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N A N O P R O B E S     E - N E W S

Vol. 8, No. 7          July 27, 2007

Updated: July 27, 2007

In this Issue:

This monthly newsletter is to inform you about techniques to improve your immunogold labeling, highlight interesting articles and novel applications of metal nanoparticles, and answer your questions. We hope you enjoy it and find it useful; as always, let us know if we can improve anything.

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Enzyme Metallography: New Paper and Coming Products

The evolution of Nanoprobes detection technologies for in situ hybridization is described in a review paper in the current issue of Human Pathology. This feature discusses the development, features and comparative merits of a number of detection methods, including Nanogold® with silver enhancement, Nanogold with gold enhancement (GOLDFISH), and finally enzyme metallography. These are discussed in the context of the development of a chromogenic assay for HER2 gene amplification; this is a significant indicator of malignant behavior, and one of the criteria used to assess patients for humanized monoclonal antibody therapy (Herceptin / Trastuzumab).

Enzyme Metallography is a biological labeling and staining method in which a targeted enzymatic probe is used to selectively deposit metal at sites of interest. It has proven to very clean and sensitive both for in situ hybridization, where it readily visualizes endogenous copies of single genes, and immunohistochemistry (IHC) detection. In the electron microscope, it produces dense, highly specific staining with very high penetration. It has also been applied successfully as an electrical detection method for biochips: this application, in which multiple independent electrical contacts are fabricated upon target binding, offers the potential for highly multiplexed target detection in a robust, miniaturized and highly portable format.

The sharply resolved black signal, used for in situ hybridization, is readily distinguished from other stains, and has been combined with fast red K immunohistochemistry to provide a concomitant brightfield gene and protein assay (EnzMet GenePro). Enzyme metallography has many advantages as a detection method. Because it is used in the conventional brightfield electron microscope, it does not require either expensive fluorescent optics, or dark adaptation on the part of the user. The signal is permanent, and does not have the photobleaching problems associated with fluorescent stains. Furthermore, because the deposited metal is electron-dense, it provides high contrast for electron microscopy, making enzyme metallography both a highly effective electron microscopic method, and a potential correlative light and electron microscopic method.

[Enzyme Metallography (EnzMet) Montage (144k)]

Upper row: Comparison between (left) conventional DAB detection with H&E counterstain and (center) EnzMet with nuclear fast red counterstain: detection of HER2 gene copies in paraffin-embedded infiltrating ductal carcinoma of the breast (micrographs courtesy of Raymond R. Tubbs, Cleveland Clinic Foundation) (right) immunohistochemical staining of cytokeratin: human prostate cancer slide from Dako, stained using Cytokeratin AE1/3 primary antibody and enzyme metallographic secondary, counterstained with Eosin.
Lower row: (left) the enzyme metallographic process; (right) schematic of the combined immunohistochemistry and in situ hybridization protocols used for the EnzMet GenePro concomitant gene and protein assay, showing the localization of the targets and the different detection probes.

Nanoprobes recently signed a deal with Ventana Medical Systems, Incorporated, for the commercial development and use of this reagent in automated slide staining instruments and applications. As a result, the first commercial product has now been introduced in Europe; it is called SISH (Silver In Situ Hybridization). Introduction of SISH in the United States is pending FDA approval. Nanoprobes will shortly introduce a commercial EnzMetTM formulation optimized for research applications and non-automated staining. Look for an announcement on our web site later this year.


  • Powell, R. D.; Pettay, J. D.; Powell, W. C.; Roche, P. C.; Grogan, T. M.; Hainfeld, J. F., and Tubbs, R. R.: Metallographic in situ hybridization. Hum. Pathol., 38, 1145-1159 (2007).

More information:

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Nanogold® Labeling: Filtration, Purification, and Beating Aggregation

One of our customers provided an interesting observation recently: they filtered their Nanogold®-IgG conjugate, prepared with Mono-Sulfo-NHS-Nanogold, through a 0.2 micron nylon filter, and observed a radical decrease in the calculated labeling. This brings up several important considerations for successful separation of Nanogold conjugates.


If you need to use an antibacterial filter (for example, to prepare a sterile solution), we recommend cellulose acetate rather than nylon or other filters. for final purification and packaging of Nanogold conjugates, we routinely use Nalgene 0.2 micron cellulose acetate syringe filters. We have occasionally observed undesirable interactions between Nanogold conjugates and other filter materials: in the case of our customer, it appears that the Nanogold bound strongly to the nylon filter material and was partially degraded, leading to cleavage of the conjugate protein and the observed labeling reduction.

If your Nanogold conjugate becomes stuck on the filter, you may be able to redissolve it by using DMSO (dimethyl sulfoxide). DMSO is a highly effective solvent for Nanogold: use in a mixture with water at the maximum proportion of DMSO that (a) your conjugate biomolecule, and (b) your filter material can tolerate. Many antibodies and proteins will tolerate 20% DMSO, and some peptides and small molecules much higher proportions, while many filter materials can tolerate up to 70%.

Liquid Chromatography - Gel filtration: how to recognize and beat aggregation

One explanation proposed for our customer's result was that they were actually separating an aggregated Nanogold species from unlabeled material. This is unlikely, because the calculated labeling indicated close to a 1 : 1 ratio of Nanogold to antibody, and the material eluted from the column as a reasonably well-defined peak. But this raises the question: how do you recognize and beat aggregation?

Below, we show what a successful separation looks like when a Nanogold-labeled protein (in this case antibody Fab' fragments) is separated from excess Nanogold over a gel filtration column.

[Gel filtration separation of Nanogold-Fab' (80k)]

Separation of a Nanogold-Fab' conjugate by liquid chromatography gel filtration, showing separation of species both larger and smaller than the desired product: column view shows how the separation will appear as the column elutes, and the chromatogram shows the trace obtained by monitoring at 280 nm. Peaks are colored to reflect the actual colors most likely to appear in the corresponding output fractions. Aggregates can be a variety of colors, depending on the fate of the gold particles.

How will you know when you have aggregation?

  • When Nanogold aggregates, there may be a color change: the gold can form larger particles that are red or red-brown, or may precipitate to give a gray or blue-black precipitate.
  • Aggregates often stick at the top of the column. Look for a brown or black discoloration at the top of the column that does not move with the eluent.
  • Aggregated materials are generally larger than the protein-Nanogold conjugate. They will tend to elute near or at the column void volume if they make it through the column.
  • If aggregation occurs, the resulting particles tend to form a range of sizes. If part of this range overlaps with the size fractionation range of the column, they will tend to give a broad and ill-defined peak.

How can you avoid it?

  • When you separate your Nanogold conjugate after the conjugation reaction, use a column material with a separation or fractionation range that extends significantly above the expected size of the conjugate. Should larger species be formed, they will then be separated from the conjugate. Below is a table listing some of the chromatography media that are available and their separation ranges; check these and published separation ranges for other materials to find an appropriate material.

  • Sometimes precipitation can result from solubilization problems. In this case, use a proportion of DMSO (dimethyl sulfoxide) in the reaction buffer and the column elution buffer. Most proteins can tolerate up to 20% DMSO. However, use caution when adding DMSO as mixing with water is exothermic, and the heat generated can accelerate hydrolysis of the reactive group. Cool buffers in the refrigerator before mixing.

  • You may need to vary the reaction conditions in order to achieve the cleanest reaction. In this case, it is sometimes helpful to order the Nanogold labeling reagent in five aliquots rather than 30 nmol in one vial, so you can run two or three different reactions under different conditions.

We have found that the following media work well for gel filtration of different Nanogold-labeled conjugates:

Labeled Biomolecule Suggested Gels MW Separation Range
Antibodies: whole IgG
Medium - large proteins (MW 30,000 - 300,000)
Superose-12 (GE Healthcare)
Pre-packed columns or bulk media.
1,000 - 300,000
Antibodies: Fab' fragments
Small - medium proteins (MW 10,000 - 50,000)
Superdex-75 (GE Healthcare)
Pre-packed columns or bulk media.
3,000-70,000 (globular proteins)
Large proteins and protein complexes (MW 100,000 - 1,000,000) Superose-6 (GE Healthcare)
Pre-packed columns or bulk media.
5,000 - 500,000
Very large proteins and protein complexes (MW 200,000 - 5,000,000) A-1.5m (Bio-Rad)
Available in coarse, medium or fine grades.
10,000 - 1,500,000 (globular proteins)
Small proteins and peptides, oligonucleotides (MW 7,000 and smaller) Superdex-Peptide (GE Healthcare)
Pre-packed columns or PG-30 bulk media.
100 - 7,000
Substrate analogs and other small molecules (MW 2,000 and smaller) GH-25 desalting gel (contact Millipore). Exclusion limit: 3,000
Substrate analogs and other small molecules (MW 3,000 and smaller) Sephadex G-25 (GE Healthcare)
Hi-Trap columns or Bulk media.
Exclusion limit: 5,000

More information:

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NTA-Ni(II)-Nanogold® Reveals Lipoprotein-Secretin Interactions

The results with NTA-Ni(II)-Nanogold® keep coming and coming! As described recently, NTA-Ni(II)-Nanogold® is a different gold probe. Instead of an antibody or protein, it is targeted by nitrilotriacetic acid (NTA) nickel (II), which is a small metal chelate that binds highly selectively to His (polyhistidine) tags. His tags may be readily engineered into most expressed proteins, so NTA-Ni(II)-Nanogold is a potential universal secondary reagent which can be used to gold-label any synthetic or expressed protein and peptide probes.

Neisseria meningitidis is a causative agent of meningitis and septicemia. It is a bacterium which expresses type IV pili, which mediate functions including autoagglutination, twitching motility, biofilm formation, adherence, and DNA uptake during transformation. The secretin PilQ supports type IV pilus extrusion and retraction, but also requires auxiliary proteins for assembly and localization in the outer membrane. In the current issue of the Journal of Bacteriology, Collins and group describe the use of NTA-Ni(II)-Nanogold labeling of a His-tagged PILP fusion protein to study the physical properties of the lipoprotein PilP and its interaction with PilQ. Results from solid phase overlay assays, mutagenesis and blotting studies showed that PilP was an inner membrane protein, required for pilus expression and transformation, since pilP mutants were nonpiliated and noncompetent. In vitro experiments using recombinant fragments of PilP and PilQ showed that the N-terminal region of PilP interacted with the middle part of the PilQ polypeptide, and NTA-Ni(II)-Nanogold labeling and cryoelectron microscopy were then used to elucidate the structure of the complex.

A meningococcal in-frame pilP-His fusion was designed to encode a six-histidine tag in the predicted loop region at amino acid 74. The PilQ complex was purified from meningococcus outer membranes by detergent solubilization and size exclusion. For native PilP purification, meningococcus M1080-PilP74-6xHis cells were grown on blood agar plates in 5% CO2 at 34°C overnight, harvested in cold phosphate-buffered saline (PBS) and collected by centrifugation at 4,000 x g for 15 minutes. The cell pellet was resuspended in cold Tris-buffered saline (pH 7.5) containing 1 x EDTA-free protease inhibitor cocktail and passed twice through a French press at 25,000 lb/in2. Cell debris was removed by centrifugation twice at 4,000 x g for 10 minutes. The supernatant was collected and membrane fraction pelleted by centrifugation at 35,000 rpm for 35 minutes. The pellet was dissolved in lysis buffer (10 mM imidazole, 300 mM NaCl, 50 mM NaH2PO4, 0.5% Triton X-100, 10 mM beta-mercaptoethanol, 3 mM MgCl2, pH 8.0) with 1 x EDTA-free protease inhibitor cocktail and incubated with rotation at 4°C overnight, then the clear fraction was centrifuged at 4,000 x g for 15 minutes. The supernatant was purified over a Ni-NTA agarose column using elution buffer (200 mM imidazole, 300 mM NaCl, 50 mM NaH2PO4, pH 8.0) then immediately dialyzed against 50 mM sodium phosphate buffer (pH 7.0) containing 300 mM NaCl and 10% glycerol.

Natively purified recombinant PilPDelta119 (100 µg/mL) was incubated with PilQ complex purified from outer membranes, with or without NTA-Ni(II)-Nanogold with agitation for 24 hours at 4°C, then centrifuged at 13,000 rpm in a benchtop centrifuge for 5 minutes and prepared for cryonegative staining. Grids were placed in a cryostage, and data recorded at 100 K in conjunction with a 4K charge-coupled-device (CCD) camera. Individual gold-labeled particles were interactively selected in 64-pixel boxes (Å/pixel 3.1) using BOXER. After contrast transfer function correction, the data were contrast normalized, centered, and low-pass filtered to 25-Å resolution. A 190-Å circular mask was applied, six rounds of iterative refinement performed in C4 symmetry using the previously calculated 3D structure of PilQ filtered to a 25-Å resolution as a start model.

]NTA-Ni(II)-Nanogold structure and labeling of the PILQ-PILP complex (56k)]

Left: Structure of NTA-Ni(II)-Nanogold, showing interaction with a His-tagged protein. Upper Center: Structure of NTA-Ni(II)-Nanogold-labeled His-tagged PILQ-PILP complex, showing location of the Nanogold particles. Right: Knob protein from adenovirus cloned with 6x-His tag, labeled with Ni-NTA-Nanogold, column purified from excess gold, and viewed in the scanning transmission electron microscope (STEM) unstained (Full width approximately 245 nm).

The three-dimensional reconstruction of the PilQ-PilP interacting complex obtained at low resolution by transmission electron microscopy showed that PilP localizes around the cap region of the PilQ oligomer. This suggests a role for PilP in pilus biogenesis. Although PilQ does not need PilP for stabilization or membrane localization, the specific interaction between these two proteins indicates that they may have another coordinated activity in pilus extrusion and retraction or in related functions.


  • Balasingham, S. V.; Collins, R. F.; Assalkhou, R.; Homberset, H.; Frye, S. A.; Derrick, J. P., and Tonjum, T.: Interactions between the Lipoprotein PilP and the Secretin PilQ in Neisseria meningitidis. J. Bacteriol., 189, 5716-5727 (2007).

More on sample preparation, labeling and microscopy:

  • Collins, R. F.; Frye, S. A.; Balasingham, S.; Ford, R. C.; Tonjum, T., and Derrick, J. P.: Interaction with type IV pili induces structural changes in the bacterial outer membrane secretin PilQ. J. Biol. Chem., 280, 18923-18930 (2005).

More information:

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Nanogold®, Synaptic Vesicle Membrane Retrieval and Synapse Development

While we prefer it if you use our products for silver enhancement and gold enhancement with our Nanogold® conjugates, we are also please to hear that other manufacturer's silver enhancers work with Nanogold probes. Koh and colleagues, reporting in the current Journal of Cell Biology, provide a case in point. The authors take advantage of the high labeling density that may be obtained with the small Nanogold probes to quantitate labeling sites in different ultrastructural regions during endocytosis.

Epidermal growth factor receptor pathway substrate clone 15 (Eps15) has been found in protein complexes with multiple endocytic proteins, including dynamin and the vertebrate homologue of Dap160, intersectin. This protein has been implicated in endocytosis, endosomal protein sorting, and cytoskeletal organization, but its role is still unclear for several reasons, including limitations of dominant-negative experiments, and apparent redundancy with other endocytic proteins. The authors used a combination of immunofluorescence, immunoprecipitation and electrophysiological experiments with Drosophila eps15-null mutants, and showed that Eps15 is required for proper synaptic bouton development and normal levels of synaptic vesicle (SV) endocytosis.

Pre-embedding immunogold was used to visualize the distribution of eps15 at points during endocytosis. Muscles were dissected from third instar Drosophila larvae, fixed in 4% paraformaldehyde and embedded in agarose. Vibratome slices of the agarose blocks were incubated with guinea pig anti-Eps15 antiserum, followed by Nanogold-conjugated secondary antibodies. The immunogold labeling was silver enhanced using an IntenSE Silver Enhancement kit from GE Healthcare; samples were then embedded in Durcupan ACM for ultrathin sectioning. Serial ultrathin sections were counterstained with uranyl acetate and lead citrate, then examined in a transmission electron microscope. Images were quantified using NIH ImageJ, and statistical evaluation was performed using Excel (Microsoft). It was noted that the silver enhancement procedure, which works by the precipitation of silver around the gold particles, resulting in the formation of irregularly shaped black precipitates; our HQ Silver silver enhancement reagent is formulated for the most uniform and regular particle development, and may help produce more regular enhanced particles in this application.

Consistent with a role in SV endocytosis, electron microscopic analysis and quantitation of Eps15 within cellular compartments showed that it moves from the center of synaptic boutons to the periphery in response to synaptic activity. The endocytic protein, Dap160/intersectin, is a major binding partner of Eps15; eps15 mutants phenotypically resemble dap160 mutants. Analyses of eps15 dap160 double mutants suggest that Eps15 and Dap160 play a coordinated role in SV endocytosis. The authors infer that Eps15 and Dap160 promote the efficiency of endocytosis from the plasma membrane by maintaining high concentrations of multiple endocytic proteins, including dynamin, at synapses.


  • Koh T. W.; Korolchuk V. I.; Wairkar Y. P.; Jiao W.; Evergren E.; Pan H.; Zhou Y.; Venken K. J, Shupliakov O.; Robinson I. M.; O'kane C. J., and Bellen H. J.: Eps15 and Dap160 control synaptic vesicle membrane retrieval and synapse development. J. Cell Biol., 178, 309-322 (2007).

More information:

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The Right Contacts: Ordering, Technical Questions, and Custom Synthesis

With the recent addition of our international distributors to our mailing list, now is a good moment to remind you of the right contact information. Note that not all of our contacts have access to all your information; therefore, it is worth taking a moment to make sure that your question will go directly to the person with access to the information you are looking for.

If you have a question about an order, such as whether it has been received or shipped, you should contact our main office (nano¤; telephone 1-877-447-6266 in North America, ++ (631) 205-9490 from elsewhere; fax (631) 203-9493. You should not contact technical support: details of individual orders, shipping, and billing are not accessible to technical support personnel.

If you are looking for Nanogold® conjugate or reagent that is similar to our catalog items and the chemistry has already been established - such as a multiply functionalized Nanogold particle, or a Nanogold-labeled primary antibody - we can usually prepare such products as custom syntheses. Please contact technical support (tech¤ if you have a specific custom synthesis request, or fill out our custom synthesis request form. If your request includes steps that we do not do regularly, such as labeling a different type of protein or biomolecule, we can often consider it, but may need to treat it as a short-term contract research project in which payment is required whether or not the synthesis is successful.

For your information, contact information is summarized below:

Question: Contact Telephone E-mail
Ordering, order status, shipping or payment Sales office 1-877-447-6266 or (631) 205-9490
Product availability or delivery time Sales office 1-877-447-6266 or (631) 205-9490
Technical question or custom synthesis Technical support 1-877-447-6266 or (631) 205-9492
Problem with product Technical support 1-877-447-6266 or (631) 205-9492
Business inquiry or general information General business office 1-877-447-6266 or (631) 205-9490

More information:

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Other Recent Publications

A number of papers have described the use of FluoroNanogold probes for electron microscopy in combination with immunofluorescent probes for fluorescence microscopy: the fluorescent component of the FluoroNanogold probe may be used to confirm successful labeling before processing for electron microscopy, while the small size of the FluoroNanogold-Fab' conjugate allows dense labeling and facilitates quantitation. The latest paper is presented by Zeniou-Meyer and group in the Journal of Biological Chemistry, who used pre-embedding immunoelectron microscopy to localize an EGFP fusion protein in Chromaffin cells as part of their study of the ultrastructural distribution of phosphatidic acid (PA) in secretory cells. Chromaffin cells transfected with the plasmid encoding for yeast soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein Spo20p PA-binding domain (wtPABD) coupled to enhanced green fluorescent protein (EGFP) were kept under resting conditions in Lockes solution or stimulated for 3 minutes in a solution containing 2 mM BaCl2, 150 mM NaCl, 5 mM KCl and 10 mM HEPES at pH 7.4. Cells were fixed for 30 minutes with 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M sodium/phosphate, pH 7.3, permeabilized with 0.05% Triton X-100 (15 minutes) or 0.1% saponin (30 minutes), then immunostained using anti-GFP primary antibodies (1/50) and Alexa Fluor 594 FluoroNanogold secondary antibody (1/60), followed by silver enhancement.

Quantitative analysis of the ultrastructural distribution of the granules and of the immunogold particles bound to the wtPABD-EGFP probe was performed in 21 transfected chromaffin cells after stimulation. Granules as well as gold particles were considered to be associated with cellular structures (plasma membrane, subplasmalemmal shell (SPM), and granule membrane) when separated by <50 nm. The culture and fixation conditions used in this study yielded SPM thicknesses of 184.5 ± 3.9 nm. The percentage of granule in the SPM region and non-SPM internal cytoplasm of the cells was counted to estimate the current secretory activity of the stimulated cells compared with resting cells, which did not display any granule in SPM shell. The density of immunogold particles in the SPM region and in the internal cytoplasm was measured using the image analysis software Axiovision AC Rel. 4.5; it was determined as the number of particles/µm2 of area. Percentages of plasma membrane- and granule-bound immunogold particles in SPM were calculated relative to total immunogold particles counted in SPM; and finally, to estimate the particular accumulation of PA at the plasma membrane involved in granule docking, we compared the numbers of immunogold particles/µm of plasma membrane at docking sites versus non-docking sites of the plasma membrane (t test).

Phospholipase D1 was found to be activated in secretagogue-stimulated cells, and it produces PA at the plasma membrane at secretory granule docking sites. Phospholipase D1 activation and PA production were shown to represent key events in the exocytotic progression. Membrane capacitance measurements indicate that reduction of endogenous PA impairs the formation of fusion-competent granules. These findings demonstrate that PA synthesis is required during exocytosis to facilitate a late event in the granule fusion pathway, and the authors propose that the underlying mechanism is related to the ability of PA to alter membrane curvature and promote hemi-fusion.


  • Zeniou-Meyer, M.; Zabari, N.; Ashery, U.; Chasserot-Golaz, S.; Haeberle, A. M.; Demais, V.; Bailly, Y.; Gottfried, I.; Nakanishi, H.; Neiman, A. M.; Du G.; Frohman, M. A.; Bader, M. F., and Vitale, N.: Phospholipase D1 Production of Phosphatidic Acid at the Plasma Membrane Promotes Exocytosis of Large Dense-core Granules at a Late Stage. J. Biol. Chem., 282, 21746-21757 (2007).

Reference for silver enhancement procedure:

Although we have often reported the fluorescence quenching properties of Nanogold and other gold nanoparticles and their application to constructs such as molecular beacons, larger metal nanoparticles can also enhance the fluorescence of molecules positioned at specific distances from their surface. Joseph Lakowicz and group, who have pioneered this field, extend it to multiple metal particle constructs in a recent paper in Nano Letters. 20 nm silver particle dimers with single Cy5 molecules localized between coupled metal particles were prepared by hybridization of 20 nm silver particle-conjugated single-stranded oligonucleotides with double-length single-stranded oligonucleotides containing single Cy5 molecules. Image analysis revealed that the single-molecule fluorescence was enhanced 7-fold on the metal monomer and 13-fold on the metal dimer relative to the free Cy5-labeled oligonucleotide in the absence of metal. Lifetimes were shortened on the silver monomers and further shortened on the silver dimers, demonstrating the near-field interaction mechanism of fluorophore with the metal substrate. Finite-difference time-domain (FDTD) calculations were employed to study the distribution of electric field near the metal monomer and dimer, providing a method to study the coupling effect of metal particle on the fluorescence enhancement.


  • Zhang, J.; Fu, Y.; Chowdhury, M. H., and Lakowicz, J. R.: Metal-Enhanced Single-Molecule Fluorescence on Silver Particle Monomer and Dimer: Coupling Effect between Metal Particles. Nano Lett., 7, 2101-2107 (2007).

Priddy and colleagues, meanwhile, were doing correlative microscopy in reverse, using a structure previously determined by scanning transmission electron microscopy (STEM) and labeling with Nanogold®-labeled calmodulin as the basis for the design of fluorescence resonance energy transfer experiments to show the conformational substates of calmodulin bound to the phosphorylase kinase complex. As they describe in a recent Protein Science paper, they exchanged the delta subunits of the phosphorylase kinase (PhK) complex with a CaM double-mutant derivatized with a fluorescent donoracceptor pair (CaM-DA) to assess the conformational substates of PhKd by single molecule fluorescence resonance energy transfer (FRET) with and without Ca2+. The exchanged subunits were determined to occupy distinct conformations, depending on the absence or presence of Ca2+, as signaled by alterations of the compact, mid-length, and extended populations of their FRET distance distributions. The combined predominant mid-length and less common compact conformations of PhKd became less abundant in the presence of Ca2+, with the delta subunits assuming more extended conformations. This behavior contrasts with the compact forms commonly observed for many of CaMs Ca2+-dependent interactions with other proteins. Furthermore, the conformational distributions of the exchanged PhKdelta subunits were distinct from those of CaM-DA free in solution, ±Ca2+, as well as from exogenous CaM bound to the PhK complex as delta'. delta' is distinct from delta in that it binds only in the presence of Ca2+, but stoichiometrically and at a different location in the complex.


  • Priddy, T. S.; Price, E. S.; Johnson, C. K., and Carlson, G. M.: Single molecule analyses of the conformational substates of calmodulin bound to the phosphorylase kinase complex. Protein Sci., 16, 1017-1023 (2007).

Original Nanogold labeling and Scanning Transmission Electron Microscopy Structure:

  • Traxler, K. W.; Norcum, M. T.; Hainfeld, J. F., and Carlson, G. M.: Direct visualization of the calmodulin subunit of phosphorylase kinase via electron microscopy following subunit exchange. J. Struct. Biol., 135, 231238 (2001).

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