doi:10.1016/j.jmb.2005.10.026 
Copyright © 2005 Elsevier Ltd All rights reserved.
Gold Nanocluster Formation using Metallothionein: Mass Spectrometry and Electron Microscopy
Christopher P. Mercoglianoa and David J. DeRosier
, a, 
aDepartment of Biology, Brandeis University, 415 South Street, Waltham, MA 02454, USA
Received 6 July 2005;
revised 4 October 2005;
accepted 13 October 2005.
Edited by W. Baumeister.
Available online 8 November 2005.
References and further reading may be available for this article. To view references and further reading you must
purchase this article.
Clonable contrasting agents for light microscopy, such as green fluorescent protein, have revolutionized biology, but few such agents have been developed for transmission electron microscopy (TEM). As an attempt to develop a novel clonable contrasting agent for TEM, we have evaluated metallothionein, a small metal-binding protein, reacted with aurothiomalate, an anti-arthritic gold compound. Electro spray ionization and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry measurements show a distribution of gold atoms bound to individual metallothionein molecules. Unlike previous reports, these data show gold binding occurred as the addition of single atoms without retention of additional ligands. Moreover, under certain conditions, MALDI spectra show gold binding ratios of greater than 1:1 with the cysteine residues of metallothionein. Together, this may hint at a gold-binding mechanism similar to gold nanocluster formation. Finally, metallothionein–gold complexes visualized in the TEM show a range of sizes similar to those used as current TEM labels, and show the potential of the protein as a clonable TEM label in which the gold cluster is grown on the label, thereby circumventing the problems associated with attaching gold clusters.
Keywords: metallothionein; mass spectrometry; transmission electron microscopy; nanocluster; gold tag
Abbreviations: TEM, transmission electron microscopy; MT, metallothionein; EXAFS, extended X-ray absorption fine structure; ESI, electrospray ionization; MS, mass spectrometry; MALDI, matrix-assisted laser desorption/ionization
Figure 1. ESI mass spectroscopy of gold-incubated metallothioneins. Sample raw mass spectra and their corresponding calculated zero-charged spectra are displayed in (a) and (b) for apo-MT, (c) and (d) for Zn7-MT, and in (e) and (f) for aurothiomalate-incubated MT. In (a), (c) and (e), peak labels designate the oligomeric state and charge-state of the protein. A peak labelled with an M corresponds to a monomer, while a peak labelled with a D corresponds to a dimer. The number in parentheses reflects the ionized charge-state of the protein in that peak. In the zero-charge spectra in (b), (d) and (f), mass values corresponding to the maximum peak amplitudes are printed just to the right of each of the peaks of interest, except for 8868 amu, which is just to the left of the peak.
Figure 2. MALDI mass spectroscopy of gold-incubated metallothioneins. MALDI mass spectra collected (a) for apo-MT and (b)–(d) aurothiomalate-incubated MT are displayed. In each case, the labelled major peak corresponds to a monomeric species and the labelled minor peak corresponds to the dimeric species. The values shown signify the mass value at the maximum peak amplitude of each peak. Upon an increase in gold to cysteine molar ratio from (b) 1:1 to (c) and (d) 10:1, the peak and distribution shift to higher mass including values corresponding to greater than one gold atom per cysteine residue within the protein.
Figure 3. MALDI mass spectra suggesting the mode of gold binding. Distributions recorded by MALDI mass spectroscopy of aurothiomalate-incubated MT occasionally displays an underlying high frequency signal ((a) and (c)). To better identify the nature of this weak, underlying signal, Fourier transforms of each of these selected spectra were calculated and are shown in (b) and (d), respectively. During both incubation with a (a) and (b) 1:1 (A and B) and (c) and (d) a 10:1 (C and D) gold to cysteine molar ratio, a similar high-frequency component results in a peak at about 0.005 in each of these transform ((b) and (d)). This corresponds to steps of about every 200 mass units, which, given the lesser accuracy of MALDI-ES, corresponds well to the 196 amu steps witnessed with ESI-MS.
Figure 4. An example of aurothiomalate inhibition and penicillamine re-activation. The ability of aurothiomalate to inhibit nucleoprotein filament formation of RecA and the ability of penicillamine to re-activate the protein was evaluated using a mobility-shift assay. Lane 1 shows a control sample of 1000 bp dsDNA that migrates more quickly through the 0.8% agarose gel than the RecA-coated DNA in lane 6. Pre-incubation of RecA with aurothiomalate results in improper nucleoprotein filament formation, as shown by the extended DNA smear seen in lane 5. Incubation of aurothiomalate-reacted RecA with increasing concentrations of penicillamine can restore proper nucleoprotein filament formation as witnessed in lanes 2–4.
Figure 5. Limited effects of penicillamine on aurothiomalate-incubated metallothionein. To evaluate the effects of penicillamine and the stability of aurothiomalate-incubated metallothionein samples, MALDI mass spectrometry was used to monitor the amount of gold bound by the protein. The values shown signify the mass value at the maximum peak amplitude of each peak. Apo-MT and aurothiomalate control samples are shown in (a) and (b), respectively. (c) The shift of MT to higher molecular mass upon gold binding. (d) Incubation of this sample with 20 mM penicillamine followed by desalting shifts the peak only slightly toward a lower molecular mass.
 |
Figure 6. TEM images of aurothiomalate-incubated metallothionein. Samples of aurothiomalate-incubated MT were evaluated as a potential TEM label by imaging them on a thin carbon foil suspended on a copper TEM grid. In these images, gold clusters appear as black spots, since they scatter electrons more strongly compared to the lower atomic number atoms found in proteins or the carbon support layer. As negative controls, samples prepared from (a) buffer alone, (b) and (c) buffer with aurothiomalate, and (e) Zn7-MT were prepared, and these show no noticeable black, spots except for the occasional large cluster witnessed in (c) (see the text for an explanation). (f) Conversely, micrographs of aurothiomalate-incubated MT reveal numerous small, varying sized clusters, thus showing the potential of MT as a clonable TEM label. For comparison, (d) shows a positive control sample containing the commercially available TEM label, Nanogold®, in which an individual cluster should be 1.4 nm. Note that the clusters in (f) are both smaller and larger than those in (d).
Table 1.
Expected mass values for gold-containing metallothionein
This Table contains mass values for metallothionein containing different numbers of gold atoms. The values are calculated by the formula: 
where Mapo-metallothionein=6125, Ngold is the number of gold atoms, Mgold=197, and Mhydrogen=1.
Abstract
Purchase PDF (648 K)
E-mail Article
Add to my Quick Links
Cited By in Scopus (8)
Purchase PDF (318 K)
400°C), silicon diffusion towards the surface is promoted and a thicker SiO2 layer can be grown on top of the substrate. In the case of the oxidation performed at temperature higher than 400°C, the results are similar to the one obtained on a clean surface. At these temperatures, the metallic film agglomerates into tridimensional crystallites on top of a very thin Au---Si alloyed layer. The fact that the latter has no influence on the oxidation is attributed to the different local arrangement of atoms at the sample surface.
