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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
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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].
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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
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amelogenin self-assembly.
44 45
INTRODUCTION
46 47
Tooth enamel, as a highly organized mineralized tissue, is the result of interactions between
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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
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Crystal Growth & Design
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secretion2 and self-assembly3 of matrix proteins, protein-mineral interactions,4 and proteolysis.5-7.
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Amelogenin (Amel) constitutes more than 90% of the total proteins found in the matrix in
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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
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Highly conserved full-length Amel is mainly composed of hydrophobic residues and a relatively
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shorter hydrophilic C-terminus.11,12 This charged C-terminus has significant Ca-P-binding
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affinity,13-16 suggesting that it is able to interact with certain crystal surfaces, thereby controlling
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the morphology of growing enamel crystals. Iijima et al. have shown that the Amel’s C-termini
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can
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Ca8(HPO4)2(PO4)·5H2O),17 causing apparent “elongation” and “thickening” of the crystals.18 A
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combination of the single-molecule force and molecular simulations has demonstrated that the
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C-terminal fragment exhibits a higher binding ability to the (100) face compared to the (001)
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face of hydroxyapatite (HAP, Ca10(PO4)6(OH)2), accounting for the c-axial elongated growth of
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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
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enamelin provide the microenvironment for the nucleation and crystal growth19 through the
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protein–mineral interactions that are a crucial factor underlying the hierarchical structure of
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the organized and elongated ribbons. Previous results have shown that the multi-steps involve
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the formation of HAP from amorphous calcium phosphate (ACP) to OCP to the final product
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of HAP.20-23 As an intermediate metastable phase, OCP structurally resembles HAP24,25
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because HAP epitaxially grows on the (100) face of OCP.26-28 An in situ dissolution study of
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the (100) face of OCP revealed a possibility of the phase transformation from OCP to HAP by
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a pseudomorphic transformation,29 providing a direct clue about the HAP formation via the 3
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OCP intermediate phase.
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Although extensive methods to probe these processes are well established for matrix
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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
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C-termini in controlling surface growth of OCP, a precursor phase of enamel crystals.24 In this
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context, we hypothesize, that such a role is important, but that the nature of assembled or
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disassembled Amel’s C-termini will strongly influence its significance. To test this hypothesis,
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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
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mimetic-physiological conditions. We demonstrated that a nonclassical crystallization
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pathway was exhibited for the OCP surface growth, and a clearly elongated aggregation of
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Ca-P nanoparticles induced by the assemblies of Amel’s C-termini was observed, i.e., the
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crystallographic c axes of OCP were aligned with the long axes of the peptide assemblies.
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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
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Synthesis and Fluorescence Labeling of 13-Mer Amel’s C-Terminal Peptides. The
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C-terminus peptide fragments (Trp-Pro-Ala-Thr-Asp-Lys-Thr-Lys-Arg-Glu-Glu-Val-Asp) of
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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
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liquid chromatography (HPLC).14,30 Moreover, the 13-mer C-terminus peptides containing 4
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Crystal Growth & Design
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fluorescein
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(Trp-Pro-Ala-Thr-Asp-Lys-Thr-Lys-Arg-Glu-Glu-Val-Asp-(FITC)-NH2) were synthesized
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and cleaved by 5 mL of TFA/thioanisole/ethandithiol/anisole (90/5/3/2) and were purified by
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C18 reversed-phase HPLC.31
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OCP Crystal Synthesis. OCP crystals were synthesized by previously reported methods,29,32
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and were characterized as single phase by X-ray diffraction (Bruker D8, Billerica,
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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
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situ AFM surface growth experiments.
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Supersaturated Solutions for OCP Surface Growth. OCP surface growth experiments in
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the absence and presence of Amel’s C-terminus peptides were made in supersaturated
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solutions at 25 °C. The relative supersaturation σ for OCP can be defined as
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isothiocyanate
𝜎=
#$% &'(
−1=𝑆−1
(FITC)
(1)
113
where IAP is the actual ionic activity product, Ksp is its value at equilibrium (the
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thermodynamic solubility product for the given OCP phase,35 -log (𝐾-. ) = 96.6 for OCP at 25
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°C) and 𝑆 is the supersaturation ratio. The thermodynamic database and software of SPEC 01
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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
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sodium chloride (NaCl) (Sigma-Aldrich, St. Louis, Missouri), calcium chloride (CaCl2)
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(Fluka, St. Louis, Missouri) and potassium dihydrogen phosphate (KH2PO4) (Sigma-Aldrich,
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St. Louis, Missouri). The Amel’s C-terminus peptide stock solutions were added to each
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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
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adjusted to 6.5 with 0.8 mol L−1 KOH solution using Metrohm 888 Dosimat Plus (Herisau, 5
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All
supersaturated
solutions
were
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Switzerland).
prepared
using
pure
water
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(resistivity >18MΩ-cm at 25 °C, pH 5.8-6.0) from a two-step purification treatment including
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triple distillation (YaR, SZ-93, Shanghai, China) and deionization (Milli-Q, Billerica, MA,
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USA). The experimental conditions are summarized in Table S1.
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Imaging OCP Surface Growth by In Situ Atomic Force Microscopy (AFM). All in situ
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OCP surface growth experiments were performed using a Bruker MultiMode VIII AFM
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(Santa Barbara, CA) operating either in contact mode or ScanAsyst mode. An optically clear
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OCP crystal was cleaved to expose a fresh (100) surface. The supersaturated (σOCP=1.77, 1.86
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or 1.98, IS = 0.15 mol L-1, pH 6.5) solutions in the absence and presence of Amel peptides
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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
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Albans, Vermont) to ensure surface-controlled reaction rather than diffusion control.36 This
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flow rate does not influence the adsorption of the peptides (1, 50 or 100 nM) on the crystal
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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
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reproducibility of the results, and the images were analyzed using the NanoScope analysis
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software.
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In Situ AFM for Self-Assembly and Disassembly of Amel’s C-Terminus Peptides on
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OCP. Pure peptide solutions (1, 50, or 100 nM in 25 mM Tris-HCL buffer, 25 °C, pH 6.5)
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were passed over OCP crystal surfaces and all in situ AFM observations were imaged in
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ScanAsyst mode. Experiments at each peptide concentration were repeated three times.
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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
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and 100 nM on the OCP (100) surfaces pre-immersed in a supersaturation solution (σ = 1.98).
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In all cases, a 40 × water-immersion objective and a 90/10 mirror as a beam splitter were used.
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All procedures were carried out in the dark.37
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Single-Molecule Force Spectroscopy (SMFS). Force measurements were made with a
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Bruker MultiMode VIII AFM (Bruker, Santa Barbara, CA) using Si3N4 cantilevers with
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triangular levers (Bruker SNL-10, spring constants of 0.06 N/m) in all force determination
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experiments. Details of AFM tip functionalization can be found in ref.13 In brief, new tips
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were immersed in acetone for 30 min, rinsed in ethanol, and then dried under room
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temperature.13 These cleaned tips were coated with 30 nm Au by thermal evaporation, and
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then were immersed in a N, N-dimethylformamide (DMF) (Sigma, St. Louis, Missouri)
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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
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an N-hydroxysuccinimide (NHS) ester that reacts with the N-terminal residue of the peptide
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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
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NHS ester-bearing tips in PBS at pH 6.5. It is necessary to use limited concentration of
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peptide for functionalizing tips in order to acquire a single molecule linking due to the
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possibility of self-assembly of the 13-mer Amel’s C-terminal peptides.13 The functionalized
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tips were rinsed in pure water prior to use.
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Force measurements between modified tips and OCP crystals or mica were performed in
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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
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surface. 7
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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.
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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).
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Our real-time observations reveal that the OCP surface growth is through particle 8
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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
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crystallization.45
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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
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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 :.;
