Impact of multivalent charge presentation on peptide–nanoparticle aggregation

Summary Strategies to achieve controlled nanoparticle aggregation have gained much interest, due to the versatility of such systems and their applications in materials science and medicine. In this article we demonstrate that coiled-coil peptide-induced aggregation based on electrostatic interactions is highly sensitive to the length of the peptide as well as the number of presented charges. The quaternary structure of the peptide was found to play an important role in aggregation kinetics. Furthermore, we show that the presence of peptide fibers leads to well-defined nanoparticle assembly on the surface of these macrostructures.


Experimental procedures
Au/MUA nanoparticle synthesis [1,2] Dodecanethiol (22 µL; 3.75 mmol) and AuClPPh 3 (20 mg; 0.04 mmol) were dissolved in benzene (3.2 mL). Borane-tert-butylamine complex (35 mg; 0.40 mmol) was added and the solution was stirred at room temperature for 2 hours. Methanol (10 mL) was added and the black precipitant was separated by centrifugation. The precipitate was washed 3 times with methanol (15 mL) and then dried under reduced pressure. The obtained powder was dissolved in 1.5 mL chloroform and slowly added to a solution of mercaptoundecanoic acid (MUA) (200 mg; 0.92 mmol) in THF (5 mL). After stirring for two days at room temperature a black precipitate was formed which was separated by centrifugation. The precipitate was washed with THF to remove the excess of MUA s2 and dried under reduced pressure. The obtained black powder was dissolved in Tris/HCl pH 11 (5 mL) and further purified by dialysis for three times.

Peptide synthesis and sample preparation
The peptides were synthesized by hand using standard Fmoc solid-phase peptide synthesis. Every amino acid was double coupled using 8 equiv HOBt, 8 equiv DIC and 8 equiv of the corresponding amino acid. To determine the peptide concentration the N-terminus was labeled with UV-active aminobenzoic acid (Abz). The peptides were cleaved from the resin using 95% TFA, 2.5% TIPS and 2.5% water, precipitated with ice cold ether and purified by reversed phase HPLC. The identification of the peptide was carried out by ESI-TOF.
Peptides were dissolved in freshly filtered Tris/HCl buffer (10 mM, pH 9). The peptide concentration was determined using UV-vis spectroscopy by measuring the absorbance at a wavelength of 314 nm. A calibration curve of glycine coupled to aminobenzoic acid (Abz-Gly) dissolved in the same buffer was used as a reference.

UV-vis spectroscopy
UV-vis spectra were measured on a CARY 50 Bio spectrophotometer (VARIAN, Darmstadt, Germany). The concentration of Au/MUA nanoparticles was calculated by measuring the absorption at 506 nm [3], was set to 50 nM. The final peptide concentration was set to 5 to 30 µM. UV-vis spectra were recorded right after addition of peptide in Tris/HCl buffer at pH 9 at room temperature using disposable semi-micro PMMA cuvettes (PlastiBRAND ® , Brand GmbH, Wertheim, Germany). The spectra were taken from 450 to 750 nm in steps of 0.25 nm. All spectra were corrected by the buffer spectra. The images were on Kodak SO-163 negative film. The accelerating voltage was 100 kV. A primary magnification of 60 k and a defocus of -1200 nm or -1800 was used.
s4 CD spectroscopy CD spectra were recorded on a J-810 spectrophotometer (Jasco GmbH, Gross-Umstadt, Germany) using a 2 mm quartz cuvette. All spectra are taken as the average of 3 single scan from 190 to 240 nm in steps of 0.5 nm at 20 °C. All spectra were corrected by the buffer spectra.

Isothermal titration calorimetry (ITC)
ITC measurements were carried out on a VP-ITC MicroCal instrument (

Agarose gel electrophoresis
Agarose gel electrophoresis was carried out Gel XL Ultra V-2 Electrophoresis System (Labnet International, Windsor, Great Britain) using 0.5 % agarose gel in Tris/HCl buffer (10 mM, pH 9) with a accelerating voltage of 100 V. The electrophoresis was stopped after 15 to 20 minutes.