Hydroxylamine

Tetraethylorthosilicate (TEOS), tetrakis (hydroxymethyl) phosphonium chloride (THPC), 3-aminopropyltrimethoxysilane (APTMS), sodium hydroxide, hydroxylamine (50% in H₂O), potassium carbonate and chloroauric acid tetrahydrate (HAuCl₄^.4H₂O) were obtained from Sigma-Aldrich. Ammonium hydroxide solution (30%) was purchased from Merck Company. All the reagents were analytical grade and used as received.

The Site 2 and Sites 2,4 peptides were fluorescently labeled at the amidated C-terminus. Both peptides were labeled with the donor probe Alexa Fluor 488 NHS Ester, whereas the Site 2 peptide was additionally labeled with the acceptor probe Alexa Fluor 568 NHS Ester (Thermo Fisher Scientific). The labeling reaction was performed at 25 °C for 1 h in 20 mM MES pH 6.8 and 50 mM NaCl and at a peptide concentration of 5 mg mL⁻¹ with a 2-fold molar excess of fluorescent dye. The labeling reaction was stopped with the addition of a 10-fold molar excess of hydroxylamine (Sigma-Aldrich Corporation). Labeled peptides were separated from any remaining unlabeled peptide using a Symmetry300 C₁₈ reverse phase HPLC column (Waters Corporation, Milford, MA). The peptides were lyophilized, resuspended in 100 mM HEPES pH 7.5, and dialyzed against 25 mM HEPES pH 7.5, 50 mM NaCl and 1 mM MgCl₂. The donor-labeled peptides were pre-incubated with 14-3-3θ at a 1:1 molar ratio (i.e., one peptide per 14-3-3θ dimer). The acceptor-labeled Site 2 peptide (at 5 μM concentration) was then titrated into the preformed complexes. The steady-state fluorescence emission spectra were recorded at 25 °C, with an excitation wavelength of 495 nm (5-nm slit width) and an emission wavelength of 520 nm (5-nm slit width).