   observed for polymers can be attributed to the
                                                                                                                   entropic benefit associated with binding of mul-
                                                                                                                   tiple carboxylate groups on a single chain. Sur-
                                                                                                                   prisingly, the concentrations at which L-CDME
                                                                                                                   becomes effective for L-cystine inhibition (~2
                                                                                                                   mg/liter) is comparable with those observed for
                                                                                                                   poly(amino acid) inhibitors of COM crystalli-
      Fig. 6. (Top left) Schematic representation of an L-cystine hillock, color-coded to denote the distinct      zation (28). The importance of molecular rec-
      projections of L-cystine along each flank of the 5.6-nm-high hillock. The six flanks are depicted in the     ognition between L-CDME and the L-cystine
      unraveled version of the hillock on the right. Equivalent projections on adjoining flanks are related by     step planes is underscored by our observations
      the 61 screw axis. Diad axes, denoted by black ovals, generate two identical projections that differ only    that other additives with proximal carboxylate
      with respect to their up or down orientation (denoted by white arrows). (Bottom right) L-CDME binding
                                                                                                                   and amine groups, such as those in L-cystine,
      at a (001)∩{010} ledge site is frustrated by one of the ester methyl group, emphasized here by the
                                                                                                                   had a negligible influence on L-cystine growth.
      white arc. (Bottom left) L-CDME binding to the (010) step: The ester methyl groups of L-CDME,
      depicted by purple spheres, block attachment of L-cystine to adjacent sites. Binding to the C1 and C4        For example, L-cysteine, the thiol relative of
                                                                                                                   L-cystine, reduced the size of L-cystine crystals
      sites on the {010} step allows S…S and hydrogen-bonding interactions between L-cystine molecules
      projecting from the step surface and L-CDME. In contrast, in the up orientation the carboxylic acid          somewhat (at 10 mg/liter), but its effect on
      terminus of L-CME can bind to the ledge sites in a manner like L-cystine, through hydrogen-bonding           crystallization yield was negligible (fig. S9).
      interaction I, at the (001) terrace, as well as S…S interactions and hydrogen bonding at the (010) step.     Urinary proteins such as osteopontin, human
      Crystallographically equivalent (001)∩(010) ledge sites, depicted here for the C6″-C1 and C6-C1′             serum albumin, and Tamm-Horsfall protein af-
      combinations, are spaced at intervals of c, generating a vicinal surface equivalent to a (011) plane. This   forded only modest reductions in crystallization
      plane intersects the (001) plane at an angle of 85°, which is consistent with the tapered faces observed     yield at concentrations comparable with phys-
      in the presence of L-CME.                                                                                    iological values (2 mg/liter), suggesting a neg-


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                                                                                                                                                      RESEARCH ARTICLES
ligible role for these substances in the regu-         umes a L-CDME dose of 10 to 50 mg per day—                        21. R. J. Davey et al., J. Chem. Soc. Faraday Trans. 88,
lation of cystine stone formation.                     far below toxic levels but greater than the amount                    3461 (1992).
                                                                                                                         22. A. S. Michaels, F. W. Tausch Jr., J. Phys. Chem. 65,
    Collectively, the AFM and bulk crystalliza-        needed for crystal growth inhibition in vitro—                        1730 (1961).
tion behavior for L-cystine suggest that L-CDME        may prove sufficient to achieve adequate L-CDME                                                    ¨
                                                                                                                         23. R. Buller, M. L. Peterson, O. Almarsson, L. Leiserowitz,
is a viable therapeutic agent for the prevention       concentrations in urine for crystal growth inhibi-                    Cryst. Growth Des. 2, 553 (2002).
of L-cystine kidney stones. This approach to stone     tion in vivo.                                                     24. I. Solomonov et al., J. Am. Chem. Soc. 129,
                                                                                                                             2615 (2007).
prevention uses a potentially benign crystal growth                                                                      25. Y. C. Liou, A. Tocilj, P. L. Davies, Z. Z. Jia, Nature 406,
inhibitor at low concentrations rather than drugs          References and Notes
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that rely on a chemical reaction with L-cystine            82, 799 (2006).                                               26. C. A. Orme et al., Nature 411, 775 (2001).
(L-cystine–binding thiol drugs), increases in urine     2. D. J. Dolin, J. R. Asplin, L. Flagel, M. Grasso,              27. A. E. Stephenson et al., Science 322, 724 (2008).
                                                                                                                         28. S. W. Guo, M. D. Ward, J. A. Wesson, Langmuir
alkalinity (which are often accompanied by un-             D. S. Goldfarb, J. Endourol. 19, 429 (2005).
                                                                                                                             18, 4284 (2002).
desirable side effects), or dramatic increases in       3. A. Mattoo, D. S. Goldfarb, Semin. Nephrol. 28,
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                                                                                                                         ally distinct states. These states are often charac-
Atomic-Level Characterization                                                                                            terized as “basins” separated by barriers on an
                                                                                                                         “energy landscape” (5).
of the Structural Dynamics of Proteins                                                                                       Substantial progress has been made, using
                                                                                                                         both experimental (1, 6) and computational (7, 8)
                                                                                                                         techniques, in characterizing conformational basins
David E. Shaw,1,2* Paul Maragakis,1† Kresten Lindorff-Larsen,1† Stefano Piana,1†                                         and the ways that proteins move within and among
Ron O. Dror,1 Michael P. Eastwood,1 Joseph A. Bank,1 John M. Jumper,1 John K. Salmon,1                                   them. It has proven difficult, however, to structur-
Yibing Shan,1 Willy Wriggers1                                                                                            ally characterize sparsely populated or disordered
                                                                                                                         states and to elucidate the “basin-hopping” mech-
Molecular dynamics (MD) simulations are widely used to study protein motions at an atomic                                anisms involved in the interconversion of various
level of detail, but they have been limited to time scales shorter than those of many biologically                       states.
critical conformational changes. We examined two fundamental processes in protein                                            All-atom molecular dynamics (MD) simula-
dynamics—protein folding and conformational change within the folded state—by means of                                   tions are designed to provide a high-resolution
extremely long all-atom MD simulations conducted on a special-purpose machine. Equilibrium                               view of the motions of biological macromole-
simulations of a WW protein domain captured multiple folding and unfolding events that                                   cules (9), producing continuous trajectories with
consistently follow a well-defined folding pathway; separate simulations of the protein’s constituent                    the potential to connect static structural snap-
substructures shed light on possible determinants of this pathway. A 1-millisecond simulation                            shots generated from experimental data. Compu-
of the folded protein BPTI reveals a small number of structurally distinct conformational states                         tational constraints, however, have limited such
whose reversible interconversion is slower than local relaxations within those states by a factor
of more than 1000.
                                                                                                                         1
                                                                                                                          D. E. Shaw Research, 120 West 45th Street, New York, NY
                                                                                                                         10036, USA. 2Center for Computational Biology and Bioinfor-
        any biological processes involve func-         folding (1), signal transduction (2), the catalytic

M       tionally important changes in the three-
        dimensional structures of proteins.
Conformational changes associated with protein
                                                       cycles of enzymes (3), and the operation of mo-
                                                       lecular machines and motor proteins (4) often
                                                       involve transitions among two or more structur-
                                                                                                                         matics, Columbia University, New York, NY 10032, USA.
                                                                                                                         *To whom correspondence should be addressed. E-mail:
                                                                                                                         David.Shaw@DEShawResearch.com
                                                                                                                         †These authors contributed equally to this work.


                                        www.sciencemag.org             SCIENCE           VOL 330          15 OCTOBER 2010                                                                  341

				
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