Gold Nanocluster Formation using Metallothionein: Mass Spectrometry and Electron Microscopy

Mercogliano, Christopher P.; DeRosier, David J.

doi:10.1016/j.jmb.2005.10.026

Cited by 67 publications

(54 citation statements)

References 34 publications

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“…Extended X-ray absorption fine structure (EXAFS) studies on the mercury complex suggested that metal atoms were bridged between two sulphurs with no additional ligand and that the protein may have refolded into a single domain (Jiang et al, 1994). Gold appears to do the same, and indeed we found that up to 40 gold atoms may at times be bound to a single MT subunit (Mercogliano and DeRosier, 2006).…”

Section: Introductionsupporting

confidence: 56%

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Concatenated metallothionein as a clonable gold label for electron microscopy

Mercogliano

DeRosier

2007

Journal of Structural Biology

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109

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Localization of proteins in cells or complexes using electron microscopy has mainly relied upon the use of heavy metal clusters, which can be difficult to direct to sites of interest. For this reason, we would like to develop a clonable tag analogous to the clonable fluorescent tags common to light microscopy. Instead of fluorescing, such a tag would initiate formation of a heavy metal cluster. To test the feasibility of such a tag, we exploited the metal-binding protein, metallothionein (MT). We created a chimeric protein by fusing one or two copies of the MT gene to the gene for maltose binding protein. These chimeric proteins bound many gold atoms, with a conservative value of 16 gold atoms per copy of metallothionein. Visualization of gold-labeled fusion proteins by scanning electron microscopy required one copy of metallothionein while transmission electron microscopy required two copies. Images of frozen-hydrated samples of simple complexes made with anti-MBP antibodies hint at the usefulness of this method.

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Section: Introductionsupporting

confidence: 56%

“…In our previous work, we have suggested that Zn 7 -MT was able to use its 20 cysteines to bind gold by a mechanism like that used to synthesize gold clusters such as the popular Nanogold Ò (Mercogliano and DeRosier, 2006). These findings encouraged us to test concatenated MT as a clonable electron microscopy label.…”

Section: Introductionmentioning

confidence: 99%

Concatenated metallothionein as a clonable gold label for electron microscopy

Mercogliano

DeRosier

2007

Journal of Structural Biology

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109

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“…If the protein content of photosynthetic membranes is well documented, then this is not the case in the Golgi stacks of the hypodermis of C. elegans, where lattices of membrane proteins are evidenced with no clue as to their nature and function. The development of clonable tags that can bind to electron-dense elements, such as metallothionein (33) or heavy metal binding peptides (34), is of current interest, although there is still no CEMOVIS labeling achievement.…”

Section: Discussionmentioning

confidence: 99%

Contribution of cryoelectron microscopy of vitreous sections to the understanding of biological membrane structure

Leforestier

Lemercier

Livolant

2012

Proc. Natl. Acad. Sci. U.S.A.

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Using cryoelectron microscopy of vitreous sections, we investigated in situ the ultrastructure of biological membranes, selected from several cell types for their diverse biological functions. Here we describe how to visualize the two membrane leaflets and tightly apposed membranes, lying as close as 1.1 nm apart, by tuning the imaging conditions. We show how defects in membrane stacks may be clues to resolving their structure. Details of membrane proteins are also resolved, as well as protein lattices with correlations between stacked membranes. Imaging the cell in its native hydrated state can now be done in the nanometer resolution range, which should open unique routes for investigating structure-function relationships.thylakoid | transmembrane proteins | multilamellar body | Golgi | cortical alveoli B iological membranes are highly specialized dynamic entities that limit structural and functional compartments and control the exchange of matter and energy. They may exhibit a wide diversity of shapes with variable curvature, including spherical, tubular, planar, sponge, or cubic bicontinuous organizations (1). They are composed of hundreds of lipid and protein species, in some cases modified by sugars. Their structure and composition vary among species, cell types, and organelles and, even within a given membrane type, in time and space. This structural and chemical variability is directly related to their functional properties (2). Lipids (mostly phospholipids) represent more than 50% of membrane components. Their amphiphilic nature leads to the formation of bilayers. The hydrophobic chains are buried inside the membrane, and the polar heads are exposed to the outer, hydrated environment. Proteins may be bound to the lipid bilayer through electrostatic interactions or partially embedded within the bilayer.Transmission electron microscopy provides various techniques for exploring cell ultrastructure. Among these, cryoelectron microscopy of vitreous sections (CEMOVIS) provides snapshots of unstained frozen-hydrated cells and tissues in their native state. The analysis of ordered fibrillar objects like DNA in sperm cells (3), bacteria (4), and dinoflagellates (5) has benefited from this approach. Nevertheless, cryoelectron tomography (CET) is often preferred over CEMOVIS to overcome limitations arising from superimpositions on 2D projections and to obtain 3D views. CET has been used to successfully image entire small cells, organelles, and thin cellular extensions (6, 7), as well as thicker objects sliced into cryosections (8-11). Spectacular results have been obtained with CET in combination with image analysis; for example, the Ccadherin atomic structure has been fit into averaged subtomograms of epidermal desmosomes (8). However, the relatively low resolution of CET (generally between 5 and 8 nm; 3 nm in the optimum cases) remains insufficient to address many biological questions, especially those dealing with membrane structure.Fine structural details, particularly the bilayer nature of membranes, have been...

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“…The main difficulty in using these noncovalent labels is low label occupancy, which is further compounded by the flexible nature of the labels. To overcome this difficulty, the use of proteins with encoded molecular tags for EM contrasting was reported (26). The fusion protein approach eliminates the occupancy problem and was used previously in the context of 3D difference mapping (27).…”

Section: Mbp Fusion Combined With 2d Image Classification Is An Effecmentioning

confidence: 99%

The architecture of the DNA replication origin recognition complex in Saccharomyces cerevisiae

Chen

Speck

Wendel

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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The origin recognition complex (ORC) is conserved in all eukaryotes. The six proteins of the Saccharomyces cerevisiae ORC that form a stable complex bind to origins of DNA replication and recruit prereplicative complex (pre-RC) proteins, one of which is Cdc6. To further understand the function of ORC we recently determined by single-particle reconstruction of electron micrographs a low-resolution, 3D structure of S. cerevisiae ORC and the ORC-Cdc6 complex. In this article, the spatial arrangement of the ORC subunits within the ORC structure is described. In one approach, a maltose binding protein (MBP) was systematically fused to the N or the C termini of the five largest ORC subunits, one subunit at a time, generating 10 MBP-fused ORCs, and the MBP density was localized in the averaged, 2D EM images of the MBP-fused ORC particles. Determining the Orc1-5 structure and comparing it with the native ORC structure localized the Orc6 subunit near Orc2 and Orc3. Finally, subunit-subunit interactions were determined by immunoprecipitation of ORC subunits synthesized in vitro. Based on the derived ORC architecture and existing structures of archaeal Orc1-DNA structures, we propose a model for ORC and suggest how ORC interacts with origin DNA and Cdc6. The studies provide a basis for understanding the overall structure of the pre-RC.electron microscopy ͉ structure ͉ ATPase I n Saccharomyces cerevisiae origins of DNA replication contain conserved A, B1, and B2 elements, where the A element and part of B1 define the binding sequence for the origin recognition complex (ORC) (1-3). ORC binds to origin DNA in an ATPdependent manner and recruits other essential proteins, such as the initiation factors Cdc6, Cdt1, and the presumptive DNA helicase MCM, to the autonomously replicating sequence (ARS) to form a prereplicative complex (pre-RC) before the initiation of DNA replication that occurs in S phase (4-7). ORC consists of six proteins named in the descending order of their relative mass: Orc1 (120 kDa), Orc2 (71 kDa), Orc3 (62 kDa), Orc4 (56 kDa), Orc5 (53 kDa), and Orc6 (50 kDa). The calculated mass of ORC is Ϸ412 kDa. Only two of the ORC subunits (Orc1 and Orc5) are known to bind ATP (8), although the largest five subunits are predicted to contain an AAAϩ fold and a DNAbinding winged helix domain (WHD) within their C-terminal halves (9, 10).The prokaryotic origin recognition proteins consist of a single polypeptide that can form oligomeric structures (11-17), which raises the question of why in eukaryotes ORC has six subunits and why Cdc6 also contributes to origin recognition (18,19). Structural studies of the replication initiator proteins have begun to shed light on the mechanism of origin recognition, but how the individual initiator proteins cooperate to promote initiation of DNA replication is not clear. The eubacterial DnaA structure suggested that the origin DNA might wrap around a super helical assembly of multiple subunits of this replication initiator (11,12). In contrast, the recent structures of archaeal Orc1/C...

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Gold Nanocluster Formation using Metallothionein: Mass Spectrometry and Electron Microscopy

Cited by 67 publications

References 34 publications

Concatenated metallothionein as a clonable gold label for electron microscopy

Concatenated metallothionein as a clonable gold label for electron microscopy

Contribution of cryoelectron microscopy of vitreous sections to the understanding of biological membrane structure

The architecture of the DNA replication origin recognition complex in Saccharomyces cerevisiae

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