N A N O P R O B E S     E - N E W S

Vol. 6, No. 3          March 9, 2005

Cellmatrix adhesions in migrating cells are usually mediated by integrins. These are alpha-beta, heterodimeric transmembrane proteins that link extracellular matrix molecules such as fibronectin to the cytoskeleton. In order to identify its binding site, the authors synthesized the cytoplasmic domain of the beta1-integrin (residues H738K778) with a histidine tag at its N-terminus. The binding of this peptide to a lipid monolayer containing a chelated nickel (II) atom (dimyristoylphosphatidyl cholinesuberimidenitriloacetic acid nickel salt) mimics the native environment at the cytoplasmic leaflet of the plasma membrane. Preliminary results using a binding assay indicated the presence of two binding sites on the beta1-integrin: cryo-EM studies were pursued in order to localize these.

The cytoplasmic domain of beta1-integrin is a 41 amino acid peptide, molecular weight about 5,000. It was synthesized with two modifications: (a) four additional histidine residues incorporated at the N-terminus to form a "His5-tag;" and (b) two different cysteine modifications: in the first, a cysteine was inserted in the middle of the sequence just after T757, and in the second, a cysteine was added at the C-terminus. Following synthesis, the His-tagged cytoplasmic domain was purified on a YM C-18 semipreparative reverse-phase HPLC column eluted with a gradient of 0.1% trifluoroacetic acid (TFA) in water to 0.1% TFA in 30% acetonitrile: composition of the purified peptide was confirmed by mass spectrometry.

Monomaleimido Nanogold was conjugated to these cysteines. In both cases, the Nanogold label was attached by mixing it with the target molecule (without reducing agents) for approximately 16 hours at room temperature. The extent of labeling was determined from the UV/visible spectrum of the conjugate: Nanogold has a molar extinction coefficient of 2.25 x 10⁵ at 280 nm and of 1.12 x 10⁵ at 420 nm, and the extinction coefficient of the modified peptide is 1.25 x 10⁵ at 280 nm; therefore, the ratio of optical densities at 280 nm : 420 nm would be 3.37 for 100% labeling. Using this method, labeling efficiency was calculated as close to 92% for the peptide containing C758, and 85% for the peptide with the C-terminal cysteine (C779). Conjugates were chromatographically purified by gel filtration using Sephadex G-25 (Sigma). As a control, the His-tagged integrin peptide lacking any cysteine residues was also subjected to the gold-labeling reaction, but failed to yield any gold-labeled peptide.

Nickel-modified lipid monolayers were set up in wells 5 mm in diameter and 1 mm deep milled into Teflon blocks. Dilaurylphosphatidylcholine (DLPC) was used as a filler lipid, with different percentages of dimyristoylphosphatidyl cholinesuberimidenitriloacetic acid Ni(II) salt (nickel-chelating lipid), and different amounts of DDMA to facilitate crystallization. Monolayers were cast upon aqueous buffer containing His-tagged beta1-integrin peptide. Alpha-actinin was then injected into the aqueous phase. Arrays were obtained using 0.0114 nmol of alpha-actinin and 0.148 nmol of the beta1-integrin peptide, using 20 mM Tris buffer (pH 7.5) with 50mM NaCl and 1mM MgCl₂. Crystals formed overnight at 4°C. Specimens were recovered from the monolayer using hydrophobic reticulated carbon films on 300 Mesh copper grids, and were either negatively stained with 2% aqueous uranyl acetate or plunge frozen without stain in liquid ethane for cryomicroscopy. Low-dose EM data were collected on a Philips CM300-FEG, a 300 kV electron microscope equipped with a field emission gun. Frozen hydrated specimen grids were transferred to a cryoholder and examined at a temperature of -180°C. Images were recorded at magnifications of 3040,000 under low-dose conditions; film was digitized at a step size of 0.7 nm with respect to the original object.

The 2-D arrays of the beta-1-integrinalpha-actinin complex were examined with and without the gold label. Averaged projections were calculated for each specimen along with a difference map to determine the relative position of the gold-labeled beta-1-integrin peptide. The beta1-integrin peptide with gold label at two sites gives difference peaks consistent with binding of the beta-1-integrin cytoplasmic domain to alpha-actinin between the first and second of the four 3-helix motifs in the central rod domain (at the R1R2 junction). The observed differences in binding for the two gold-labeled peptides were attributed to the asymmetrical arrangement of the R1R4 domain with respect to the lipid monolayer, steric factors due to packing of adjacent molecules within the 2-D array, and flexibility of the C-terminal region of the beta1-integrin cytoplasmic domain.

The beta1-integrin cytoplasmic domain also contains two talin binding sites. The investigators examined the possible effect of talin binding, and how the talin:beta3-integrin structure could impact the beta1-integrin:alpha-actinin interaction, by aligning the F2F3 domain structure relative to their beta-integrinalpha-actinin model. The resulting model implies that simultaneous binding of both alpha-actinin and the talin F2F3 fragment to the beta1-integrin domain places both proteins in proximity but on opposite planes of the beta1-integrin cytoplasmic domain. It is suggested that in vivo both the talin F2F3 domain and alpha-actinin may bind simultaneously to the beta1-integrin cytoplasmic domain: the talin F2 domain may bind the beta1-integrin at the low-affinity site leaving the entire integrin cytoplasmic domain locked in an inactive state. Recent evidence shows that when the talin head-tail association is relieved, the F3 domain is then free to bind the beta-integrin at the high-affinity site. This disrupts the association with alpha-integrin and in turn triggers integrin activation; the authors propose that this activation step also exposes the once masked alpha-actinin binding site on the beta-integrin, allowing the alpha-actinin R1R4 domain to bind to the unmasked site on the beta-integrin to further stabilize integrin associations with the actin cytoskeleton.

Reference:

Kelly, D. F., and Taylor, K. A.: Identification of the beta1-integrin binding site on alpha-actinin by cryoelectron microscopy. J. Struct. Biol., 149, 290-302 (2005).

• Abstract (courtesy of Science Direct).

More information:

• Nanogold labeling reagents: catalog information
• Monomaleimido Nanogold: product information and protocols for use
• Application feature: Nanogold-peptides
• Technical Help: Nanogold labeling reagents
• Guide to Gold Cluster labeling (detailed technical discussions)
• References: Nanogold labeling with ultrathin cryosections
• References: Nanogold labeling for other applications

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Labeling Other Functional Groups: Hydroxyls

In fact, some questions and situations come up quite often, so it's worth using this space to summarize how you might go about labeling if you are in one of them. Hydroxyl groups are one: if all you have is a hydroxyl, how can you label it? There are several possible approaches:

• Use a hydroxyl-reactive cross-linker.

  There is a hydroxyl-reactive cross-linker: p-Maleimidophenylisocyanate. Unfortunately, the maleimide group, which this reagent introduces, is not directly reactive towards Nanogold labeling reagents: but simple treatment with mercaptoethylamine hydrochloride will add an amino- group, which you can then label with Mono-Sulfo-NHS-Nanogold.

• Make it more reactive towards amines, so you can label with Monoamino Nanogold®.

  This can be done relatively easily: react it with tosyl chloride (p-toluene-sulfonato chloride). This converts it to a tosyl group, which is now strongly reactive towards amines: simply react with Monoamino Nanogold.

  Scheme 1: Tosylation of hydroxylated molecule and reaction with Monoamino Nanogold.

• Oxidize it to a ketone or aldehyde, then react with a bifunctional hydrazido cross-linker with a Nanogold-reactive functionality at the other end.

  Many methods are available for the oxidation of hydroxyls to carbonyls. Once this is achieved, you can use a heterobifunctional hydrazide cross-linker to introduce a reactive functional group. Succinimidyl 6-(3-[2-pyridyldithio]-propionamido) hydrazide (SPDP hydrazide) introduces a disulfide, which is readily reduced to a sulfhydryl and labeled with Monomaleimido Nanogold:

  Scheme 2: Oxidation of hydroxyl to carbonyl, followed by reaction with hydrazido cross-linker, activation of disulfide, and labeling with Monomaleimido Nanogold.

  Many carbonyl-reactive hydrazido- maleimide cross-linkers are available: you can use these to introduce maleimides, which may then be reacted with mercaptoethylamine hydrochloride to convert them to amines and labeled with Mono-Sulfo-NHS-Nanogold. For examples, the list of heterobifunctional cross-linkers from Molecular Biosciences, cross-linker selection guide from Pierce are useful references.

  If you have a primary hydroxyl and can convert it to an aldehyde, you have a simpler option: you can react it directly with Monoamino Nanogold. This is the same reaction used, after periodate oxidation, to label RNA and glycoproteins, and details are in our application note on RNA labeling.

If you have a cis-1,2-dihydroxy group, you can oxidize with sodium periodate to generate the dialdehyde, then react with Monoamino Nanogold. The full procedure is given in our application note on RNA labeling: you can also use this procedure for any molecule containing a cis-1,2-dihydroxy group, such as a glycoprotein or other carbohydrate:

Periodate oxidation of carbohydrate (cis-1,2-diol) followed by reaction with Monoamino Nanogold.

More information:

• Catalog information: Nanogold labeling reagents
• Monomaleimido Nanogold: Product information and protocols
• Monoamino Nanogold: Product information and protocols
• Application note: RNA labeling with Nanogold
• Alternative chemistry for oligonucleotide labeling
• Cross-linker selection guide (Pierce)
• Molecular Biosciences (cross-linker supplier and resource)

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NanoVan for Viral Visualization

It is helpful, when combining negative staining with ultrastructural gold labeling, if the negative stain is not too electron-dense, so that the overall contrast allows easy visualization of both the gold labeling and the negative stain. NanoVan is a novel negative staining reagent, which is based on vanadium. It is recommended for use with Nanogold® because the lower atomic number of vanadium means that the stain is less electron-dense than heavy metal-based stains such as uranyl acetate or lead citrate. NanoVan is very fine-grained and highly amorphous, and has been used for a number of high-resolution STEM and TEM studies of virus and protein ultrastructure. Nanoprobes also offers a second negative stain reagent, Nano-W, based on the heavier element tungsten. This gives a more dense stain, and is more suited to use with larger gold labels.

Advantages of these reagents:

• NanoVan and Nano-W are completely miscible: they may be mixed in different proportions to give desired intermediate stain density.
• Near-neutral pH results in better ultrastructural preservation.
• NanoVan is less susceptible to electron beam damage than uranyl acetate.
• Fine grain allows high imaging resolution.

Isherwood and Patel now describe the use of NanoVan with 10 nm colloidal gold staining to investigate the processing and transmembrane topology of the E2p7 protein of hepatitis C virus. Hepatitis C virus (HCV) contains a positive-strand genomic RNA encoding a single polyprotein of approximately 3010 amino acid residues that is processed into at least 10 different proteins (C, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B). The HCV structural proteins, core (C) and the two envelope glycoproteins E1 and E2, are located within the N terminus of the polyprotein, and non-structural proteins (NS2 to NS5B) reside within the C-terminal part. The status of the p7 protein is unknown, although it is known to have ion channel activity and is essential for HCV infectivity. The C terminus of each structural protein is composed of a hydrophobic amino acid sequence, which acts as a signal peptide to target the proteins located downstream to the endoplasmic reticulum (ER). Although cleavage at the C/E1 and E1/E2 sites is completed rapidly after translation, cleavage at E2/p7 and p7/NS2 is delayed, resulting in an E2p7NS2 species; and cleavage at the E2/p7 site is incomplete, resulting in two E2-specific species, E2 and E2p7 of unknown significance.

The investigators investigated the topology and processing of the p7 protein using 10 nm colloidal gold labeling with negative staining. Since the virus has not been successfully cultured, virus-like particles (VLPs) from Sf21 cells infected with rbacs were prepared in insect cells using a recombinant baculovirus containing the cDNA of the HCV structural proteins. The purity and quality of the VLP preparation were verified by negative-stained transmission electron microscopy (EM): VLPs (5 ml) were loaded on to Formvar-coated nickel grids, stained with Nanovan and examined in the electron microscope. Samples were then labeled with colloidal gold on Formvar-coated nickel grids: these were incubated in primary antibody (anti-E2 antibodies AP33 or ALP98, or the rabbit polyclonal antiserum R646) for 23 hours at room temperature. The grids were washed three times in distilled water, then incubated for 2 h with anti-mouse IgG conjugated to 10 nm gold. After three distilled water washes as above, samples were stained with Nanovan and examined by EM. Interestingly, they report that the colloidal gold probe was from us; although we currently don't offer 10 nm colloidal gold, we are working on an improved, covalently linked alternative.

Partial processing was seen at the E2/p7 site in mammalian cells, the efficiency of which improved in the presence of nucleotide sequences downstream of p7. In insect cells, no processing at the E2/p7 site occurred and the uncleaved E2p7 species was incorporated into virus-like particles when expressed in the context of CE1E2p7c-myc. Radiolabeling and immunoprecipitation studies confirmed that E2p7c-myc formed a heterodimer with E1, indicating that the E1E2p7 heterodimer may play a functional role in virus replication. Given that partial or no processing occurs between E2 and p7 in mammalian or insect cells, it is important to determine whether E2p7 as an unprocessed form is incorporated into virus particles. This was confirmed in the EM experiments: anti-E2 mAb was found to bind to all VLPs, but an antibody against c-myc showed specific labeling of c-myc on the outer surface of the VLPs derived from CE1E2p7c-myc, but not from CE1E2p7. Comparison of the p7 signal peptide sequences of strains BK and H77c revealed 3 amino acid differences (positions 720, 733 and 742). Mutational analysis showed that the V720L change in the H77c sequence substantially increased E2/p7 site processivity. The p7 protein adopts a double membrane-spanning topology with both its N and C termini orientated luminally in the endoplasmic reticulum. The transmembrane topology of E2p7 species was examined by trypsin protection assay and western blotting, and by immunofluorescence: in both cases, the C terminus of p7 in E2p7 was found to be cytoplasmically orientated, indicating that p7 adopts a dual transmembrane topology.

Reference:

Isherwood, B. J., and Patel A. H.: Analysis of the processing and transmembrane topology of the E2p7 protein of hepatitis C virus. J. Gen. Virol., 86, 667-676 (2005).

• Abstract (courtesy of the Journal of General Virology).

Reference for VLP preparation:

Baumert, T. F.; Ito, S.; Wong, D. T., and Liang, T. J.: Hepatitis C virus structural proteins assemble into viruslike particles in insect cells. J. Virol., 72, 3827-3836 (1998).

• Abstract (courtesy of the Journal of Virology).
• Reprint (PDF) (courtesy of the Journal of Virology).

More information:

• Catalog information: Negative stain reagents
• NanoVan: Product information and instructions
• Nano-W: Product information and instructions
• Technical help: Negative staining
• Negative staining: Other references
• 10 nm covalent gold-Fab' conjugate: Our 1999 Microscopy & Microanalysis paper

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Protein Array with Silver-Enhanced Gold Detects Myocardial Infarction

The colloidal gold labeled antibody used for detection was prepared in advance. Monoclonal anti-human cTnI antibodies were adsorbed to 13±1.5 nm colloidal gold particles, prepared by the conventional citrate reduction of tetrachloroaurate. Likewise, the substrate for the assay was also prepared in advance: a suitable size (4 cm x 4 cm) cellulose nitrate membrane was soaked in a TrisHCl buffer solution (TB, pH 8.2) solution containing 30% glycerol for 1 min. After aspiration of the TB solution, 2 microliter spots of individual capture antibody (IgG1) at a concentration of 100 micrograms/ml were spotted onto the membrane, which was then kept in a humidity chamber for 2 h. Unbound IgG1s were washed out using 0.2M TB containing 0.5% Tween 80 and 10% NaCl). Arrays were then blocked with a low-fat dry milk solution, washed with TB and aspirated.

For the analytical procedure, 20 microliters of detecting cTnI serum samples and 10 microliters of colloidal goldIgG were spotted onto the appropriate region of the pretreated supporting membrane, followed by incubation 20 min in TBST. After a stringent wash with deionized water, the arrays were immersed in the silver enhancement solution for 5 minutes and washed with deionized water again. The array image was recorded with a flatbed scanner or judged by eye.

The detection procedure of the assay could be completed within 40 minutes, considerably faster than the routine enzyme-linked immunosorbent assay (ELISA) that usually requires 3 hours or more for turnaround. The detection limit of cTnI was found to be 1 ng/ml, similar to those detected by ELISA.

As part of the work, the reaction conditions were varied in order to identify those giving the highest sensitivity and lowest non-specific or 'background' binding. Out of phosphate or Tris buffers at pH 7.4, 8.2 or 9.0, that at pH 8.2 was found to give the highest signal-to-noise ratio, with background arising at other pH values. Comparison of different amounts (25, 50, 100, 150 and 200 micrograms/mL) of capture antibody indicated that 100 micrograms/mL was necessary for best sensitivity. Low-fat dried milk was found to be more effective than bovine serum albumin as a blocking agent, with best results at 5% concentration. Different immunologic reaction medium conditions were also examined, including TB, TB containing Tween 80, TB containing sodium chloride and TB containing both Tween 80 and sodium chloride. 0.5% Tween 80 and 10% NaCl gave much lower background and higher signal than other conditions: this was attributed to the fact that combination of surfactant and salt prevented most of the nonspecific binding, while having little effect on the specific immunoreaction. A 20 min incubation time was found to be optimum, with shorter times giving weaker signals and longer times giving little further improvement.

Reference:

Guo, H., Zhang, J.; Yang, D.; Xiao, P., and He, N.: Protein array for assist diagnosis of acute myocardial infarction. Coll. Surf. B: Biointerfaces, 40, 195-198 (2005).

• Abstract (courtesy of Science Direct).

More information:

• Colloidal gold conjugation procedure (Research Diagnostics)
• Colloidal gold: technical help (Nanoprobes)
• Silver enhancement: technical help
• Silver enhancement: catalog information
• Gold in rapid diagnostics tests (courtesy of IVD Technology)

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Other Recent Publications

• Abstract (courtesy of Science Direct).

MacMillan and group present more results using self-assembling arrays of bacterial heat shock protein, TF55-beta, as templates for the arrangement of metal nanoparticles, in this case transition metal nickel and cobalt-platinum nanoparticles which are likely candidates for catalytic activity. A loop that occludes the central pore of the assembled chaperonin was genetically removed, and a polyhistidine (His10) sequence added to its amino terminus. With these modifications, the solvent-accessible cores of assembled chaperonins possess 180 additional His residues, creating a region with enhanced affinity for metal ions that is spatially constrained by the interior dimensions of the chaperonin. When incubated with Pd(II), the chaperonin cores become activated sites which selectively initiate chemical reduction of magnetic transition metal ions (Ni²⁺ or Co²⁺) from precursor salts to yield arrays of bimetallic (Ni-Pd or Co-Pd) nanoparticles with dimensions defined by the chaperonin.

Reference:

McMillan, R. A.; Howard, J.; Zaluzec, N. J.; Kagawa, H. K.; Mogul, R.; Li, Y.-F.; Paavola, C. D., and J. D. Trent: A Self-Assembling Protein Template for Constrained Synthesis and Patterning of Nanoparticle Arrays. J. Amer. Chem. Soc., 127, 2800-2801 (2005).

• Article information (DOI) (courtesy of the American Chemical Society).

Raman spectroscopy is used frequently to study the properties of molecules adsorbed onto gold or silver particles. Adsorption to the metal particles causes enhancement of some Raman-active vibrational modes of organic molecules, and this phenomenon, called Surface Enhanced Raman Scattering, or SERS, provides a means both to study these molecules and to image metal nanoparticle constructs. Laurent and co-workers have investigated this phenomenon further, using Raman scattering to image the surface plasmon of arrays of gold particles adsorbed to methylene blue, and confirm that Raman scattering can be useful for studying surface plasmon properties, such as localized excitation properties at nanoparticle defects or junctions.

Reference:

Laurent, G.; Félidj, N.; Lau Truong, S.; Aubard, J.; Lévi, G.; Krenn, J. R.; Hohenau, A.; Leitner, A., and Aussenegg, F. R.: Imaging Surface Plasmon of Gold Nanoparticle Arrays by Far-Field Raman Scattering. Nano Letters, 5, 253-258 (2005).

• Article information (DOI) (courtesy of the American Chemical Society).

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