Dynamics and Molecular Mechanism of Phosphate Binding to a

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Dynamics and Molecular Mechanism of Phosphate Binding to a Biomimetic Hexapeptide Hang Zhai, Lihong Qin, Wenjun Zhang, Christine V Putnis, and Lijun Wang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b03062 • Publication Date (Web): 23 Aug 2018 Downloaded from http://pubs.acs.org on August 24, 2018

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Environmental Science & Technology

1

Dynamics and Molecular Mechanism of Phosphate Binding to a

2

Biomimetic Hexapeptide

3 4

Hang Zhai,† Lihong Qin,† Wenjun Zhang,*,† Christine V. Putnis,‡,§ and Lijun

5

Wang*,†

6 †

7

College of Resources and Environment, Huazhong Agricultural University, Wuhan

8

430070, China ‡

9

Institut für Mineralogie, University of Münster, 48149 Münster, Germany §

10

Department of Chemistry, Curtin University, Perth, WA6845, Australia

11 12 13

*

To whom correspondence should be addressed.

14 15

Email: [email protected] or [email protected]

16 17 18 19 20 21 22 23 24 25 1

ACS Paragon Plus Environment


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ABSTRACT

27

Phosphorus (P) recovery from wastewater is essential for sustainable P management. A

28

biomimetic hexapeptide (SGAGKT) has been demonstrated to bind inorganic P in P-

29

rich environments, however the dynamics and molecular mechanisms of P-binding to

30

the hexapeptide still remain largely unknown. We used dynamic force spectroscopy

31

(DFS) to directly distinguish the P-unbound and P-bound SGAGKT adsorbed to a mica

32

(001) surface by measuring the single-molecule binding free energy (DGb). Using

33

atomic force microscopy (AFM) to determine real-time step retreat velocities of

34

triangular etch pits formed at the nanoscale on the dissolving (010) face of brushite

35

(CaHPO4·2H2O) in the presence of SGAGKT, we observed that SGAGKT peptides

36

promoted in situ dissolution through an enhanced P-binding driven by hydrogen bonds

37

in a P-loop being capable of discriminating phosphate over arsenate, concomitantly

38

forming a thermodynamically favored SGAGKT-HPO42- complexation at pH 8.0 and

39

relatively low ionic strength, consistent with the DFS and isothermal titration

40

calorimetry (ITC) determinations. The findings reveal the thermodynamic and kinetic

41

basis for binding of phosphate to SGAGKT and provide direct evidence for phosphate

42

discrimination in phosphate/arsenate-rich environments.

43 44 45 46 47

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INTRODUCTION

49 50

Phosphorus (P) is one of the main limiting macronutrients for the continuous growth

51

of crops, resulting in large amounts of P applications. 1-3 As a non-renewable resource,

52

the existing P ore reserves underpin agricultural demands for P fertilizers.4,5 On the

53

other hand, dissolved P can migrate to water and subsequently increase the risk of

54

eutrophication6 and phosphate-induced mobilization of arsenic or arsenate (As),

55

chromium (Cr), and other anionic contaminants.7 Therefore, recovering P from P-rich

56

wastewaters for reuse can be a promising strategy for sustainable, agricultural and

57

environmental management.

58

The common techniques for P recovery from solutions focus on chemical

59

precipitation by the formation of struvite (MgNH4PO4·6H2O),8 calcium phosphates9

60

and iron phosphates.10 The formation of the precipitates may be not generally favored

61

due to highly thermodynamic energy barriers to reach supersaturation for the

62

precipitation11 and poor selectivity to distinguish P from As.12 In contrast, enhanced

63

biological P removal (EBPR) is more optimized using (poly)phosphate accumulating

64

macromolecules with high specificity to take up and store P from waste effluents,13 and

65

the reacted P-binding biomacromolecules in EBPR can be used directly as a P

66

fertilizer.14 Specifically, biomacromolecules bind phosphates non-covalently through

67

their P-binding sites, that form a P-loop with the consensus sequence of Gly-Xxx-Xxx-

68

Xxx-Xxx-Gly-Lys-(Ser or Thr), especially for proteins binding the β-phosphate of ATP

69

and GTP.15-18 Gruber et al. applied molecular dynamics simulations to show that

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the conformational ensemble of a small intrinsically-disordered peptide, Ser-Gly-

71

Ala-Gly-Lys-Thr (SGAGKT), was significantly stabilized by the binding of phosphate

72

anions through multiple binding modes.19 Based on the consensus sequence of a stable

73

P-loop binding nest, the SGAGKT hexapeptide possesses two glycine (G) residues to

74

promote the LRLR (L represent residues as in the left-handed, and R as in right-handed

75

of an α-helix) conformation, and a zwitterionic lysine (K) to bind phosphate anions.20

76

While the SGAGKT hexapeptide has been simulated to be capable of binding

77

phosphate anions, direct experimental characterizations of the dynamics and molecular

78

mechanisms of phosphate binding to the SGAGKT hexapeptide and the influence of

79

the environmental factors, such as pH, ionic strength (IS), and the competitive binding

80

of the chemically similar arsenate (As) on phosphate-binding are still lacking.

81

Therefore, the aim of this study was to investigate SGAGKT’s ability to bind phosphate

82

by elucidating the mechanistic basis of adsorption such that the peptide-based system

83

can be experimentally controlled via solution conditions.

84

To achieve the above goals, in situ dynamic force spectroscopy (DFS)21, 22 at the

85

single molecular level was used to make thermodynamic comparisons of the binding

86

force between the P-unbound and P-bound SGAGKT on mica. After calculating the

87

equilibrium free energy of binding (DGb), we demonstrated that HPO42- was

88

preferentially bound to the hexapeptide molecule, this subsequently weakened the

89

adhesion of the hexapeptide to mica. Furthermore, using atomic force microscopy

90

(AFM), the real-time dissolution kinetics of the brushite (010) face at the nanoscale was

91

significantly promoted by the hexapeptide through an enhanced P-binding driven by

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hydrogen bonds in a P-loop being capable of discriminating phosphate over arsenate at

93

pH 8.0 and relatively low ionic strength, consistent with the DFS and isothermal

94

titration calorimetry (ITC) determinations. These in situ direct observations confirmed

95

that the use of SGAGKT hexapeptides provides a possibility to recover P with high

96

specificity from alkaline wastewaters, and the findings can enhance the understanding

97

of the dynamics and molecular mechanism of phosphate binding to a biomimetic

98

hexapeptide with the sequence of SGAGKT.

99 100

EXPERIMENTAL SECTION

101 102

SGAGKT Hexapeptide Synthesis. Hexapeptides were synthesized according to the

103

standard procedures of the solid phase peptide synthesis from Bioyeargene Biotech.

104

(Wuhan, China).23 The synthetic hexapeptide fragments (98.16% in purity) were

105

purified by High-Performance Liquid Chromatography (Waters 600 HPLC, Waters,

106

Milford, MA)23 and the molecular weight (520.56) was verified by mass

107

spectrometry.23

108

Tip Decoration and Dynamic Force Spectroscopy (DFS). Details of the tip

109

decoration are reported in SI Materials and Methods. Briefly, the Au-coated Si3N4 tip

110

(Bruker, SNL-10) was modified with a SGAGKT hexapeptide with the

111

heterobifunctional crosslinker (succinimidyl 6-(3-[2-pyridyldithio]-propionamido)

112

hexanoate, LC-SPDP).24,

113

conducted in freshly prepared solutions using an AFM (NanoScope V-Multimode 8,

114

Bruker) equipped with a liquid cell with a constant forward velocity of 200 nm/s. Dwell

25

DFS measurements on the (001) face of mica were

5



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time of 1 s was chosen for various reverse velocities of 20 nm/s, 200 nm/s, 601 nm/s,

116

1.12 µm/s, 2.6 µm/s, 3.91 µm/s, and 7.81 µm/s. In order to acquire more accurate values,

117

the worm-like chain (WLC) model26 with a nonlinear least-squares fitting method

118

(Figure S1) was used to analyze the number of tethers being stretched and the contour

119

lengths of the hexapeptide. DFS measurements also carried out on the (010) surfaces

120

of brushite (CaHPO4·2H2O) or pharmacolite (CaHAsO4·2H2O) at pH 8.0. We chose

121

relatively flat areas (terraces) of both minerals without newly formed etch pits (Figure

122

S2), i.e., dissolution hardly occurred during the DFS measurement.

123

Brushite and Pharmacolite Synthesis. Single crystals of brushite and pharmacolite

124

were synthesized by a gel method with Ca/P (Ca/As) ratio of 1:1 at pH 6.0.27 Synthetic

125

crystals were rinsed with ethanol to remove the gel and were identified as single phase

126

by X-ray diffraction (Bruker D8, Billerica, MA, USA).

127

Brushite Dissolution Kinetics. Synthetic brushite single crystals were used for in situ

128

dissolution experiments by AFM. We used water as a reference to counter the effects

129

of the stress applied from the AFM tip.28 Reaction solutions with and without 10 µM

130

SGAGKT peptides at various pH values (4.0-8.0) (Table S1), or 10 µM SGAGKT

131

solutions with different concentrations of NaCl (10-105 µM) at pH 8.0 (Table S2), or

132

10 µM SGAGKT solutions in the presence of 0.1-10 µM NaHAsO4 at pH 8.0 (Table

133

S3) were freshly prepared from the reagents (Sigma-Aldrich, St. Louis, MO, USA)

134

dissolved in ultrahigh purity water from a two-step purification treatment, including

135

triple distillation (YaR, SZ-97A, Shanghai, China) and deionization (Milli-Q, Billerica,

136

MA, USA). The pH values of all reaction solutions were adjusted by 0.01 M NaOH or

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0.01 M HCl and measured by the glass pH electrode coupled with a single-junction

138

Ag/AgCl reference electrode (Orion 4 Star ISE meter, Thermo). In situ AFM

139

dissolution experiments were conducted in contact mode by AFM (Agilent 5500,

140

Phoenix) equipped with a fluid cell. The freshly prepared solutions were injected into

141

a 1 mL fluid cell with a pump (Cole Parmer Instrument) at the flow rate of

142

approximately 0.3 mL/min to avoid the dissolution rate being influenced by rapid flow

143

rates. All images were collected with Si3N4 tips with a force constant of 0.2 N/m at scan

144

rates of 3.0-5.0 Hz. PicoScan 5 software was used for quantitative analyses of AFM

145

images. All data were presented as mean value ± standard deviation (SD) with statistical

146

analysis (P < 0.01) by the SPSS software.

147

The Size of SGAGKT Hexapeptide Aggregates. The measurements were conducted

148

by AFM (NanoScope V-Multimode 8, Bruker) in ScanAsyst mode. 10 µM SGAGKT

149

solutions in the presence of NaCl (10-105 µM) were injected into the fluid cell.

150

Following 60 min deposition on the freshly cleaved mica (001) surface, the aggregates

151

were imaged with a Si3N4 tip (Bruker, Scanasyst-Fluid+) at room temperature and the

152

height images were analyzed by the NanoScope analysis software.

153

Isothermal Titration Calorimetry. The heat of SGAGKT hexapeptides reacted with

154

Na2HPO4 and Na2HAsO4 was measured by an isothermal microcalorimeter TAM Ⅲ

155

(TA Instruments, USA). An aliquot (700 µL) of Na2HPO4 or Na2HAsO4 solutions (1

156

mM, pH 8.0) was filled into the 1 mL ampoule with a three-blade golden propeller

157

stirring at 120 rev/min. Prior to the heat measurement, the solution in the ampoule was

158

equilibrated for at least 2 h. The hexapeptide solution (10 µM) was filled into a 250 µL

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Hamilton syringe with a stainless-steel needle tip submerged into the solution in the

160

ampoule. A total volume of 200 µL hexapeptide solution was injected into the ampoule

161

five times at a rate of 1 µL/s. The time interval between each injection was 30 min to

162

ensure the complete reaction. Three repeats were conducted for microcalorimetric

163

measurements to ensure reproducibility.

164 165

RESULTS

166 167

DFS Measurements to Detect SGAGKT-P Binding. A SGAGKT hexapeptide-

168

functionalized AFM tip was immersed in solutions without (Figure 1A) and with 50

169

µM Na2HPO4 (Figure 1B) at various pH values ranging from 4.0 to 8.0 for complete

170

reaction with P. Then the tip was approached to the (001) face of mica with a constant

171

forward velocity of 200 nm/s, and was retreated with reverse velocities of 20 nm/s to

172

7.91 µm/s. The force versus separation distance (FD) curves during the retreat process

173

(Figure S3A) and force versus time (FT) curves (Figure S3B) were recorded

174

simultaneously during DFS measurements. A plot of mean rupture force (Figure S3C)

175

versus loading rate (R = ΔF/Δt) showed an increasing rupture force with increasing

176

loading rate (Figure S3D). As a result, bond rupture dynamics approached a near-

177

equilibrium regime at low loading rates (pulling velocities). The data were fitted with

178

the analytical approximation model to determine and calculate the equilibrium free

179

energy of binding (DGb)21 (Tables S4-8), that is the quasi-static work of bond breaking

180

at zero loading rate. We determined the binding strength of the hexapeptide SGAGKT

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on mica in H2O or 50 µM Na2HPO4 at three different pH values (4.0, 6.0 and 8.0)

182

(Figure 1C). A significant difference existed in the binding energy of 22.22 ± 0.50 and

183

20.97 ± 0.43 kJ/mol (n = 3) in H2O (pH 8.0 adjusted by 0.01 M NaOH) and in 50 µM

184

Na2HPO4 (pH 8.0), respectively, suggesting that DGb was decreased following the

185

formation of the SGAGKT-HPO42- complex at pH 8.0 (Figure 1D). No differences were

186

detected at pH 4.0 and 6.0, demonstrating that the P-binding to SGAGKT peptides was

187

probably inhibited under acidic conditions (pH ≤ 6.0), consistent with the results of

188

Bianchi et al..20

189

To evaluate the effect of ionic strength (IS) on the binding energies, DFS

190

measurements of the SGAGKT hexapeptide on mica were conducted in 50 µM

191

Na2HPO4 at pH 8.0 with NaCl ranging from 10 to 105 µM. With NaCl concentrations

192

below 103 µM, there was no significant difference in DGb, whereas the binding free

193

energies DGb increased to 24.13 ± 0.58 kJ/mol (n = 3) in 103 µM NaCl solution, and

194

subsequently dropped to 22.73 ± 0.46 kJ/mol (n = 3) with increasing NaCl

195

concentrations to 105 µM (Figure 2). This suggests that the P-binding to SGAGKT

196

peptides is significantly inhibited at relatively high ionic strength ([NaCl] ≥ 103 µM).

197

To

investigate

the

selectivity

of

the

hexapeptide

SGAGKT,

arsenate

198

(Na2HAsO4·7H2O) was used as the competitive substrate for the P-binding to the

199

hexapeptide. DFS was measured on mica in 50 µM Na2HPO4, 25 µM Na2HPO4 + 25

200

µM Na2HAsO4, and 50 µM Na2HAsO4 solutions at pH 8.0, respectively, and we

201

observed the following trend of the binding energies: 50 µM Na2HAsO4 > 50 µM

202

Na2HPO4 ≈ 25 µM Na2HPO4 + 25 µM Na2HAsO4 (Figure 3A and B), demonstrating

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the hexapeptide specifically binds phosphate rather than arsenate. Furthermore, DFS

204

measurements were also conducted on the (010) face of brushite (CaHPO4·2H2O)

205

(Figure S4A and B) and pharmacolite (CaHAsO4·2H2O)29 (Figure S4C and D). The

206

calculated DGb of the hexapeptide binding to brushite and pharmacolite were -27.96 ±

207

3.10 kJ/mol (n = 3) and -21.23 ± 2.54 kJ/mol (n = 3) (Figure 3C), respectively,

208

indicating that the P-binding was greater than the As-binding to hexapeptide SGAGKT

209

at pH 8.0.

210

Brushite Dissolution Features in the Absence and Presence of the SGAGKT

211

Peptides. We conducted the brushite dissolution experiments in SGAGKT solutions at

212

pH 4.0 to 8.0. The dissolution on the brushite (010) face occurred with the formation

213

and spreading of triangular etch pits27 along the [101]Cc, [100]Cc, and [101]Cc directions

214

(Figure 4A and B). At pH 4.0 and 6.0, there was no significant difference in step retreat

215

velocities in H2O (pH adjusted by 0.01 M HCl) and 10 µM SGAGKT solutions.

216

However, as pH was increased to 8.0, the step retreat velocity along the [101]Cc

217

direction increased to 3.12 ± 0.11 nm/s (n = 3) from 2.53 ± 0.23 nm/s (n = 3) in the

218

presence of 10 µM SGAGKT hexapeptides (Figure 4C). A similar phenomenon

219

occurred on the [100]Cc steps (Figure S5A). Overall, the P-binding to the hexapeptide

220

only occurred at pH 8.0, fully consistent with the DFS results (Figure 1). Moreover, we

221

further conducted the brushite dissolution experiments in 10 µM SGAGKT solutions

222

(pH 8.0) in the presence of various concentrations of NaCl (10 to 105 µM). A 10 µM

223

NaCl solution increased the step retreat velocities along the [101]Cc (Figure 4D) and

224

[100]Cc directions (Figure S5B) to 2.87 ± 0.10 nm/s (n = 3) and 3.18 ± 0.19 nm/s (n =

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3), respectively. After adding 10 µM SGAGKT hexapeptides into 10 µM NaCl

226

solutions, the [101]Cc step retreat velocities further increased to 3.98 ± 0.10 nm/s (n =

227

3). However, a 105 µM NaCl solution did not show a significant increase of step retreat

228

velocities: the [101]Cc step retreat velocities were 5.29 ± 0.15 nm/s (n = 3) and 5.20 ±

229

0.12 nm/s (n = 3) in the absence and presence of 10 µM SGAGKT hexapeptide in 105

230

µM NaCl solutions. A similar phenomenon occurred on the [100]Cc steps (Figure S5B).

231

These results suggest that the P binding to the hexapeptide only occurred at relatively

232

low ionic strength, also consistent with the DFS results (Figure 2).

233

After 10 µM Na2HAsO4 at pH 8.0 was introduced, the step retreat velocities along

234

the [101]Cc (Figure 4E) and [100]Cc (Figure S5C) directions increased to 3.80 ± 0.16

235

nm/s (n = 3) and 4.26 ± 0.15 nm/s (n = 3). This increase did not occur following the

236

addition of 10 µM SGAGKT hexapeptides: both the [101]Cc and [100]Cc steps remained

237

at almost constant retreat rates of about 4.19 ± 0.12 nm/s (n = 3) and 5.82 ± 0.23 nm/s

238

(n = 3) by varying Na2HAsO4 concentrations from 0.1 to 10 µM, suggesting Na2HAsO4

239

has little impact on P-binding to the SGAGKT peptide, consistent with the DFS results

240

(Figure 3).

241 242

DISCUSSION

243 244

The Effect of Solution Conditions on the Hexapeptide-HPO42- Complexes.

245

Phosphate anions can bind to SGAGKT peptides by a P-loop through H-bonds, N···H-

246

O-P with the side chain є-amino group of the lysine residue and N-H···O-P with the

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successive main-chain CONH groups.20, 30 The formation of the SGAGKT-HnPO4(3-n)-

248

complexes (n depending on pH) increases the negative charge of the SGAGKT peptide.

249

Thus, the repelling force driven by charges between the hexapeptide-HnPO4(3-n)-

250

complexes and a mica (001) surface (the basal plane of mica carrying an overall

251

negative charge due to isomorphic substitutions)31 increased. This subsequently caused

252

a decrease of DGb between the SGAGKT hexapeptide and mica. Based on the

253

difference of DGb between the P-unbound SGAGKY and SGAGKT-HnPO4(3-n)- to mica

254

(Figure 1A and B), we can directly distinguish whether phosphate is bound to the

255

hexapeptide under various solution conditions.

256

a. pH. In consideration of triprotic equilibria for phosphoric acid and the SGAGKT

257

hexapeptide, pH alters the relative concentrations of the protonated forms of H3PO40,

258

H2PO4-, HPO42-, and PO43- (pK1 = 2.12, pK2 = 7.21 and pK3 = 11.77) (Figure S6A), and

259

the hexapeptide (HPep) of H3Pep2+, H2Pep+, HPep and Pep- (pK1 = 2.91, pK2 = 7.01

260

and pK3 = 10.95)20 (Figure S6B). The major chemical reactions at different pH values

261

may include:

262 263

H2PO4- + H2Pep+ → [(H2Pep)(H2PO4)]

(pH 4.0-6.0)

(1)

264

HPO42- + HPep → [(HPep)(HPO4)]2-

(pH 8.0)

(2)

265 266

The DFS results showed that phosphate binds to the SGAGKT hexapeptide only at pH

267

8.0, indicating that HPO42- rather than H2PO4- binds to HPep (Figure 1). The binding is

268

driven by hydrogen bonds (H-bonds), that is the formation of the [(HPep)(HPO4)]2-

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complexes through H-bonds of N···H-O-P with the lysine (K) amino group (NH2) and

270

N-H···O-P with the macrodipoles of CONH groups in the SGAGKT mainchain amino

271

acids.20, 30 For H2PO4- binding to H2Pep+, H-bonds of N···H-O-P will result in the

272

formation of NH+···H-O-P, and N-H···O-P will be N···H-O-P. The H-bond strength is

273

in the order of NH+···H < N···H < O···H due to the fact that O is more electronegative

274

than N. The weaker the H-bonds in the complexes, the less stable the complexes.32, 33

275

Thus, it is impossible to bind either H2PO4- or H3PO4 by the formation of the

276

[(HPep)(H2PO4)]- or [(HPep)(H3PO4)] complexes. We also predicted the formation of

277

the most stable [(HPep)(PO4)]3- complex through H-bonds, N-H···O-P at pH ≥ 10.0

278

with DG of 0.5 kcal/mol lower than the formation of the [(HPep)(HPO4)]2- complexes.20

279

However, it is impossible to carry out the DFS determinations at pH ≥ 10.0 due to

280

alkali-induced chemical reactions on mica (between SiO2 and Al2O3 with NaOH) and

281

the formation of precipitates on the dissolving brushite surface (Figure S7). Such high

282

pH values would also be unrealistic in the natural environment.

283

b. NaCl. At the single-molecule level, the inhibition of the binding of HPO42- to

284

hexapeptide induced by NaCl ( ≥ 103 µM) occurs (Figure 2). The most likely

285

explanation is that Na+ ions increase the activation energy barrier for water molecules

286

that are expelled from the HPO42- anion shell. This thus increases the energy barrier for

287

the H-bond formation between SGAGKT and HPO42- with the stabilization of the

288

hydration shell of HPO42- anions.34 As NaCl concentrations were further increased to

289

105 µM, there was a significant reduction in the binding free energy between SGAGKT-

290

HPO42- complexes and mica (Figure 2). This may be attributed to a high salt

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291

concentration decreasing the adsorption of the SGAGKT hexapeptide to mica rather

292

than influencing the P-binding. Indeed, both electrostatic interaction and H-bond

293

weakening play a key role in reducing the binding force at relatively high

294

concentrations of NaCl.35

295

At relatively high concentrations of NaCl (103-105 µM), SGAGKT hexapeptides

296

exert weak interactions on the step retreat velocities during brushite dissolution (Figure

297

4D). We also observed that in various NaCl solutions (10-105 µM) (Figure 5A-C)

298

aggregation of the SGAGKT hexapeptides occurred on mica, and the sizes of

299

aggregates increased from 2.15 ± 0.17 nm at 10 µM NaCl to 6.79 ± 0.25 nm at 105 µM

300

NaCl (Figure 5D), suggesting an increase in the degree of hexapeptide oligomerization

301

with increasing NaCl concentrations (Figure 5E). Na+/Cl- ions possibly decrease the

302

number of water molecules available to interact with the SGAGKT hexapeptides.36

303

Aggregated or oligomerized hexapeptides result in a decrease in concentration of

304

SGAGKT monomers.

305

c. Na2HAsO4. Phosphate and arsenate have nearly identical pKa values and similarly

306

charged oxygen atoms.37,

307

hexapeptides is much stronger than that of arsenate (Figure 3). High selectivity occurs

308

in many P-binding proteins.20, 35, 37, 38 Comparing HAsO42- and HPO42-, binding to the

309

hexapeptide, an extensive network of dipole–anion interactions and of repulsive

310

interactions, results in the 4% larger arsenate distorting a unique low-barrier H-bond

311

that is a key component of the recognition site.38 In addition, the length of an As–O

312

bond (165 pm) is approximately 10% longer than that of a P–O bond (153 pm), and as

38

However, binding of phosphate to the SGAGKT

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313

a consequence, the radius of arsenate is approximately 10% larger than that of

314

phosphate. This contributes to extra negative charges on the O atom of the P-O bond

315

than that of the As–O.39,

316

electronegativity, the stronger the H-bond. Therefore, the [(HPep)(HPO4)]2- complex is

317

more stable than the [(HPep)(HAsO4)]2- complex.

40

As a H-bond acceptor in O···H, the greater the

318

The formation of complexes between phosphate anions and hexapeptides is expected

319

to involve entropy changes (∆H°) due to the binding generated by the formation of H-

320

bonds.41 Consistently, isothermal titration calorimetry (ITC) results (Figure 6A and B)

321

showed that the total heat for phosphate and arsenate binding to hexapeptides was 1.92

322

± 0.17 mJ (n = 3) and 1.18 ± 0.12 mJ (n = 3), respectively (Figure 6C). The calculated

323

binding enthalpy (the binding heat per hexapeptide molecule) of phosphate and arsenate

324

were 5.99 ± 0.53 kJ/mol (n = 3) and 2.97 ± 0.30 kJ/mol (n = 3), respectively (Figure

325

6D), indicating that the P-binding to hexapeptides is more exothermic than the As-

326

binding. This indicates that HPO42- was more preferentially bound to the hexapeptides

327

than HAsO42-. This selectivity between phosphate and arsenate may be driven by a

328

small energy difference of the interactions, including H-bonds, electrostatics, van der

329

Waals forces, and steric hindrance37,

330

hexapeptides.45, 46 However, the strength difference of H-bonds between the SGAGKT

331

hexapeptide and the substrate of phosphate and arsenate having the same anionic

332

structure may be the primary cause for higher selectivity.38

333

SGAGKT Hexapeptides Promoting Brushite Dissolution through P-Binding. At

334

pH 8.0, the dissolution kinetics was significantly altered by the SGAGKT hexapeptide,

42-44

without conformation changes of the

15



Page 16 of 33

335

causing brushite to dissolve more rapidly along both the [101]Cc and [100]Cc directions

336

than in the absence of SGAGKT hexapeptide (Figure 4). This can be explained due to

337

the enhanced chelation or complexation47, 48 of HPO42- or Ca2+ by SGAGKT peptides

338

to increase the step retreat velocities. As the Ca2+-terminated polar step of brushite, the

339

[101]Cc step, showed no enhanced dissolution by SGAGKT (Figure S8), whereas the

340

retreat velocities of the [101]Cc steps with the OH/HOPO32- termination and the [100]Cc

341

steps with the mixed charges49 were significantly promoted. This further suggests P-

342

binding rather than Ca2+-binding by SGAGKT (Figure 4). In this case, the formation of

343

[(HPep)(HPO4)]2- complexes at pH 8.0 at the brushite-fluid interface enhances

344

dissolution by the following reactions:

345 346

H2O + CaHPO4(s) → Ca2+ + HPO42-

(3)

347

HPep + HPO42- → Ca2+ + [(HPep)(HPO4)]2-

(4)

348 349

If the [(HPep)(HPO4)]2- complex is unstable and dissociates to release the bound

350

HPO42-, the step retreat velocities will not be changed, such as the case of that at pH

351

4.0-6.0 (Figure 4C). However, at pH 8.0, the [(HPep)(HPO4)]2- complex can exist stably,

352

consistent with theoretical predictions that the addition of a phosphate anion stabilizes

353

the P-loop conformation.19 The loop-type nest, such as the protein/peptide-anion

354

binding mode, has been observed in various proteins and anions (SO42-, ClO4-, MoO42-,

355

F-, Cl-, Br-, and I-).50-53 Stoichiometry (1:1) of the protein/anion complexes was

356

demonstrated despite the differences in size, charge, and structure of anions.50 With

16


Page 17 of 33


357

increasing concentration of SGAGKT hexapeptides at the brushite-fluid interface, more

358

HPO42- ions released from the dissolving brushite surfaces were bound to the

359

hexapeptides, resulting in accelerating the dissolution rates of both the [101]Cc and

360

[100]Cc steps (Figure S9).

361 362

Environmental Implications. P removal from wastewaters using peptides not only

363

prevents eutrophication, but also presents a promising method to recover P for a

364

possible reuse as a non-toxic fertilizer.54 The present study has systematically shown

365

that SGAGKT hexapeptides can be used for P-binding at pH ≥ 8.0 and low

366

concentrations of NaCl (≤ 103 µM) to recover P with higher selectivity compared to

367

arsenate. Due to their relatively smaller sizes compared to proteins, SGAGKT

368

hexapeptides are lacking the second and tertiary structures so that they can retain their

369

selectivity for P-binding in diverse environments without considering structural

370

changes that could potentially influence P-binding. Moreover, following degradation

371

including oxidation, hydrolysis and deamination,55-57 biomimetic hexapeptides will not

372

create additional pollution in solutions during environmental remediation. However,

373

some practical questions still exist: (1) Given that the binding effect is specifically

374

around pH ³ 8.0, how will this be possible in an environment when pH will vary and

375

can certainly be lower. The addition of other molecules should be needed for further

376

investigations in more neutral or even acidic waters in the future study. (2) Although at

377

pH 8.0 P can be removed from the water using a hexapeptide, if As is present it still is

378

there to contaminate the water. The combined addition of the hexapeptide with the other

17



Page 18 of 33

379

remediation materials, such as calcite, could be a strategy to sequester As58 following

380

P removal. (3) Peptides in general are costly to manufacture so their use on a large scale

381

may not be cost-effective. Despite these practical questions, this fundamental study not

382

only elucidates the dynamics and molecular mechanism for phosphate bound to

383

hexapeptides but also demonstrates how DFS and in situ imaging by AFM can be

384

applied to relevant research on the biomimetic recovery of other valuable resources,

385

such as nitrogen, and the removal of other anions, such as organic pesticides from

386

polluted wastewater for environmental remediation.

387 388

ASSOCIATED CONTENT

389

Supporting Information

390

The Supporting Information is available free of charge on the ACS Publications website.

391

SI Materials and methods; AFM dissolution experimental conditions of brushite

392

(Tables S1-S3); Values of feq (equilibrium force) and Xt (Tables S4-S8); WLC model

393

fitting of a typical FD curve for pulling a single SGAGKT hexapeptide (Figures S1);

394

Flat areas (terraces) of the (010) surfaces of both minerals CaHPO4·2H2O or

395

CaHAsO4·2H2O (Figure S2); Dynamic force spectroscopy measurements (Figure S3);

396

Representative SEM images and XRD patterns of brushite and pharmacolite (Figure

397

S4); Step retreat velocities along the [100]Cc direction in H2O and 10 µM SGAGKT at

398

pH (4.0–8.0) (Figure S5); The relative speciation distributions of phosphate and the

399

SGAGKT hexapeptide (HPep) (Figure S6); Time sequence of AFM deflection images 18


Page 19 of 33


400

of brushite dissolving in H2O at pH 10.0 (Figure S7); Step retreat velocities of etch pits

401

of brushite along the [101]Cc direction (Figure S8); Step retreat velocities of etch pits of

402

brushite along (A) the [101]Cc and (B) [100]Cc directions (Figure S9).

403 404

AUTHOR INFORMATION

405 406

Corresponding Authors

407

*Phone/Fax: +86-27-87288382. E-mails: [email protected];

408

[email protected]

409

ACKNOWLEDGMENTS

410 411

This work was supported by the National Natural Science Foundation of China

412

(41471245 and 41071208) and the Fundamental Research Funds for the Central

413

Universities (2662015PY206 and 2662017PY061). C.V.P. acknowledges funding

414

through the EU seventh Framework Marie S. Curie ITNs: Minsc; CO2 react; and

415

Flowtrans.

416 417 418

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phosphorus cycle. Nature 2017, 541 (7637), 386-389.

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2. Elser, J.; Bennett, E. Phosphorus cycle: a broken biogeochemical cycle. Nature 2011, 478 (7367), 29-31.

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58. Renard, F.; Putnis, C. V.; Montes-Hernandez, G.; Ruiz-Agudo, E.; Hovelmann, J.;

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Sarret, G. Interactions of arsenic with calcite surfaces revealed by in situ nanoscale

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578 579

Figure 1. DFS measurements at the hexapeptide–mica interface. A schematic diagram

580

showing the difference between (A) the P-unbound SGAGKT and (B) the P-bound

581

SGAGKT adsorbed to a mica (001) surface. HPO42- binding to SGAGKT increases the

582

negative charge, and this subsequently results in a decrease in adsorption on mica. (C)

583

Phosphate binding to SGAGKT at different pH values. DFS shows that mean rupture

584

force measured on the (001) face of mica and calculated from the repeated force curve

585

measurements decreased with a decrease in the loading rate. The solid and dash lines

586

denote the fits to the data in the presence and absence of 50 µM Na2HPO4 at pH 4.0-

587

8.0, respectively. (D) The binding free energy calculated with corresponding fitting

588

parameters shows the SGAGKT-P binding occurred at pH 8.0. Values are mean ± SD

589

of three independent sets of experiments. Two stars indicate a significant difference at

590

P < 0.01. The SPSS software was used for data analyses.

591 27



Page 28 of 33

592 593

Figure 2. The SGAGKT-phosphate binding at different IS. (A) Dynamic force

594

spectroscopy and (B) the binding free energy calculated with the fitting parameters in

595

50 µM Na2HPO4 solutions in the presence of different concentrations of NaCl (10-105

596

µM) show an inhibition role on the P-binding at high IS ([NaCl] ≥ 103 µM). Values

597

are mean ± SD of three independent sets of experiments. Different uppercase letters in

598

B indicate significant difference at P < 0.01.

599

28


Page 29 of 33


600 601

Figure 3. Phosphate-binding in the presence of Na2HAsO4. (A) Dynamic force

602

spectroscopy and (B) the calculated binding free energy in 50 µM Na2HPO4, 50 µM

603

Na2HAsO4, or 25 µM Na2HPO4 + 25 µM Na2HAsO4 solutions showing that the

604

SGAGKT hexapeptide preferred to bind to P rather than to As at pH 8.0. Values are

605

mean ± SD of three independent sets of experiments. Different uppercase letters in B

606

indicate significant difference at P < 0.01. (C) DFS measurements carried out on the

607

(010) surfaces of minerals CaHPO4·2H2O or CaHAsO4·2H2O at pH 8.0 showing that

608

SGAGKT binding to the mineral CaHPO4·2H2O was greater than to the mineral

29



Page 30 of 33

609

CaHAsO4·2H2O. All experimental results revealed SGAGKT-P binding with higher

610

selectivity.

611 612

Figure 4. The SGAGKT hexapeptides accelerating the dissolution of the brushite (010)

613

face by enhanced phosphate-binding. (A, B) Time sequence of representative AFM

614

deflection images showing the dissolution of etch pits on the [100]Cc, [101]Cc, and

615

[101]Cc steps on a brushite (010) face after exposure to (A1, A2) H2O at pH 8.0 or (B1,

616

B2) 10 µM SGAGKT at pH 8.0 for 5 min. The dashed lines are the distances between

617

reference points (blue star) and the retreating [101]Cc steps. Dd is the distance difference

618

measured after 5 min (Δt). Step retreat velocities along the [101]Cc direction were

619

calculated by v = Δd/Δt after exposure to H2O or 10 µM SGAGKT solutions at different

620

(C) pH (4.0-8.0), or (D) 10 µM SGAGKT solutions with different concentrations of

621

NaCl (10-105 µM) at pH 8.0, or (E) 10 µM SGAGKT solutions in the presence of 0.1-

622

10 µM NaHAsO4 at pH 8.0. Two stars in C and D and different uppercase letters in E

623

indicate significant difference at P < 0.01.

624

30


Page 31 of 33


625 626

Figure 5. The aggregation of SGAGKT hexapeptides induced by NaCl. Representative

627

AFM height images of 10 µM SGAGKT solutions in the presence of (A) 10 µM, (B)

628

103 µM, and (C) 105 µM NaCl after 60 min. (D) Particle height distributions and (E)

629

the plot of the mean height versus the concentrations of NaCl (10-105 µM) in 10 µM

630

SGAGKT solutions, suggesting that the hexapeptide aggregation (indicated by particle

631

height) was promoted with an increase in NaCl concentration. Mean value ± SD of

632

three independent sets of experiments.

633 634 635

31



Page 32 of 33

636 637

Figure 6. ITC measurements for the phosphate-arsenate selectivity of SGAGKT

638

hexapeptides. The power–time curves for titrations of SGAGKT added into (A) 1 mM

639

Na2HPO4 and (B) 1 mM Na2HAsO4 at pH 8.0 (25 ℃). (C) The total heat and (D) the

640

calculated binding enthalpy for phosphate and arsenate binding to hexapeptides

641

(presented as mean value ± SD, n =3). Different uppercase letters in C and D indicate

642

significant difference at P < 0.01.

643 644 645 646 647 648 649 32


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Dynamics and Molecular Mechanism of Phosphate Binding to a

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