In Situ Atomic Force Microscopy Imaging of ... - ACS Publications

Mar 21, 2017 - The Institute for Geoscience Research (TIGeR), Curtin University, Perth, Western Australia 6102, Australia. •S Supporting Information...
1 downloads 13 Views 4MB Size
Subscriber access provided by HACETTEPE UNIVERSITESI KUTUPHANESI

Article

In Situ AFM Imaging of Octacalcium Phosphate Crystallization and Its Modulation by Amelogenin’s C-Terminus Shanshan Wu, Menghan Yu, Meng Li, Lijun Wang, Christine V Putnis, and Andrew Putnis Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.7b00129 • Publication Date (Web): 21 Mar 2017 Downloaded from http://pubs.acs.org on March 23, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Crystal Growth & Design is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

Crystal Growth & Design

In Situ AFM Imaging of Octacalcium Phosphate Crystallization and Its Modulation by Amelogenin’s C-Terminus

2 3

Shanshan Wu,† Menghan Yu,† Meng Li,† Lijun Wang,*, † Christine V. Putnis,‡,§ and

4

Andrew Putnis‡,¶

5 6 †

7

College of Resources and Environment, Huazhong Agricultural University, Wuhan

8

430070, China ‡

9 10 11

§

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

Department of Chemistry, Curtin University, Perth, Western Australia 6845, Australia ¶

The Institute for Geoscience Research (TIGeR), Curtin University, Perth, Western

12

Australia 6102, Australia

13 14

Corresponding author

15

*College of Resources and Environment, Huazhong Agricultural University, 1 Shizishan St.,

16

Wuhan 430070, China. Phone/Fax: +86-27-87288382. E-mail: [email protected].


17 18 19 20 21 22 23 24 1

ACS Paragon Plus Environment

Crystal Growth & Design

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

25

Page 2 of 36

ABSTRACT

26 27

Amelogenin proteins play a critical role in controlling crystal growth and orientation into the

28

highly organized calcium phosphate (Ca-P) minerals during tooth enamel formation. However,

29

real-time observations for understanding the kinetics and mechanisms of Ca-P surface

30

crystallization and its modulation by amelogenin have been lacking. We monitor the kinetics of

31

the (100) surface growth of octacalcium phosphate (OCP) with precisely defined

32

thermodynamic driving forces in the presence of amelogenin’s C-terminus peptides inside a

33

fluid cell of an atomic force microscope (AFM) with a controlled near-physiological

34

environment. During in situ growth via a nonclassical particle attachment pathway, an obviously

35

elongated aggregation of Ca-P nanoparticles induced by the assembly of amelogenin’s C-termini

36

was observed. The nanostructured fibrous assemblies, reminiscent of extracellular matrix, are

37

able to bind Ca-P nanoparticles and direct OCP mineralization. This was analyzed and

38

rationalized through single-molecule determination of the binding free energy of the C-terminal

39

fragment adsorbed to the (100) face of OCP. Combining in situ growth kinetics with force

40

spectroscopy reveals the shape evolution from spherical particles to elongated nanorods

41

resembling the nanostructure of enamel crystallites. The findings improve the fundamental

42

understanding of natural biomineralization through nonclassical crystallization routes and

43

amelogenin self-assembly.

44 45

INTRODUCTION

46 47

Tooth enamel, as a highly organized mineralized tissue, is the result of interactions between

48

matrix proteins and calcium phosphate (Ca-P) mineral surfaces, and its formation occurs in the

49

extracellular matrix of a growing tooth through complex cellular and molecular events,1 such as 2

ACS Paragon Plus Environment

Page 3 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Crystal Growth & Design

50

secretion2 and self-assembly3 of matrix proteins, protein-mineral interactions,4 and proteolysis.5-7.

51

Amelogenin (Amel) constitutes more than 90% of the total proteins found in the matrix in

52

developing enamel.1,8 Much evidence shows that Amel proteins self-assemble into nanospheres

53

and nanochains, playing a potential role in guiding nucleation and growth of Ca-P crystals.9,10

54

Highly conserved full-length Amel is mainly composed of hydrophobic residues and a relatively

55

shorter hydrophilic C-terminus.11,12 This charged C-terminus has significant Ca-P-binding

56

affinity,13-16 suggesting that it is able to interact with certain crystal surfaces, thereby controlling

57

the morphology of growing enamel crystals. Iijima et al. have shown that the Amel’s C-termini

58

can

59

Ca8(HPO4)2(PO4)·5H2O),17 causing apparent “elongation” and “thickening” of the crystals.18 A

60

combination of the single-molecule force and molecular simulations has demonstrated that the

61

C-terminal fragment exhibits a higher binding ability to the (100) face compared to the (001)

62

face of hydroxyapatite (HAP, Ca10(PO4)6(OH)2), accounting for the c-axial elongated growth of

63

enamel crystals.13

dramatically

change

the

crystal

shape

of

octacalcium

phosphate

(OCP,

64

At the early stages of the enamel formation, initial enamel crystals were detected

65

separate from the adjacent dentine, and electron-microprobe analyses revealed that early

66

enamel crystals were OCP or tricalcium phosphate.19 Supramolecular aggregates of Amel and

67

enamelin provide the microenvironment for the nucleation and crystal growth19 through the

68

protein–mineral interactions that are a crucial factor underlying the hierarchical structure of

69

the organized and elongated ribbons. Previous results have shown that the multi-steps involve

70

the formation of HAP from amorphous calcium phosphate (ACP) to OCP to the final product

71

of HAP.20-23 As an intermediate metastable phase, OCP structurally resembles HAP24,25

72

because HAP epitaxially grows on the (100) face of OCP.26-28 An in situ dissolution study of

73

the (100) face of OCP revealed a possibility of the phase transformation from OCP to HAP by

74

a pseudomorphic transformation,29 providing a direct clue about the HAP formation via the 3

ACS Paragon Plus Environment

Crystal Growth & Design

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

75

Page 4 of 36

OCP intermediate phase.

76

Although extensive methods to probe these processes are well established for matrix

77

proteins and their control over Ca-P crystallization, real-time observations for understanding

78

the kinetics and mechanisms of in situ OCP surface crystallization and its modulation by

79

Amel have been lacking. The aim of the present study is to explore the role of Amel’s

80

C-termini in controlling surface growth of OCP, a precursor phase of enamel crystals.24 In this

81

context, we hypothesize, that such a role is important, but that the nature of assembled or

82

disassembled Amel’s C-termini will strongly influence its significance. To test this hypothesis,

83

a series of in situ AFM experiments combined with the single-molecule force determination

84

were performed, in which an OCP (100) surface interacted with different concentrations of

85

Amel’s C-terminus peptides in various supersaturated solutions with respect to OCP under

86

mimetic-physiological conditions. We demonstrated that a nonclassical crystallization

87

pathway was exhibited for the OCP surface growth, and a clearly elongated aggregation of

88

Ca-P nanoparticles induced by the assemblies of Amel’s C-termini was observed, i.e., the

89

crystallographic c axes of OCP were aligned with the long axes of the peptide assemblies.

90

However, relatively high concentrations of Amel’s C-terminus peptides had no effect due to

91

the disassembly of peptide oligomers/particles induced by the OCP crystal surfaces.

92 93

EXPERIMENTAL SECTION

94 95

Synthesis and Fluorescence Labeling of 13-Mer Amel’s C-Terminal Peptides. The

96

C-terminus peptide fragments (Trp-Pro-Ala-Thr-Asp-Lys-Thr-Lys-Arg-Glu-Glu-Val-Asp) of

97

Amel were synthesized according to standard procedures of solid phase peptide synthesis (GL

98

Biotechem, Shanghai, China) and were purified by C18 reversed-phase high-performance

99

liquid chromatography (HPLC).14,30 Moreover, the 13-mer C-terminus peptides containing 4

ACS Paragon Plus Environment

Page 5 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Crystal Growth & Design

100

fluorescein

101

(Trp-Pro-Ala-Thr-Asp-Lys-Thr-Lys-Arg-Glu-Glu-Val-Asp-(FITC)-NH2) were synthesized

102

and cleaved by 5 mL of TFA/thioanisole/ethandithiol/anisole (90/5/3/2) and were purified by

103

C18 reversed-phase HPLC.31

104

OCP Crystal Synthesis. OCP crystals were synthesized by previously reported methods,29,32

105

and were characterized as single phase by X-ray diffraction (Bruker D8, Billerica,

106

Massachusetts) (Figure S1). Rietveld refinement was performed using the structural model of

107

OCP (JCPDS, PDF# 44-077833, 34). Synthetic OCP crystals as seed substrates were used for in

108

situ AFM surface growth experiments.

109

Supersaturated Solutions for OCP Surface Growth. OCP surface growth experiments in

110

the absence and presence of Amel’s C-terminus peptides were made in supersaturated

111

solutions at 25 °C. The relative supersaturation σ for OCP can be defined as

112

isothiocyanate

𝜎=

#$% &'(

−1=𝑆−1

(FITC)

(1)

113

where IAP is the actual ionic activity product, Ksp is its value at equilibrium (the

114

thermodynamic solubility product for the given OCP phase,35 -log (𝐾-. ) = 96.6 for OCP at 25

115

°C) and 𝑆 is the supersaturation ratio. The thermodynamic database and software of SPEC 01

116

were used for the calculations of the activities. A range of supersaturated solutions (𝜎/01 =

117

1.77–1.98, pH = 6.50, and an ionic strength (IS) = 0.15 M) were prepared by slowly mixing of

118

sodium chloride (NaCl) (Sigma-Aldrich, St. Louis, Missouri), calcium chloride (CaCl2)

119

(Fluka, St. Louis, Missouri) and potassium dihydrogen phosphate (KH2PO4) (Sigma-Aldrich,

120

St. Louis, Missouri). The Amel’s C-terminus peptide stock solutions were added to each

121

supersaturated solution to make the peptide concentrations at 1, 50, or 100 nM prior to pH

122

adjustment. The peptide stock solution was prepared by dissolving 1 mg lyophilized peptides

123

in 100 mL water and then was diluted into 10 mM Tris-HCl buffer. The pH value was finally

124

adjusted to 6.5 with 0.8 mol L−1 KOH solution using Metrohm 888 Dosimat Plus (Herisau, 5

ACS Paragon Plus Environment

Crystal Growth & Design

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

All

supersaturated

solutions

were

Page 6 of 36

125

Switzerland).

prepared

using

pure

water

126

(resistivity >18MΩ-cm at 25 °C, pH 5.8-6.0) from a two-step purification treatment including

127

triple distillation (YaR, SZ-93, Shanghai, China) and deionization (Milli-Q, Billerica, MA,

128

USA). The experimental conditions are summarized in Table S1.

129

Imaging OCP Surface Growth by In Situ Atomic Force Microscopy (AFM). All in situ

130

OCP surface growth experiments were performed using a Bruker MultiMode VIII AFM

131

(Santa Barbara, CA) operating either in contact mode or ScanAsyst mode. An optically clear

132

OCP crystal was cleaved to expose a fresh (100) surface. The supersaturated (σOCP=1.77, 1.86

133

or 1.98, IS = 0.15 mol L-1, pH 6.5) solutions in the absence and presence of Amel peptides

134

were passed over the cleaved OCP crystals inside the AFM fluid cell at a constant flow rate of

135

0.5-1.0 mL/min using a syringe pump (Razel Scientific Instruments model R100-E, Saint

136

Albans, Vermont) to ensure surface-controlled reaction rather than diffusion control.36 This

137

flow rate does not influence the adsorption of the peptides (1, 50 or 100 nM) on the crystal

138

faces. AFM images were collected using Si3N4 tips (Bruker DNP-S10, spring constants of

139

0.12-0.35 N/m or ScanAsyst-Fluid + with a spring constant of 0.7 N/m) with scan rates of 2-4

140

Hz. Measurements were made on more than three crystals per solution composition to ensure

141

reproducibility of the results, and the images were analyzed using the NanoScope analysis

142

software.

143

In Situ AFM for Self-Assembly and Disassembly of Amel’s C-Terminus Peptides on

144

OCP. Pure peptide solutions (1, 50, or 100 nM in 25 mM Tris-HCL buffer, 25 °C, pH 6.5)

145

were passed over OCP crystal surfaces and all in situ AFM observations were imaged in

146

ScanAsyst mode. Experiments at each peptide concentration were repeated three times.

147

Scanning Confocal Interference Microscopy (SCIM). Leica TCS SP8 SCIM (Wetzlar,

148

Hesse, Germany) using a helium/neon laser (λ= 632.8 nm) or a krypton/argon laser (λ= 488

149

nm) was used to image the adsorption of FITC labeled Amel’s C-terminal peptides at 1, 50 6

ACS Paragon Plus Environment

Page 7 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Crystal Growth & Design

150

and 100 nM on the OCP (100) surfaces pre-immersed in a supersaturation solution (σ = 1.98).

151

In all cases, a 40 × water-immersion objective and a 90/10 mirror as a beam splitter were used.

152

All procedures were carried out in the dark.37

153

Single-Molecule Force Spectroscopy (SMFS). Force measurements were made with a

154

Bruker MultiMode VIII AFM (Bruker, Santa Barbara, CA) using Si3N4 cantilevers with

155

triangular levers (Bruker SNL-10, spring constants of 0.06 N/m) in all force determination

156

experiments. Details of AFM tip functionalization can be found in ref.13 In brief, new tips

157

were immersed in acetone for 30 min, rinsed in ethanol, and then dried under room

158

temperature.13 These cleaned tips were coated with 30 nm Au by thermal evaporation, and

159

then were immersed in a N, N-dimethylformamide (DMF) (Sigma, St. Louis, Missouri)

160

solution containing 0.2 mM of the heterobifunctional cross-linker LC-SPDP (Thermo

161

Scientific, Waltham, Massachusetts) consisting of a pyridyl disulfide that adsorbs to Au, and

162

an N-hydroxysuccinimide (NHS) ester that reacts with the N-terminal residue of the peptide

163

through nucleophilic attack to form a stable covalent bond.13 After rinsing in DMF, followed

164

by ethanol, the tips were immersed overnight in an Amel’s C-terminal peptide solution at 40

165

nM in phosphate buffer solutions (PBS).13 Finally, the peptide was anchored linking to the

166

NHS ester-bearing tips in PBS at pH 6.5. It is necessary to use limited concentration of

167

peptide for functionalizing tips in order to acquire a single molecule linking due to the

168

possibility of self-assembly of the 13-mer Amel’s C-terminal peptides.13 The functionalized

169

tips were rinsed in pure water prior to use.

170

Force measurements between modified tips and OCP crystals or mica were performed in

171

PBS at pH 6.5 using a Bruker MultiMode VIII AFM (Santa Barbara, CA). Details of force

172

measurements and the theoretical analyses of binding free energies can be found in refs.13,38

173

Force curves were measured for each velocity at 256 locations in 2×2 µm2 on the crystal

174

surface. 7

ACS Paragon Plus Environment

Crystal Growth & Design

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 36

175

Dynamic Light Scattering (DLS). DLS was performed using a peptide sample that was

176

filtered into a flow cell of the instrument Zatasizer Nano ZS90 (Malvern, Worcestershire, UK)

177

at room temperature. Peptide solutions at concentrations of 1, 50 or 100 nM were prepared by

178

dissolution of the lyophilized 13-mer Amel’s C-terminal peptides in 10 mM Tris-HCl buffer

179

(Aldrich, St. Louis, Mossorui) (IS = 0.15 M, and pH 6.5) in the absence and presence of 4.1

180

mM CaCl2 and stored at 4 °C prior to the experiments.39-41 The data were analyzed by using

181

Zetasizer sotfware.

182 183

RESULTS AND DISCUSSION

184 185

Growing OCP Crystals by Attaching Particles in Pure Supersaturated Solutions. We

186

used in situ AFM in ScanAsyst mode to observe the OCP (100) growth (Figure 1A) in order

187

to minimize the potential dislodgement/removal of particles caused by the movement of the

188

AFM tip. At σ = 1.77, AFM images showed that the sizes of spherical particles in both width

189

and length gradually increased with reaction times, from 78.12 ± 39.19 nm to 178.31 ±

190

35.34 nm in length; from 79.70 ± 18.62 nm to 167.90 ± 11.24 nm in width after 100 and

191

500 min of growth, respectively (Figure 2B). This demonstrated that the ratio of length over

192

width of forming particles remained at about 1 (Figure 2C). Some aggregated

193

pancake-shaped particles formed after 150 min of growth (Figure 1A). When ScanAsyst

194

mode was changed to contact mode, these aggregated pancake-shaped particles were

195

extensively detected even after 540 min of growth (Figure 1B). The height (about 2.0 nm) of

196

the particles grown on the OCP (100) surface within the AFM experimental time frame

197

(0-540 min), regardless of the imaging mode, remained almost constant at different

198

supersaturations ranging from σ = 1.77 to 1.98 (Figure 2A).

199

Our real-time observations reveal that the OCP surface growth is through particle 8

ACS Paragon Plus Environment

Page 9 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Crystal Growth & Design

200

attachment and aggregation in pure supersaturated solutions (Figure 1A and B). Crystals

201

grow in a number of ways, including pathways involving the assembly of other particles and

202

multi-ion complexes.42 In the present investigations, in situ AFM results revealed that

203

primary particles (amorphous or crystalline) exist throughout crystallization processes,

204

implying that crystallization by particle attachment (CPA)42-44 is a prevalent growth

205

mechanism, especially at early stages of OCP surface crystallization. The consistency of

206

particle height further suggests a characteristic of OCP crystallization by the attachment and

207

fusion of primary particles with a height of about 2-3 nm, in a good agreement with the

208

observation of the heights of the primary particles formed during HAP surface

209

crystallization.45

210

Elongated Growth in the Presence of Amel’s C-Terminal Peptides. At concentrations of

211

1, 50 and 100 nM Amel’s C-terminal peptides in supersaturated solutions (σ = 1.77, 1.86 and

212

1.98), we also observed, at the earliest stages, the formation of stable Ca-P nanoparticles

213

with heights of about 2 nm (Figures 3A and 4A). After 360 min, particle elongation with an

214

aspect ratio of about 2:1 occurred in the presence of 50 nM Amel’s C-terminal peptides

215

(Figures 3A and 4C). Compared to the presence of 1 or 100 nM Amel’s C-terminal peptides

216

(Figures S2 and S3), the particle lengths grown in the presence of 50 nM Amel’s C-terminal

217

peptides at σ = 1.77, 1.86 and 1.98 increased to 182.7±46.0 nm, 246.3±93 nm and 298.1±

218

37.4 nm, respectively, whereas the widths remained at 103.8 ±33.1 nm, 114.0 ± 37.0 nm

219

and 143.5 ± 25.6 nm, respectively (Figures 4B, S4 and S5). During the growth, the heights

220

of particles remained constant at about 2.0 nm at all peptide concentrations and

221

supersaturations tested (Figure 4A). Using contact mode, elongated particles became more

222

evident after 300 min (Figures 3B, 4C and S6) in all supersaturated solutions containing 50

223

nM Amel’s C-terminal peptides.

224

The Role of Amel’s C-Terminal Peptides in Particle Attachment. To further understand 9

ACS Paragon Plus Environment

Crystal Growth & Design

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 36

225

how elongated Ca-P particles formed in the presence of Amel’s C-terminal peptides only at

226

50 nM, we used in situ AFM to observe the size and morphology of pure Amel’s C-terminal

227

peptides adsorbed onto the (100) face of OCP (in the absence of OCP supersaturated

228

solutions). As shown in Figure 5, discrete peptide nanoparticles with heights of about 1.1 nm,

229

3.0-4.0 nm and 2.0-3.0 nm formed at concentrations of 1, 50 and 100 nM, respectively (Figure

230

5D and E). Interestingly, only at 50 nM concentrations, these particles connected to each other

231

(as shown within the blue rectangles in Figure 5B2) to form elongated nanorod-like

232

assemblies (Figure 5B3 and B4) with an aspect ratio of about 2:1 (Figure 5F). The

233

disassembly of relatively large spherical peptide particles was observed at 100 nM (Figure 5C)

234

and the height of particles gradually decreased to about 1.2 nm from 3.0 nm while the aspect

235

ratio was kept at about 1:1 (Figure 5F). Following the adsorption of 1 nM peptides on the

236

OCP (100) surface, no aggregated particles formed (Figure 5A). This was identified by

237

fluorescence imaging using SCIM to observe the adsorption of the Amel’s C-terminal

238

peptides modified by FITC on the OCP (100) crystal surfaces (Figure 6), and results showed

239

that only at 50 nM, elongated and oriented peptides with green fluorescence were observed

240

(Figure 6B), whereas at 1 nM and 100 nM no elongated peptide assembles on the OCP (100)

241

crystal surface were seen (Figure 6A and C).

242

Real-time AFM images as seen in Figure 5B show the adsorption of 50 nM Amel

243

peptides on OCP, and then these adsorbed peptides assembled into well-aligned nanorods. The

244

height of primary peptide particles (about 2.2 nm) corresponds to the theoretical

245

hydrodynamic radius RH of the dimer and trimer of the 13-mer Amel’s C-termini (26 residues)

246

according to the equation 𝑅3 = 4.75 ± 1.11 𝑁 :.;