Phil. Trans. R. Soc. A (2012) 370, 1478–1498doi:10.1098/rsta.2011.0236

Ab initio modelling of protein–biomaterialinteractions: influence of amino acid polar sidechains on adsorption at hydroxyapatite surfaces

BY ALBERT RIMOLA1,2, MARTA CORNO2, JORGE GARZA3

AND PIERO UGLIENGO2,*1Departament de Química, Universitat Autònoma de Barcelona,

Bellaterra 08193, Spain2Dipartimento di Chimica IFM, NIS Centre of Excellence and INSTM

(Materials Science and Technology National Consortium),Università di Torino, Via Pietro Giuria 7,

10125 Torino, Italy3Departamento de Química, División de Ciencias Básicas e Ingenería,

Universidad Autónoma Metropolitana Iztapalapa, Distrito Federal,09340, Mexico

The adsorption from the gas phase of five different amino acids (AAs), namely Gly, Ser,Lys, Gln and Glu, on three surface models of hexagonal hydroxyapatite (HA) has beenstudied at B3LYP level with Gaussian type basis set within a periodic approach. TheAA adsorption was simulated on the (001) and (010) stoichiometric surfaces, the latterboth in its pristine and water-reacted form. Low/high AA coverage has been studied bydoubling the HA unit cell size. The AAs have been docked to the HA surfaces followingthe electrostatic complementarity between the electrostatic potentials of AA and thebare HA. Gly adsorbs as a zwitterion at the (001) surface, whereas at the (010) ones,the proton of the COOH group is transferred to the surface resulting in an HA+/Gly−ion pair. For the other AAs, the common COOH−CH−NH2 moiety behaves like in Gly,while the specific side-chain functionalities adsorb at the HA surfaces by maximizingelectrostatic and H-bond interactions. The interactions between the side chains and theHA surface impart a higher stability compared with the Gly case, with Glu being thestrongest adsorbate owing to its high Ca affinity and H-bond donor propensity. For AAsof large size, the adsorption is more favourable in conditions of low coverage as repulsionbetween adjacent AAs is avoided. For all considered AAs, the strongest interaction isalways established on the (010) faces rather than on the (001) one, therefore suggestingan easier growth along the c-direction of HA crystals from AA solutions.

Keywords: ab initio calculations; hydroxyapatite; amino acids; adsorption at surfaces

*Author for correspondence (piero.ugliengo@unito.it).

Electronic supplementary material is available at http://dx.doi.org/10.1098/rsta.2011.0236 or viahttp://rsta.royalsocietypublishing.org.

One contribution of 11 to a Theme Issue ‘Structure and biological activity of glasses and ceramics’.

This journal is © 2012 The Royal Society1478

on March 25, 2016http://rsta.royalsocietypublishing.org/Downloaded from

Amino acids on hydroxyapatite surfaces 1479

1. Introduction

The study of biomolecule/biomaterial interactions is an actual gaining-ground research field because of the potentiality of nanotechnology applied tobiotechnological processes and in biomedical applications [1–5]. Among variousbiomaterials, hydroxyapatite (HA, Ca10(PO4)6(OH)2) is the natural majorinorganic constituent of bone and teeth and thus often used as a reference tostudy biomolecule/biocompatible surface interactions [6,7]. Among biomolecules,proteins are by far the most interesting, owing to their adhesion to HA surfaces.This is particularly the case for bone tissue regenerators, since protein adhesionto implants and prostheses is crucially mediated by processes occurring attheir surfaces. Indeed, functionalizing HA-based materials—as for the case ofthe bioglass surfaces—to enhance their biocompatibility is a common target[8,9]. Furthermore, understanding the protein/HA contact is of relevance inbiomineralization processes [10], since HA grows spontaneously outside the Hench45S5 Bioglass (45% SiO2–24.5% Na2O–24.5% CaO–6% P2O5), when the latter iscontacted with simulated body fluids (SBFs) [11]. In that respect, the HA growthrate is well known to be strongly influenced by the biological composition of theSBF, i.e. different amino acids (AAs), peptides and proteins may inhibit the HAgrowth to a different degree. Additionally, calcium phosphate-based nanoparticleshave been proposed as suitable materials for loading and securing different drugs,to be delivered afterwards in a controlled way inside a living body [12,13].

The ultimate goals when studying protein/HA surface interactions are: (i) toknow how the AA side-chains interact with the HA surfaces and (ii) based onpoint (i), to understand how proteins change their native conformation as a resultof the adsorption process. As for point (i), adsorption isothermal experiments aswell as conventional high-resolution spectroscopy studies have been performedto determine both macroscopic quantities and the actual interaction interface ofstatherin [14–20], myoglobin [21,22], amelogenin [23,24], bovine serum albumin[24–26], bovine serum fibrinogen [26], lysozome [25,26] and proline-rich proteinPRP1 [27] upon HA adsorption. Most of these papers conclude that proteins aregenerally adsorbed by electrostatic forces of different strength depending on theprotein structure and AA sequence. It is worth mentioning that merging severalof the mentioned techniques has allowed one to identify the statherin amino acidicresidues involved in the interaction with HA, as is beautifully reviewed in Goobeset al. [19]. As for point (ii), it is usually observed that proteins denature owingto contact with inorganic solid substrates. However, recent findings highlight HAsurfaces as ideal platforms to stabilize (and even to induce) well-defined protein-folded conformations upon adsorption [17,28], a fact that enables the retentionof the peptide biological functionality.

Despite the wide experimental investigations dedicated to protein/HA systems,detailed atomistic features related to the binding mechanisms and the anchoringpoints occurring at HA surfaces remain relatively scarce. In this respect, severalclassical molecular dynamics-based studies are present in the literature [29–31], focusing on the dynamic behaviour of proteins in water and when drivento surfaces. The adoption of classical force fields (FFs) needed to handle thelarge number of atoms involved inevitably lowers the accuracy of the results,as actual FFs cannot cope with bond breaking/making which may indeed occurbetween side-chain functionalities and the HA surface. Resorting to quantum

Phil. Trans. R. Soc. A (2012)

on March 25, 2016http://rsta.royalsocietypublishing.org/Downloaded from

1480 A. Rimola et al.

mechanical methods ideally solves this problem: however, modelling protein–HAinteractions at this level remains almost impossible. Nevertheless, by followinga hierarchical approach, the interaction with single AA allows one to learn howside-chains interact with the HA surface. The outcome of this approach is twofold:(i) to understand the influence of AA on the HA surface growth, as recentexperiments have addressed [32–35] and (ii) to provide accurate benchmark datafor the development of specific FFs devoted to simulate protein–HA interactions.We have recently published ab initio results for the gas-phase adsorption of glycine(Gly), at (001) and (010) HA surfaces, providing a detailed molecular pictureof the Gly–HA interface in absence of water [36]. The present paper extendsthat study by reporting the interaction (structure and energetics) of polar non-aromatic AAs (serine Ser, lysine Lys, glutamine Gln and glutamic acid Glu) onHA surfaces by means of periodic B3LYP calculations. The simulation of theAA interaction onto HA surfaces in complete dried conditions is particularlyinteresting because it allows understanding the intrinsic AA–HA features, freefrom the influence of water, although it was recently shown by some of us thatthe Gly–HA adducts with the presence of some water molecules are very similarto the water-free ones, since Gly interacts directly towards the surface with waterbehaving as spectator [37]. It is worth noting that, indeed, a number of AAs maybe easily sublimated [38] and their spectral features in the gas phase have beenstudied at very high resolution [39,40]. This feature has been used to adsorb Glyby chemical vapour deposition on amorphous silica [41] and by some of us on HAnanocrystals [42]. In both cases, only the combination with quantum mechanicalresults allowed one to resolve the atomistic and vibrational details of Gly on bothsurfaces at an unprecedented level of detail. In the present work, we only focusedon the structural and energetic features as the vibrational analysis would havebeen rather cumbersome to describe without the experimental counterpart tocompare with, which is, to our knowledge, still missing.

2. Computational details

In the following, only essential computational details are provided leaving fullaccount to the electronic supplementary material. All the periodic calculationsof the interaction of the considered AAs with the HA (001) and (010) faces havebeen performed with the ab initio code CRYSTAL06 [43] using a Gaussian basisset of polarized double-zeta quality already adopted in previous studies [44]. Allthe SCF calculations and geometry optimizations were performed with B3LYPdensity functional method [45,46] using 10 reciprocal k-points to sample theBrillouin zone. Geometries were optimized by relaxing the internal coordinateswithin P1 symmetry keeping the lattice parameters fixed at the values of theoptimized bare surfaces.

In a periodic treatment, the adsorption energy DE per unit cell per adsorbateis defined as

DE = E(SM//SM) − E(S//S) − Em(M//M),

where E(S//S) is the energy of the bare HA slab S in its optimized geometry,Em(M//M) is the molecular energy of the free AA molecule M in its optimizedgeometry and E(SM//SM) is the energy of the considered AA–HA system inits optimized geometry (the symbol following the double slash identifies the

Phil. Trans. R. Soc. A (2012)

on March 25, 2016http://rsta.royalsocietypublishing.org/Downloaded from

Amino acids on hydroxyapatite surfaces 1481

geometry at which the energy has been computed). DE is a negative quantityfor a bound system and can be recast in terms of deformation cost of the surfacedES, the adsorbate DEM, the lateral adsorbate–adsorbate interactions DEL, andthe interaction DE∗ between the pre-deformed constituents. The final expressionfor DEC, inclusive of the basis set superposition error (BSSE) correction, reads

DEC = DE + BSSE = DE∗C + dES + DEM + DEL; BSSE = DE∗C − DE∗.

Because B3LYP did not include dispersion energy, the post-DFT dispersivecorrection suggested by Grimme [47] and re-parametrized by some of us forperiodic systems (referred to as D∗) [48] has been added to the BSSE-correctedadsorption energies DEC in a posteriori fashion to obtain the final DEC + D∗. Inthis way, the dispersive contribution is inserted in a non-self-consistent way tothe adsorption energy, which, somehow, underestimates its relevance.

3. Results and discussion

This section is organized as follows: (i) a brief description of the structural featuresfor the (001), (010) and (010)w stoichiometric HA surfaces; (ii) the most relevantstructural information derived from the interaction of each AA on the three HAsurfaces; (iii) the adsorption energy values of the AA–HA systems; and (iv) acomparison of the affinity of different AAs for each surface.

(a) Hydroxyapatite surface models

For the sake of brevity, here we only report the most relevant structuraland energetic features of the stoichiometric (001) and (010) HA surfaces, asfurther details have been already reported in recent papers [49–51]. The HA(001) and (010) surfaces are the most relevant from a biological point of view:the (001) plane is the dominant surface in the thermodynamic morphology[52–54], whereas the crystal growth occurs overall through the c-direction duringbiomineralization, thereby the (010) face being very extended in the final HAcrystal and often responsible for interaction with molecules [55–57]. These twosurfaces were modelled within the slab approach by selective cuts of the optimizedbulk structure. They are both perfect planes, without defects (vacancies, stepsor kink sites), and their thicknesses are 14 Å for the (001) case (fully optimizedwithin the P3 layer group) and 13 Å for the (010) case (P1 symmetry). Thegeometry optimizations for AA adsorption were carried out within the P1symmetry for both cases. To simulate the effect of both low and high AA surfacecoverage, two unit cells were adopted (figure 1): the single-cell (SC), consistentwith the unit cell of HA bulk, and a double-cell (DC), derived from the SC bydoubling the a and the c values for the (001) and the (010) surfaces, respectively.

The (001) surface unit cell exhibits two Ca ion types (Ca1 and Ca3), whereasfor the (010) surface, three Ca types (Ca1, Ca2 and Ca3) exist (figure 1). Theanalysis of the electrostatic potential features (not shown) reveals high positivevalues of potential on top of Ca ions (suggesting compatible sites for AA electrondonor groups), whereas deep negative zones are located in the proximity ofoxygen atoms of the PO4 groups (suggesting H-bond interactions with AA H-bonddonor groups).

Phil. Trans. R. Soc. A (2012)

on March 25, 2016http://rsta.royalsocietypublishing.org/Downloaded from

1482 A. Rimola et al.

Ca1

Ca1

Ca2

Ca3

Ca3c Ca3a

Ca2a Ca2b

Ca2a Ca2b

Ca3

Ca1

Ca3b

P

P

O

O

OH

OH

OHP

O

OHw

Hw

a

b

c

a

b

c

a

b

c

HA (010)w

HA (001)

HA (010)

Figure 1. B3LYP-optimized geometries of the double-cell HA surfaces used in the present work.Single-cell slab models defined as shaded regions. (Online version in colour.)

The (001) slab exhibits a smaller surface energy than the (010) one (1.043 and1.709 J m−2, respectively, at B3LYP level) [50,51] because the number and thetype of bonds that should be cut to define the (010) surface impart a higher costin comparison with the (001) surface. Therefore, the (010) model is expected to bemore reactive. This was indeed the case for water adsorption, which is molecularlyadsorbed at the (001) surface, whereas it spontaneously dissociates on the Ca1and Ca2 sites of the (010) surface while it is molecularly adsorbed on Ca3 ion[51,58]. Owing to this fast reaction with water (ubiquitously present during crystalgrowth), the pristine HA (010) surface is unlikely to exist ‘as cut’ from the HA

Phil. Trans. R. Soc. A (2012)

on March 25, 2016http://rsta.royalsocietypublishing.org/Downloaded from

Amino acids on hydroxyapatite surfaces 1483

bulk, and therefore the AA adsorption at the water-reacted HA (010) surface(hereafter named as (010)w surface) was also considered in this work. The moststable reconstructed (010)w surface is shown in figure 1, in which the Hw and OHwlabel the new functionalities resulting from water dissociation and giving rise toPOH and CaOH groups, respectively. The newly formed OHw group is sharedby Ca3, Ca2a and Ca2b ions with a local geometry resembling that surroundingthe OH group of the HA (001) surface (figure 1). It is clear that this particulargeometrical arrangement gives stability to the final structure and it is worthnoting that H2O does not dissociate on Ca1 (vide supra) because the surface OHcannot be similarly stabilized. The reactivity of the pristine (010) and the (010)wsurfaces can be appreciated by considering the energy of adsorption with respectto H2O computed at B3LYP level with the same basis set adopted here [51]:−315 and −249 kJ mol−1 for H2O chemisorbed on Ca3 and Ca2 ions of the (010)surface, respectively, to be compared with the much lower value of −92 kJ mol−1

for H2O physisorbed on Ca1 of the (010)w surface. It is then expected that asimilar difference in reactivity will be shown for the AA adsorption (vide infra).

(b) Strategies for amino acid adsorption

AAs were manually docked to the HA surfaces in agreement with theelectrostatic complementarity between the two partners and the resulting adductsused as initial structures for the full B3LYP optimization. In the present study,the AAs were adsorbed only at the top face of the slabs. This yields a permanentdipole perpendicular to the surface owing to the asymmetry introduced betweenthe two surfaces (top–bottom), which may affect the adsorption energy values.We check the dependency of the adsorption energy of Gly when adsorbed at the(001), (010) and (010)w HA surfaces for top and top–bottom cases. Results areavailable in the electronic supplementary material (figure S1 and table S1) andshow differences in the adsorption energy between top and top–bottom cases of7 kJ mol−1 at the most, which is very small considering the large computed values(around −250/−450 kJ mol−1). This allowed simulation of the AA adsorption atthe top face only, with a significant saving in computer time.

(c) The glycine–hydroxyapatite surface systems

As reported in previous simulations [36,59], Gly adsorbs as a zwitterion on the(001) surface (001-Gly, figure 2). The stabilization is due to the simultaneouspresence of COO−/Ca+ electrostatic interactions and H-bonds between NH+

3protons and surface oxygen atoms of the PO4 group. This structure is the moststable in both SC and DC slab models. It is worth noting that, contrary to themetal ion [Ca-Gly]2+ complex occurring in gas phase [60], with only one Ca ion asa coordinator centre, in 001-Gly two different Ca ions are simultaneously involvedin the Gly adsorption, resulting in a larger stabilization owing to the enhancedelectrostatic interactions.

At the (010) surface, Gly interacts through a Gly−/HA+ ion pair (010-Gly,figure 2) as the proton is transferred to the surface (occurring from COOH or NH+

3for neutral and zwitterion forms, respectively), in agreement with previous works[36,59]. The remaining COO− group interacts with three Ca ions. In particular,the O1 of the COO− group is shared, almost equivalently, with Ca2a (Ca2a–O1:

Phil. Trans. R. Soc. A (2012)

on March 25, 2016http://rsta.royalsocietypublishing.org/Downloaded from

1484 A. Rimola et al.

001-Gly

010w-Gly010-Gly

Ca1

Ca3

Ca1 Ca1

Ca3

OHw

Hw

Ca3c

Ca2bCa2a

Ca2b Ca1

Ca2a

Ca2a

H

H

H

C

O1

O1

O1

C

C

O2

O2

O2

N

N

N

P

P

P

a

a

a

b

b

b

Figure 2. B3LYP-optimized geometries of Gly with the considered HA surfaces. Geometryparameters are reported in table 1. (Online version in colour.)

2.584 Å), Ca2b (Ca2b–O1: 2.426 Å) and Ca3 (Ca3–O1: 2.411 Å) ions; the secondoxygen O2 of the COO− group interacts more strongly with Ca2a (Ca2a–O2:2.349 Å, figure 2) while the NH2 acts as H-bond acceptor from the POH surfacegroups formed by the protonation process (H...N: 1.611 Å).

The proton transfer towards the HA surface also occurred on the water-reactedHA (010)w surface (010w-Gly, figure 2), the basic PO4 surface group becomingprotonated. Because at the pristine (010)w surface the Ca2a, Ca2b and Ca3 ions(those anchoring Gly in 010-Gly) are already engaged in the interaction with theOHw resulting from the dissociated H2O (vide supra), the COO− group interactswith the remaining Ca1 (Ca1–O2: 2.384 Å), Ca2a (Ca2a–O1: 2.566 Å) and Ca2b(Ca2b–O1: 2.435 Å) ions. The NH2 group interacts with the newly created POHfunctionality (derived from the Gly deprotonation) by a rather strong H-bond(H…N: 1.683 Å). A comparison of the adsorption energies between the 010-Glyand 010w-Gly adducts (table 2) reveals that Gly is adsorbed more strongly onthe pristine surface than on the water reacted one, as anticipated (vide supra).This is due to: (i) a higher reactivity of the Ca2a, Ca2b and Ca3 ions of the (010)surface compared with the Ca1, Ca2a and Ca2b ions of the water reacted (010)wone and (ii) the decreased basic character of the PO4 groups on the (010)w surfacein comparison with the (010) one.

Phil. Trans. R. Soc. A (2012)

on March 25, 2016http://rsta.royalsocietypublishing.org/Downloaded from

Amino acids on hydroxyapatite surfaces 1485

Tab

le1.

B3L

YP

-opt

imiz

edbo

ndle

ngth

sof

the

differ

ent

AA

–HA

stru

ctur

es.B

are

valu

esbe

long

toSC

syst

ems,

valu

esin

pare

nthe

ses

toth

eD

Con

es.

Uni

tsin

ångs

tröm

s.

HA

(001

)

stru

ctur

eC

a1− O

1C

a3c−

O2

Ca3

b−O

3C

a1− N

Ca1

− O3

ab

c

001-

Gly

2.33

4(2

.330

)2.

354

(2.3

66)

1.62

2(1

.618

)1.

956

(1.9

45)

001-

Ser

2.37

2(2

.339

)2.

375

(2.3

79)

2.58

2(2

.599

)1.

709

(1.6

95)

1.87

5(1

.897

)1.

507

(1.4

85)

001-

Lys

2.40

5(2

.396

)2.

334

(2.3

46)

2.44

3(2

.436

)1.

705

(1.6

76)

1.76

0(1

.799

)00

1-G

ln2.

369

(2.3

48)

2.37

8(2

.360

)2.

444

(2.3

80)

1.62

9(1

.665

)2.

011

(1.8

43)

1.72

7(1

.754

)00

1-G

lu2.

359

(2.3

34)

2.36

2(2

.358

)2.

427

(2.4

01)

1.66

2(1

.669

)2.

093

(1.9

22)

1.45

0(1

.379

)

HA

(010

)

stru

ctur

eC

a3− O

1C

a2a−

O1

Ca2

b−O

1C

a2a−

O2

Ca2

b−O

3C

a1− N

Ca2

a−O

3a

bc

010-

Gly

2.41

1(2

.408

)2.

584

(2.5

42)

2.42

6(2

.380

)2.

349

(2.3

49)

1.61

1(1

.501

)1.

857

(1.8

38)

010-

Ser

2.39

2(2

.390

)2.

523

(2.4

76)

2.40

6(2

.381

)2.

410

(2.3

83)

2.48

8(2

.500

)1.

620

(1.5

62)

2.08

6(2

.103

)1.

635

(1.6

63)

010-

Lys

2.44

5(2

.429

)2.

589

(2.5

31)

2.47

2(2

.407

)2.

393

(2.3

56)

2.63

3(2

.679

)1.

617

(1.5

32)

2.07

1(2

.030

)01

0-G

ln2.

406

(2.4

35)

2.83

5(2

.566

)2.

446

(2.4

16)

2.37

6(2

.410

)2.

374

(2.4

36)

2.98

8(2

.484

)1.

627

(1.5

39)

1.98

9(2

.086

)1.

662

(—)

010-

Glu

2.42

8(2

.435

)2.

826

(2.5

63)

2.46

3(2

.399

)2.

395

(2.3

74)

2.39

5(2

.462

)2.

850

(2.6

69)

1.61

0(1

.535

)2.

012

(2.1

83)

1.48

7(—

)

HA

(010

)w

stru

ctur

eC

a1− O

2C

a2a−

O1

Ca2

b−O

1a

bc

010w

-Gly

2.38

4(2

.385

)2.

566

(2.4

98)

2.43

5(2

.410

)1.

683

(1.6

67)

2.12

7(2

.073

)01

0w-S

er2.

301

(2.2

78)

2.46

9(2

.402

)2.

498

(2.5

08)

1.82

7(1

.806

)2.

115

(2.3

03)

1.70

2(1

.706

)01

0w-L

ys2.

342

(2.3

15)

2.41

5(2

.521

)C

a2a-

N(2

.559

)a2.

444

(2.4

66)

1.98

3(1

.706

)1.

577

(1.9

52)

2.07

1(—

)

010w

-Gln

2.30

2(2

.328

)2.

528

(2.5

27)

Ca2

a-O

3(2

.357

)a2.

455

(2.4

50)

1.88

1(1

.762

)1.

733

(1.9

31)

—(2

.009

)

010w

-Glu

2.28

8(2

.329

)2.

571

(2.5

22)

Ca2

a-O

3(2

.368

)a2.

417

(2.4

42)

1.89

2(1

.748

)2.

105

(1.9

34)

—1.

834

a See

stru

ctur

es01

0w-L

ys(d

c),01

0w-G

ln(d

c)an

d01

0w-G

lu(d

c)of

figur

es4,

5an

d6,

resp

ecti

vely

.

Phil. Trans. R. Soc. A (2012)

on March 25, 2016http://rsta.royalsocietypublishing.org/Downloaded from

1486 A. Rimola et al.

Table 2. Computed adsorption energies for the different AA–HA systems with SC and DC slabmodels: non-corrected (DE) and BSSE-corrected (DEC) adsorption energies; estimated DEC valuesconsidering the percentage of the BSSE upon DE of 45% (DEC−est, see text for details), contributionof the dispersive forces (D∗) by single-point energy evaluation on the B3LYP optimum structuresand DEC and DEC−est including the term D∗ (DEC + D∗ and DEC−est + D∗, respectively). Unitsin kJ mol−1.

SC DC

system −DE −DEC −D∗ −(DEC + D∗) −DE −DEC −D∗ −(DEC + D∗)

001-Gly 248 136 54 190 251 141 43 184001-Ser 312 160 77 237 316 168 65 233001-Lys 324 173 86 259 347 191 71 262001-Gln 329 168 94 262 343 180 76 255001-Glu 342 179 90 269 368 203 75 278

010-Gly 449 247 70 317 449 245 68 313010-Ser 484 266 79 345 491 270 85 355010-Lys 528 290 99 389 534 293 126 419010-Gln 543 299 105 404 555 305 110 415010-Glu 571 314 120 434 579 318 107 425

010w-Gly 323 178 63 240 318 175 57 232010w-Ser 329 181 77 258 324 178 68 245010w-Lys 373 205 102 307 371 204 115 324010w-Gln 328 181 96 277 372 205 98 302010w-Glu 314 172 91 263 380 209 95 300

The features of the Gly–HA structures are then adopted as a reference for theother AAs, as the common COOH–NH2 moiety was assumed to behave similarlyto what is found for Gly, while the side chains specifically interact with theremaining surface functionalities. As such, the common features to all AAs willno longer be discussed.

(d) Structure of the polar amino acid–hydroxyapatite surface systems

The B3LYP-optimized geometries of Ser, Lys, Gln and Glu interacting withthe HA surfaces are illustrated in figures 3–6, respectively. Table 1 reports themost relevant structural features.

(i) Serine

Ser (exhibiting a CH2OH side-chain functionality) adsorbs on the (001) surfaceforming a H-bond between the surface PO4 and the OH groups (001-Ser, figure 3)for both SC and DC slab models. The optimized structure also exhibits anelectrostatic interaction between a Ca ion and the side-chain oxygen atom(Ca3b−O3), although it is weaker (Ca3b−O3 distance of 2.5 Å) compared withthat involving the backbone Ca−COO moiety (around 2.3 Å).

Ser interacts with the (010) surface via an additional H-bond and the Ca2b−O3bond (010-Ser, figure 3), this latter missed with the (010)w surface (010w-Ser,figure 3) owing to the engagement of the Ca ions by the pristine OHw.

Phil. Trans. R. Soc. A (2012)

on March 25, 2016http://rsta.royalsocietypublishing.org/Downloaded from

Amino acids on hydroxyapatite surfaces 1487

001-Ser

010-Ser 010w-Ser

Ca3c

Ca2b

Ca2b

Ca2a

Ca2a

Ca2a

Ca1

Ca3Ca3

OHw

Hw

Ca1 Ca1

Ca3b

C

C

C

H

H

H

N

N

N P

P

P

Ca1

a

a

b

b

c

O1

O1O1

O2

O2

O2

O3

O3

Figure 3. B3LYP-optimized geometries of Ser with the considered HA surfaces. Geometryparameters are reported in table 1. (Online version in colour.)

(ii) Lysine

Lys (with a (CH2)4NH2 side-chain functionality) adsorbs on the (001) surfacevia the N atom through the Ca1 ion, without forming any H-bond interactions(001-Lys, figure 4). This occurs in both the SC and the DC models, the Ca1−Nbond distance being moderately different. In the SC slab model (simulating highLys loading), the Ca1 already engaged with the O1 backbone carboxylate atomalso interacts with the N side-chain atom of an image Lys of the adjacent unitcell, giving rise to Lys–Lys repulsive lateral interaction. This is illustrated infigure 4, where the terminal NH2 groups of the image Lys molecule are depictedin sticks. In contrast, in the DC slab model (simulating low Lys loading),two independent Ca1 ions exist, so that one interacts with O1 and the otherwith the N side-chain atom (picture not reported). Accordingly, for the SCslab, the Ca1−O1 and Ca1−N bond distances are longer than for the DC oneby about 0.010 Å.

With the (010) HA surface, the interactions, in both slab models, are throughthe N side-chain atom and the free Ca1 ion (010-Lys, figure 4). For the SC (010)wslab, the NH2 side-chain H-bonds with the POHw surface and also with the imageLys (010w-Lys, figure 4). For the DC (010)w slab, the side-chain N atom bindsto the free Ca2a ion (see 010w-Lys(dc) of figure 4), as occurs for Gln and Glucases (vide infra).

Phil. Trans. R. Soc. A (2012)

on March 25, 2016http://rsta.royalsocietypublishing.org/Downloaded from

1488 A. Rimola et al.

001-Lys

010-Lys 010w-Lys

010w-Lys(dc)

Ca2a

Ca1

Ca1 Ca1

Ca1Ca1

Ca2a

Ca2aCa2a

Ca2a

Ca2b

Ca2a

Ca2b

Ca2b

Ca2b

Ca3

Ca3 Ca3

Ca3

Ca3c

P

P

P

P

C

CH

H

H

N

N

N

HN

C

N

Ca3c

Ca1Ca1

a

b

b

b

bb

a

a

a

c

c

OHw

OHw

OHw

O1O1

O1

O2C

O2

O2

Hw

Hw

Hw

O2O2

O1O1

Figure 4. B3LYP-optimized geometries of Lys with the considered HA surfaces. The (dc) labelidentifies the DC slab model. Geometry parameters are reported in table 1. (Online versionin colour.)

(iii) Glutamine and glutamic acid

The features of the Gln and Glu adsorbates are similar, despite thedifferences in their side-chain functionalities ((CH2)2CONH2 and (CH2)2COOH,respectively). For both AAs, the C=O side-chain groups interact with Ca1(Ca1−O3 bond), whereas the side-chain protons (NH and OH for Gln andGlu, respectively) H-bond with the PO4 surface groups (001-Gln and 001-Glu,

Phil. Trans. R. Soc. A (2012)

on March 25, 2016http://rsta.royalsocietypublishing.org/Downloaded from

Amino acids on hydroxyapatite surfaces 1489

001-Gln

010-Gln 010w-Gln(sc)

010w-Gln(dc)

Ca3c Ca3c

Ca2b

Ca2b Ca2b

Ca2b

Ca2b

Ca2aCa2a

Ca2a

Ca2a Ca2a

Ca2a

Ca2a Ca1

Ca1

Ca3

Ca3

Ca3

Ca3

Ca1

Ca1

Ca1

P

P

P

b

b

c

c

c

b

b

a

a

a

a

cc

Ca1

OHw

OHw OHw

Hw

Hw

Hw

O2

O1

O1

O1

O1

O2

O2

O2

O3

O3

H

HH

H

C

CC

C

N

N

N

N

O3

O2

O2O3

O1

O1

Figure 5. B3LYP-optimized geometries of Gln with the considered HA surfaces. The (dc) labelidentifies the DC slab model. Geometry parameters are reported in table 1. (Online versionin colour.)

figures 5 and 6, respectively). The H-bond established by the Glu side chainis notably stronger than the one established by the Gln side chain (1.4 versus1.7 Å, respectively) because of the stronger acidic character of the COOHgroup.

Similar facts are observed for the adsorption of Gln and Glu on the (010)surface, modelled by means of a SC slab (010-Gln, 010-Glu, 010w-Gln, 010w-Gluof figures 5 and 6, respectively). Nonetheless, significant changes are observedwith a DC slab model: (i) at the (010) surface, the Ca2a−O3 bonds becomestronger (with a shortening of about 0.5 and 0.2 Å for Gln and Glu, respectively,

Phil. Trans. R. Soc. A (2012)

on March 25, 2016http://rsta.royalsocietypublishing.org/Downloaded from

1490 A. Rimola et al.

001-Glu

010-Glu 010w-Glu(sc)

010w-Glu(dc)

Ca1

Ca3c

Ca2b

Ca2b

Ca2b

Ca2b

Ca2b

Ca2a Ca2a

Ca2a

Ca2a

Ca2aCa2a

Ca2a

Ca3

Ca3c

Ca3

Ca3

Ca1 Ca1Ca1

Ca1Ca1

C

C

C

C

H

H

H

Hw

Hw

Hw

OHw

OHw

OHw

Ca1O1 O1

O2O2

O2

O2

O2

O2

O1O1

O1

O1

O3

O3 O3

O3

N

N

N

N

P

P

P

H

c

cc

c

b

bb

b

c

a

a

a

a

Figure 6. B3LYP-optimized geometries of Glu with the considered HA surfaces. The (dc) labelidentifies the DC slab model. Geometry parameters are reported in table 1. (Online version incolour.)

table 1); (ii) the H-bonds (values c in figures 5 and 6 and table 1) between AAimages disappear with the DC; and (iii) at the (010)w surface, the Glu and GlnC=O side-chain groups interact with the Ca2a atom (see 010-Gln(dc) and 010-Glu(dc) of figures 5 and 6, respectively). An additional H-bond is established(H-bond labelled as c in figure 5), in which, for the particular case of Glu, theacidic proton is transferred to a PO4 surface group.

Phil. Trans. R. Soc. A (2012)

on March 25, 2016http://rsta.royalsocietypublishing.org/Downloaded from

Amino acids on hydroxyapatite surfaces 1491

–150001

010

Gly Lys Gln GluSer

010w–250

–350

–450

DEC

+D

* (k

J mol

–1)

Figure 7. AA affinity ladders towards the HA surfaces according to the computed adsorptionenergies DEC + D∗. Solid lines refer to SC slab models, dashed lines to DC ones.

(e) Adsorption energies for all considered models

Values of the adsorption energy, non-corrected (DE), BSSE-corrected (DEC),and including dispersive forces (DEC + D∗) are reported in table 2, whereasfigure 7 depicts the AA affinity ladders towards the HA surfaces and figure 8the relative increment in the interaction energies with respect to Gly, assumed asa reference (vide infra). For periodic systems, the BSSE can only be evaluated forcases in which both the adsorbate and the surface remain electroneutral, so allprocesses in which bond making/breaking occur are excluded. Only adsorptionat the (001) surface brings about neutral constituents and the resulting BSSEwas about 45–49% of the interaction energies. For the other surfaces, we simplyassumed a fixed 45 per cent BSSE resulting from averaging over the BSSE%for the (001) cases and the resulting BSSE-corrected interaction energies arelabelled DEC−est in table 2. A different possibility is to assume a fixed BSSEfor each AA, also derived from the analysis of the interaction energies forthe (001) case and to correct all cases from the (010) and (010)w surfaces.This approach will change the absolute values of DEC + D∗ compared withthe previous strategy (in particular for the (010) case), but will essentiallypreserve the relative trend (see the electronic supplementary material, figure S3).Nevertheless, this different approach brings some inconsistency in the data, as theDEC + D∗ data for Ser on the (010)w surface are less negative (smaller bindingenergy) than for Gly, a behaviour difficult to understand owing to the OH groupinvolved in a rather strong H-bond with the (010)w surface for Ser, which ismissing in Gly.

(i) Hydroxyapatite (001) surface

The affinity of the AAs for the HA (001) surface monitored by the (DEC + D∗)adsorption energies (reported in table 2) shows the Gly < Ser < Lys ≈ Gln < Glutrend (figure 7). The DEC + D∗ values are higher (in absolute terms) comparedwith those for other molecules (approx. −100 kJ mol−1 for water [51,58] or approx.−80 kJ mol−1 for citric acid [61]) and of the same order as those for proline and

Phil. Trans. R. Soc. A (2012)

on March 25, 2016http://rsta.royalsocietypublishing.org/Downloaded from

1492 A. Rimola et al.

120(a)

(b)

Ser Lys Gln Glu Ser Lys Gln Glu

DT (

kJ m

ol–1

)100

80

60

40

20

0

120

DT (

kJ m

ol–1

)

100

80

60

40

20

0

DD*

(kJ m

ol–1

)DD

* (k

J mol

–1)

Figure 8. DT = [DEC + D∗]Gly − [DEC + D∗]AA and DD∗ = D∗Gly − D∗

AA for (a) SC and (b) DCslab models. Dotted bars, (001); striped bars, (010); grey bars, (010)w.

hydroxyproline (approx. −300 and approx. −500, respectively) [59], showing thatpolar AA side-chains interact strongly with HA surfaces. The dependency of theinteraction energy as a function of the cell size is rather limited (see table 1 andfigure 7). The largest differences are shown by the DEC values, but they becomeless important owing to the subtle interplay of the dispersive contribution, whichis larger for the SC model owing to the favourable lateral interactions.

The DE energy contributions (available in the electronic supplementarymaterial, table S2) show that the highest surface energy deformation dES valuesoccur for AAs with the largest number of surface-specific interactions, which, inturn, increases energy deformation cost DEM of the adsorbate.

(ii) Hydroxyapatite (010) and (010)w surfaces

The affinity of the AAs for the HA (010) and (010)w surfaces monitored by the(DEC−est + D∗) adsorption energies (reported in table 2 and in figure 7) shows:(i) for HA (010), the Gly < Ser < Lys < Gln < Glu (SC) and Gly < Ser < Gln <Lys < Glu (DC) trends and (ii) for HA (010)w, the Gly < Ser < Glu < Gln <Lys (SC) and Gly < Ser < Glu ≈ Gln < Lys (DC) trends. The differences areessentially connected to the positions between Lys, Gln and Glu, so it seemsthat their strength of adsorption is a delicate balance between the intrinsicaffinity of the AA towards HA (modulated by electrostatic, H-bond and dispersiveinteractions) as well as the surface morphology (pristine or water-reacted (010)

Phil. Trans. R. Soc. A (2012)

on March 25, 2016http://rsta.royalsocietypublishing.org/Downloaded from

Amino acids on hydroxyapatite surfaces 1493

surface) and the amount of AA already adsorbed. Despite that, results clearlyindicate that Gly and Ser exhibit the lowest adsorption energies, whereas Lys,Gln and Glu exhibit the largest ones.

(iii) Additional notes on adsorption energies

In order to have a deeper insight into the driving forces for AA adsorption, wecomputed the interaction energies of the same AAs with a free Ca2+ ion, formingthe doubly charged [Ca-AA]2+ ion complex. It is well known that the most stablegas-phase [Ca-Gly]2+ isomer exhibits Gly in its zwitterionic state [60]; however,structural information on the remaining [Ca-AA]2+ ion complexes is scarce. Thus,we simulated the [Ca-AA]2+ ion complexes, in which the AAs are both in theirneutral and zwitterionic state. For both cases, we computed the interactionenergy in order to obtain a consistent electrostatic trend of the lateral chainsin interaction with Ca ions. Structures and energies of the computed [Ca-AA]2+ion complex are available in the electronic supplementary material (figure S2and table S3). For both AA forms, the obtained trends follow Gly < Ser < Glu ≈Gln < Lys, which is in agreement with the Lewis basic character of the lateralchains (OH < CO < NH2). This scale is somehow similar to those obtained for theAA adsorption onto HA surfaces, the main differences arising from the positionsof Lys, Gln and Glu. Therefore, it seems that, on the one hand, the possibleH-bonds between Gln and Glu with the surfaces and, on the other hand, thelarge dispersive interactions between Lys and the surfaces alter the magnitude ofinteraction, giving rise to the different scale affinities reported above.

All AAs have in common with Gly the NH+3 CHCOO− moiety that exhibits

the same interactions with the HA surfaces for all considered AAs. Because ofthat, it is possible to compute the excess of interaction energy of a given AAwith respect to Gly, as owing to the specific affinity of its side chain for the HAsurfaces. This quantity, named DT = [DEC + D∗]Gly − [DEC + D∗]AA, is shown infigure 8 for both SC and DC models. Because [DEC + D∗]Gly is less negative than[DEC + D∗]AA for all AAs, their difference is always a positive quantity. The puredispersion contribution to DT is also shown as DD∗ = D∗

Gly − D∗AA in the same

figure and again is always a positive quantity. The SC model represents a highloading of AA at the HA surfaces and it turns out that: (i) the smallest incrementis due to CH2OH (Ser) side chain for all surfaces; (ii) DT is almost constant at75 kJ mol−1 for the (001) surface for all AAs but for Ser; and (iii) at the (010),DT increases monotonically along the Ser > Lys > Gln > Glu series, whereas forthe (010)w, the behaviour is dramatically different, Lys being the highest andSer and Glu showing almost the same value. The dispersion contribution DD∗to DT is totally dominant for Gln and Glu on the (010)w showing that thespecific interactions with the lateral chains are inhibited for these AAs, whereasit amounts about a half of DT for all other cases and surfaces. The DC case mimicsa low AA loading. For the (001) surface, the DT trend is similar to SC, with DTvalues being all slightly higher. For the (010) surface, the three Lys, Gln and Gluexhibit a very similar DT value at variance with the SC case. The most dramaticchange is for the (010)w surface that shows higher DT increments compared withthe SC as now the side-chain groups can exploit the surface functionalities in amore efficient way. The DD∗ values are similar to those of the SC case, with Lyshaving the highest contributions for the (010) and (010)w surfaces.

Phil. Trans. R. Soc. A (2012)

on March 25, 2016http://rsta.royalsocietypublishing.org/Downloaded from

1494 A. Rimola et al.

It is worth analysing the computed adsorption energies as a function of thesurface type and of the kind of AA. Results indicate that the pristine (010)surface is more reactive than the hydrated (010)w surface which, in turn, exhibitsmore favourable adsorption energies than the (001) one. This fact allows one tounderstand the growth and the final surface morphology of the HA crystals inthe presence of physiological fluids. That is, by extending our results to biologicalsystems, proteins would prefer to adsorb at the (010) face so that the growthrate along its direction would be inhibited, in favour of the growth along otherdirections. Additionally, the fact that the computed adsorption energies are sofavourable suggests that proteins containing the tested amino acidic residues,especially carrying Ca binding or H-bond donor groups, will serve as inhibitorsof the HA growth. In particular, our results estimate that C=O- and NH2-containing lateral chains of proteins may be of use to control the growth of calciumphosphate materials.

4. Conclusions

The adsorption of five AAs with different physico-chemical properties (Gly asnon-polar, Ser as polar–neutral, Lys as polar–basic, Gln as polar–amidic andGlu as polar–acidic) on models of stoichiometric (001), (010) and (010)w HAsurfaces under strict gas-phase conditions has been studied with B3LYP andGaussian type basis set in a periodic approach using the CRYSTAL06 code.AAs were manually docked towards the HA surface following the electrostaticcomplementarity and by maximizing H-bond interactions. High and low AAloadings have been simulated by adopting two unit cell sizes. The most interestingpoints emerging from the present work are summarized as follows.

— Gly is adsorbed as a zwitterion at the HA (001) surface, the COO− groupinteracting with two Ca ions and the NH+

3 protons H-bonding the oxygensurface atoms, whereas on both the pristine (010) surface and that reactedwith water (010)w, a Gly−/HA+ ion pair is established.

— For the other AAs, the adsorption occurs keeping the commonNH+

3 CHCOO− moiety in the same configuration as for Gly, while leavingthe side-chain groups to maximize the interaction with the availablesurface sites.

— For Gly and Ser, no significant differences are observed between SC andDC models, whereas the adsorption for Lys, Gln and Glu is much moresensitive to lateral interactions. For these latter, a general increase in thestrength of interaction is computed when moving from SC to DC.

— The driving forces for AA adsorption are the electrostatic affinity for Caions, H-bonds towards the basic PO4 surface groups and the dispersiveinteractions. When the contribution of the common NH+

3 CHCOO− moietyis subtracted from the interaction energy (DT values), it emergesthat the AA affinity averaged over all HA surfaces, for the lowAA loading (DC case) follows the trend: CH2OH (Ser 35 kJ mol−1) <

(CH2)2CONH2 (Gln 81 kJ mol−1) < (CH2)2COOH (Glu 91 kJ mol−1) ≈(CH2)4NH2 (Lys 92 kJ mol−1). Clearly, Lys is able to sample the Caions with the greatest affinity and the long hydrocarbon chain gains

Phil. Trans. R. Soc. A (2012)

on March 25, 2016http://rsta.royalsocietypublishing.org/Downloaded from

Amino acids on hydroxyapatite surfaces 1495

dispersion energy. These incremental values can constitute a database forpredicting the protein affinity towards HA by the knowledge of the exposedside chains.

— For all cases, the interaction with the (010) faces was found to bemore favourable than the (001) one, indicating a major reactivity of theformer surfaces. According to these results, protein amino acidic residuesprefer to adsorb on the (010) faces, thereby inhibiting more strongly thecrystal growth of these surfaces and consequently leading to an elongatedmorphology in the c-direction of the apatite crystal.

— Purely geometrically defined surfaces may not be representative of thecorresponding surface in the real material, as reaction with water maydeeply alter the adsorption character of the pristine surface. Here, thewater-reacted (010)w surface exhibits much weaker adsorption energieswith respect to those computed for the pristine (010) surface.

Results of the present paper are aimed at contributing with significant clues tothe structure and energetics that drive the protein–calcium–apatite interactions,such as to provide a detailed atomistic picture of the interface between AAsand HA, or to estimate an affinity scale for the interaction of polar amino acidicresidues with HA surfaces. Along this line, it is worth mentioning that such water-free systems are indeed possible to study by means of IR spectroscopy owingto the development of the fast thermal heating technique [39,40], which allowsexperimentalists to adsorb neutral AAs on HA by chemical vapour deposition.In that respect, new experimental measurements under highly controllableconditions would be welcome and could be used for direct comparisons and tovalidate the present results.

A.R. is indebted to ‘Comissionat per a Universitats I Recerca del Deprtament D’Innovació,Universitats I Empresa de la Generalitat de Catalunya’. Financial support from MIUR (ProjectCOFIN2006, Prot. 2006032335_005), from the Regione Piemonte (Bando ricerca scientificaPiemonte 2004, Settore: Nanotecnologie e nanoscienze, ‘Materiali nanostrutturati biocompatibiliper applicazioni biomediche’), and from MICINN through the CTQ2008-06381/BQU projectis gratefully acknowledged. P.U. kindly acknowledges BSC-MN for the generous allowance ofcomputing time through the ‘BCV-2008-2-0013: Simulation of peptide folding induced by inorganicmaterials’ and ‘BCV-2008-3-0013: Simulation of peptide folding induced by inorganic materialsII’ projects.

References

1 De, M., Ghosh, P. S. & Rotello, V. M. 2008 Applications of nanoparticles in biology. Adv.Mater. 20, 4225–4241. (doi:10.1002/adma.200703183)

2 Gray, J. J. 2004 The interaction of proteins with solid surfaces. Curr. Opin. Struct. Biol. 14,110–115. (doi:10.1016/j.sbi.2003.12.001)

3 Hudson, S., Cooney, J. & Magner, E. 2008 Proteins in mesoporous silicates. Angew. Chem. Int.Ed. 47, 8582–8594. (doi:10.1002/anie.200705238)

4 Vallet-Regí, M., Balas, F. & Arcos, D. 2007 Mesoporous materials for drug delivery. Angew.Chem. Int. Ed. 46, 7548–7558. (doi:10.1002/anie.200604488)

5 Vo-Dinh, T. 2007 Nanotechnology in biology and medicine: methods, devices, and applications.London, UK: Taylor and Francis Group.

6 Jones, F. H. 2001 Teeth and bones: applications of surface science to dental materials andrelated biomaterials. Surf. Sci. Rep. 42, 75–205. (doi:10.1016/S0167-5729(00)00011-X)

Phil. Trans. R. Soc. A (2012)

on March 25, 2016http://rsta.royalsocietypublishing.org/Downloaded from

1496 A. Rimola et al.

7 Ratner, B. D., Hoffman, A. S., Schoen, F. J. & Lemons, J. 2004 Biomaterial science: anintroduction to materials in medicine. Amsterdam, The Netherlands: Elsevier Inc.

8 Hench, L. L. & Polak, J. M. 2002 Third-generation biomedical materials. Science 295,1014–1017. (doi:10.1126/science.1067404)

9 LeGeros, R. Z. 2008 Calcium phosphate-based osteoinductive materials. Chem. Rev. 108,4742–4753. (doi:10.1021/cr800427g)

10 Palmer, L. C., Newcomb, C. J., Kaltz, S. R., Spoerke, E. D. & Stupp, S. I. 2008 Biomimeticsystems for hydroxyapatite mineralization inspired by bone and enamel. Chem. Rev. 108,4754–4783. (doi:10.1021/cr8004422)

11 Hench, L. L., Splinter, R. J., Allen, W. C. & Greenlee, T. K. 1971 Bonding mechanismsat the interface of ceramic prosthetic materials. J. Biomed. Mater. Res. Symp. 2, 117–141.(doi:10.1002/jbm.820050611)

12 Palazzo, B., Iafisco, M., Laforgia, M., Margiotta, N., Natile, G., Bianchi, C. L., Walsh, D.,Mann, S. & Roveri, N. 2007 Biomimetic hydroxyapatite–drug nanocrystals as potentialbone substitutes with antitumor drug delivery properties. Adv. Funct. Mater. 17, 2180–2188.(doi:10.1002/adfm.200600361)

13 Morgan, T. T. et al. 2008 Encapsulation of organic molecules in calcium phosphatenanocomposite particles for intracellular imaging and drug delivery. Nano Lett. 8, 4108–4115.(doi:10.1021/nl8019888)

14 Shaw, W. J., Long, J. R., Dindot, J. L., Campbell, A. A., Stayton, P. S. & Drobny, G. P. 2000Determination of statherin n-terminal peptide conformation on hydroxyapatite crystals. J. Am.Chem. Soc. 122, 1709–1716. (doi:10.1021/ja9842823)

15 Long, J. R., Shaw, W. J., Stayton, P. S. & Drobny, G. P. 2001 Structure and dynamics ofhydrated statherin on hydroxyapatite as determined by solid-state NMR. Biochemistry 40,15 451–15 455. (doi:10.1021/bi010864c)

16 Gibson, J. M., Popham, J. M., Raghunathan, V., Stayton, P. S. & Drobny, G. P. 2006 A solid-state NMR study of the dynamics and interactions of phenylalanine rings in a statherin fragmentbound to hydroxyapatite crystals. J. Am. Chem. Soc. 128, 5364–5370. (doi:10.1021/ja056731m)

17 Goobes, R., Goobes, G., Campbell, C. T. & Stayton, P. S. 2006 Thermodynamics of statherinadsorption onto hydroxyapatite. Biochemistry 45, 5576–5586. (doi:10.1021/bi052321z)

18 Goobes, R., Goobes, G., Shaw, W. J., Drobny, G. P., Campbell, C. T. & Stayton, P. S.2007 Thermodynamic roles of basic amino acids in statherin recognition of hydroxyapatite.Biochemistry 46, 4725–4733. (doi:10.1021/bi602345a)

19 Goobes, G. et al. 2007 The structure, dynamics, and energetics of protein adsorption—lessonslearned from adsorption of statherin to hydroxyapatite. Magn. Reson. Chem. 45, S32–S47.(doi:10.1002/mrc.2123)

20 Chen, P.-H., Tseng, Y.-H., Mou, Y., Tsai, Y.-L., Guo, S.-M., Huang, S.-J., Yu, S. S.-F. & Chan,J. C. C. 2008 Adsorption of a statherin peptide fragment on the surface of nanocrystallites ofhydroxyapatite. J. Am. Chem. Soc. 130, 2862–2868. (doi:10.1021/ja076607y)

21 Kandori, K., Fudo, A. & Ishikawa, T. 2000 Adsorption of myoglobin onto various synthetichydroxyapatite particles. Phys. Chem. Chem. Phys. 2, 2015–2020. (doi:10.1039/a909396f)

22 Kandori, K., Murata, K. & Ishikawa, T. 2007 Microcalorimetric study of protein adsorptiononto calcium hydroxyapatites. Langmuir 23, 2064–2070. (doi:10.1021/la062562n)

23 Shaw, W. J., Campbell, A. A., Paine, M. L. & Snead, M. L. 2004 The COOH terminus ofthe amelogenin, LRAP, is oriented next to the hydroxyapatite surface. J. Biol. Chem. 279,40 263–40 266. (doi:10.1074/jbc.C400322200)

24 Wallwork, M. L., Kirkham, J., Zhang, J., Smith, D. A., Brookes, S. J., Shore, R. C., Wood,S. R., Ryu, O. & Robinson, C. 2001 Binding of matrix proteins to developing enamel crystals:an atomic force microscopy study. Langmuir 17, 2508–2513. (doi:10.1021/la001281r)

25 Kandori, K., Mukai, M., Yasukawa, A. & Ishikawa, T. 2000 Competitive and cooperativeadsorptions of bovine serum albumin and lysozyme to synthetic calcium hydroxyapatites.Langmuir 16, 2301–2305. (doi:10.1021/la9906424)

26 Brandes, N., Welzel, P. B., Werner, C. & Kroh, L. W. 2006 Adsorption-induced conformationalchanges of proteins onto ceramic particles: differential scanning calorimetry and FTIR analysis.J. Colloid Interface Sci. 299, 56–69. (doi:10.1016/j.jcis.2006.01.065)

Phil. Trans. R. Soc. A (2012)

on March 25, 2016http://rsta.royalsocietypublishing.org/Downloaded from

Amino acids on hydroxyapatite surfaces 1497

27 Elangovan, S., Margolis, H. C., Oppenheim, F. G. & Beniash, E. 2007 Conformational changesin salivary proline-rich protein 1 upon adsorption to calcium phosphate crystals. Langmuir 23,11 200–11 205. (doi:10.1021/la7013978)

28 Capriotti, L. A., Beebe Jr, T. P. & Schneider, J. P. 2007 Hydroxyapatite surface-induced peptidefolding. J. Am. Chem. Soc. 129, 5281–5287. (doi:10.1021/ja070356b)

29 Chen, X., Wang, Q., Shen, J., Pan, H. & Wu, T. 2007 Adsorption of leucine-rich amelogeninprotein on hydroxyapatite (001) surface through -coo- claws. J. Phys. Chem. C 111, 1284–1290.(doi:10.1021/jp0646630)

30 Makrodimitris, K., Masica, D. L., Kim, E. T. & Gray, J. J. 2007 Structure prediction of protein–solid surface interactions reveals a molecular recognition motif of statherin for hydroxyapatite.J. Am. Chem. Soc. 129, 13 713–13 722. (doi:10.1021/ja074602v)

31 Shen, J.-W., Wu, T., Wang, Q. & Pan, H.-H. 2008 Molecular simulation of proteinadsorption and desorption on hydroxyapatite surfaces. Biomaterials 29, 513–532. (doi:10.1016/j.biomaterials.2007.10.016)

32 Pareek, A., Torrelles, X., Angermund, K., Rius, J., Magdans, U. & Gies, H. 2009 Competitiveadsorption of glycine and water on the fluorapatite (100) surface. Langmuir 25, 1453–1458.(doi:10.1021/la802706y)

33 Rosseeva, E. V., Golovanova, O. A. & Frank-Kamenetskaya, O. V. 2007 The influence of aminoacids on the formation of nanocrystalline hydroxyapatite. Glass Phys. Chem. 33, 283–286.(doi:10.1134/S1087659607030170)

34 Shafei, G. M. S. E. & Moussa, N. A. 2001 Adsorption of some essential amino acids onhydroxyapatite. J. Colloid Interface Sci. 238, 160–166. (doi:10.1006/jcis.2001.7474)

35 Koutsopoulos, S. & Dalas, E. 2000 Hydroxyapatite crystallization in the presence of serine,tyrosine and hydroxyproline amino acids with polar side groups. J. Cryst. Growth 216, 443–449.(doi:10.1016/S0022-0248(00)00415-2)

36 Rimola, A., Corno, M., Zicovich-Wilson, C. & Ugliengo, P. 2008 Ab-initio modelling ofprotein/biomaterial interactions: glycine adsorption at hydroxyapatite surfaces. J. Am. Chem.Soc. 130, 16 181–16 183. (doi:10.1021/ja806520d)

37 Rimola, A., Corno, M., Zicovich-Wilson, C. M. & Ugliengo, P. 2009 Ab initio modelingof protein/biomaterial interactions: competitive adsorption between glycine and water ontohydroxyapatite surfaces. Phys. Chem. Chem. Phys. 11, 9005–9007. (doi:10.1039/b913311a)

38 Gross, D. & Grodsky, G. 1955 On the sublimation of amino acids and peptides. J. Am. Chem.Soc. 77, 1678–1680. (doi:10.1021/ja01611a085)

39 Linder, R., Nispel, M., Häber, T. & Kleinermanns, K. 2005 Gas-phase FT-IR-spectra of naturalamino acids. Chem. Phys. Lett. 409, 260–264. (doi:10.1016/j.cplett.2005.04.109)

40 Linder, R., Seefeld, K., Vavra, A. & Kleinermanns, K. 2008 Gas phase infrared spectra ofnonaromatic amino acids. Chem. Phys. Lett. 453, 1–6. (doi:10.1016/j.cplett.2007.12.069)

41 Meng, M., Stievano, L. & Lambert, J. F. 2004 Adsorption and thermal condensationmechanisms of amino acids on oxide supports. 1. Glycine on silica. Langmuir 20, 914–923.(doi:10.1021/la035336b)

42 Rimola, A., Sakhno, Y., Bertinetti, L., Lelli, M., Martra, G. & Ugliengo, P. 2011 Toward asurface science model for biology: glycine adsorption on nanohydroxyapatite with well-definedsurfaces. J. Phys. Chem. Lett. 2, 1390–1394. (doi:10.1021/jz200457x)

43 Dovesi, R. et al. 2006 CRYSTAL06 user’s manual. Torino, Italy: University of Torino.44 Corno, M., Rimola, A., Bolis, V. & Ugliengo, P. 2010 Hydroxyapatite as a key biomaterial:

quantum-mechanical simulation of its surfaces in interaction with biomolecules. Phys. Chem.Chem. Phys. 12, 6309–6329. (doi:10.1039/c002146f)

45 Becke, A. D. 1993 Density-functional thermochemistry. III. The role of exact exchange.J. Chem. Phys. 98, 5648–5652. (doi:10.1063/1.464913)

46 Lee, C., Yang, W. & Parr, R. G. 1988 Development of the Colle-Slavetti correlation-energyformula into a functional of the electron density. Phys. Rev. B 37, 785–789. (doi:10.1103/PhysRevB.37.785)

47 Grimme, S. 2006 Semiempirical GGA-type density functional constructed with a long-rangedispersion correction. J. Comput. Chem. 27, 1787–1799. (doi:10.1002/jcc.20495)

Phil. Trans. R. Soc. A (2012)

on March 25, 2016http://rsta.royalsocietypublishing.org/Downloaded from

1498 A. Rimola et al.

48 Civalleri, B., Zicovich-Wilson, C. M., Valenzano, L. & Ugliengo, P. 2008 B3LYP augmentedwith an empirical dispersion term (B3LYP-D∗) as applied to molecular crystals. Cryst. Eng.Commun. 10, 405–410. (doi:10.1039/B715018K)

49 Corno, M., Busco, C., Civalleri, B. & Ugliengo, P. 2006 Periodic ab initio study of structuraland vibrational features of hexagonal hydroxyapatite Ca10(PO4)6(OH)2. Phys. Chem. Chem.Phys. 8, 2464–2472. (doi:10.1039/b602419j)

50 Corno, M., Orlando, R., Civalleri, B. & Ugliengo, P. 2007 Periodic B3LYP study ofhydroxyapatite (001) surface modelled by thin layer slabs. Eur. J. Mineral. 19, 757–767.(doi:10.1127/0935-1221/2007/0019-1764)

51 Corno, M., Busco, C., Bolis, V., Tosoni, S. & Ugliengo, P. 2009 Water adsorption on thestoichiometric (001) and (010) surfaces of hydroxyapatite: a periodic B3LYP study. Langmuir25, 2188–2198. (doi:10.1021/la803253k)

52 Mkhonto, D. & de Leeuw, N. H. 2002 A computer modelling study of the effect of water onthe surface structure and morphology of fluorapatite: introducing a Ca10(PO4)6F2 potentialmodel. J. Mater. Chem. 12, 2633–2642. (doi:10.1039/b204111a)

53 de Leeuw, N. H. & Rabone, J. A. L. 2007 Molecular dynamics simulations of the interactionof citric acid with the hydroxyapatite (0001) and (011−0) surfaces in an aqueous environment.Cryst. Eng. Commun. 9, 1178–1186. (doi:10.1039/b710974a)

54 Vallet-Regí, M. & González-Calbet, J. M. 2004 Calcium phosphates as substitution of bonetissues. Prog. Solid State Chem. 32, 1–31. (doi:10.1016/j.progsolidstchem.2004.07.001)

55 Simmer, J. P. & Fincham, A. G. 1995 Molecular mechanisms of dental enamel formation. Crit.Rev. Oral Biol. Med. 6, 84–108. (doi:10.1177/10454411950060020701)

56 Kirkham, J., Brookes, S. J., Shore, R. C., Wood, S. R., Smith, D. A., Zhang, J., Chen,H. & Robinson, C. 2002 Physico-chemical properties of crystal surfaces in matrix–mineralinteractions during mammalian biomineralisation. Curr. Opin. Colloid Interface Sci. 7, 124–132.(doi:10.1016/S1359-0294(02)00017-1)

57 Sato, K., Kogure, T., Iwai, H. & Tanaka, J. 2002 Atomic-scale {101̄ 0} interfacial structure inhydroxyapatite determined by high-resolution transmission electron microscopy. J. Am. Ceram.Soc. 85, 3054–3058. (doi:10.1111/j.1151-2916.2002.tb00578.x)

58 Astala, R. & Stott, M. J. 2008 First-principles study of hydroxyapatite surfaces and wateradsorption. Phys. Rev. B 78, 075427. (doi:10.1103/PhysRevB.78.075427)

59 Almora-Barrios, N., Austen, K. F. & de Leeuw, N. H. 2009 Density functional theory studyof the binding of glycine, proline, and hydroxyproline to the hydroxyapatite (0001) and (010)surfaces. Langmuir 25, 5018–5025. (doi:10.1021/la803842g)

60 Corral, I., Mó, O., Yáñez, M., Salpin, J.-Y., Tortajada, J., Morán, D. & Radom, L. 2006 Anexperimental and theoretical investigation of gas-phase reactions of Ca2+ with glycine. Chem.Eur. J. 12, 6787–6796. (doi:10.1002/chem.200600127)

61 Filgueiras, M. R. T., Mkhonto, D. & de Leeuw, N. H. 2006 Computer simulations of theadsorption of citric acid at hydroxyapatite surfaces. J. Cryst. Growth 294, 60–68. (doi:10.1016/j.jcrysgro.2006.05.077)

Phil. Trans. R. Soc. A (2012)

on March 25, 2016http://rsta.royalsocietypublishing.org/Downloaded from