Simple Method of Synthesizing Nickel–Nitrilotriacetic Acid Gold Nanoparticles with a Narrow Size Distribution for Protein Labeling

Abstract

We developed a simple method to synthesize nickel–nitrilotriacetic acid gold nanoparticles (Ni–NTA Au NPs) with a narrow size distribution for site-specific labeling in protein complexes. Au NPs were synthesized by the reduction of HAuCl4 using trisodium citrate and tannin acid. Then, the nanoparticle surfaces were modified with NTA and subsequent complexation with Ni2+. The mean diameter of the synthesized Ni–NTA Au NPs was 4.3 nm, and the coefficient of variation was 9%. The specific binding of the Ni–NTA Au NPs to polyhistidine-tagged (His-tagged) proteins was determined by transmission electron microscopy using kinesin and the p62 subunit of dynactin. Consequently, our method is useful for analyzing the substructures of protein complexes.

Domain organization and conformational change of dynactin p150

“…p150 mutants carry the His-tag at respective locations: the N-and C-termini of p150, both sides and the hinge of CC1, and both sides of CC2 (Figure 2A, Figure 1-S1). The efficiency and specificity of nanogold labeling of the human dynactin used in this study has been reported previously (Kitai et al, 2011).…”

“…A streptavidin binding peptide (SBP) was fused to one of the subunits (Figure 1-S1) and all the other subunit components of the dynactin complex were co-purified by affinity chromatography (Figure 1-S2). We firstly used a complex including recombinant p62 (SBP-p62 in Figure 1-S1), which locates at the pointed end of the Arp1 rod (Kitai et al, 2011;Schafer et al, 1994), to obtain an intact form of the dynactin sidearm. The negative stain EM images revealed the sidearm complex projecting from one end of the Arp1 rod ( Figure 1A; Figure 1-S3A, left).…”

“…Chemically modified gold nanoparticles visualize the tagged sites within protein complexes (Ichikawa et al, 2015;Guesdon et al, 2016;Song et al, 2014). Using gold nanoparticles with diameter less than 5 nm (Kitai et al 2011), the specific sites within ~30 nm CC1-protrusion (Figure 3, Figure 3-S1) and ~20 nm CC2-neck (Figure 4) were clearly discernible. This provided sufficient information to determine the folding pattern and the domain organization of p150 in the sidearm through the observation of individual dynactin molecules.…”

Genetically Programmable Thermoresponsive Plasmonic Gold/Silk-Elastin Protein Core/Shell Nanoparticles

“…42 The responsive self-assembly characteristics of Au-S4E8Gs are based on the thermally reversible conformational switching in the elastin-like block between the hydrophilic random coil conformation and the hydrophobic β-spiral adopting repetitive type II β-turns. 26−28,31 …”

“…39−41 The synthesis strategy following the method reported by Kitai et al 42 and is shown in Figure S2. The XPS characterization of the Au NPs at various steps of the reaction is summarized in Figure S3.…”