Fibrous projections from the core of a bacteriophage T7 procapsid

A cylindrical core previously demonstrated in a bacteriophage T7 procapsid (capsid I) has been further examined by electron microscopy. Fibrous extensions of the core have been observed; these fibers appear to connect the core to the capsid I envelope. After infection of a nonpermissive host with bacteriophage T7 amber mutant in any gene coding for a core protein, the resulting lysates contained more noncapsid assemblies of capsid envelope protein than did wild-type lysates; these assemblies had a mass two to at least 500 times greater than the mass of capsid I. This suggests that the internal core and fibers assist the assembly of subunits in the envelope of capsid I.     

Tandem mass spectrometry of intact GroEL-substrate complexes reveals substrate-specific conformational changes in the trans ring

It has been suggested that the bacterial GroEL chaperonin accommodates only one substrate at any given time, due to conformational changes to both the cis and trans ring that are induced upon substrate binding. Using electrospray ionization mass spectrometry, we show that indeed GroEL binds only one molecule of the model substrate Rubisco. In contrast, the capsid protein of bacteriophage T4, a natural GroEL substrate, can occupy both rings simultaneously. As these substrates are of similar size, the data indicate that each substrate induces distinct conformational changes in the GroEL chaperonin. The distinctive binding behavior of Rubisco and the capsid protein was further investigated using tandem mass spectrometry on the intact 800-914 kDa GroEL-substrate complexes. Our data suggest that even in the gas phase the substrates remain bound inside the GroEL cavity. The analysis revealed further that binding of Rubisco to the GroEL oligomer stabilizes the chaperonin complex significantly, whereas binding of one capsid protein did not have the same effect. However, addition of a second capsid protein molecule to GroEL resulted in a similar stabilizing effect to that obtained after the binding of a single Rubisco. On the basis of the stoichiometry of the GroEL chaperonin-substrate complex and the dissociation behavior of the two different substrates, we hypothesize that the binding of a single capsid polypeptide does not induce significant conformational changes in the GroEL trans ring, and hence the unoccupied GroEL ring remains accessible for a second capsid molecule.     

Binding of intercalating and groove-binding cyanine dyes to bacteriophage t5

The interaction between four related cyanine dyes and bacteriophage T5 is investigated with fluorescence and absorption spectroscopy. The dyes, which differ in size, charge, and mode of DNA-binding, penetrate the capsid and bind the DNA inside. The rate of association decreases progressively with increasing dye size, from a few minutes for YO to more than 50 h for YOYO (at 37 degrees C). The relative affinity for the phage DNA is a factor of about 0.2 lower than for the same T5-DNA when free in solution. Comparison of groove-bound BOXTO-PRO and intercalating YO-PRO shows that the reduced affinity is not due to DNA extension but perhaps influenced by competition with other cationic DNA-binding agents inside the capsid. Although, the extent of dye binding to the phages decreases with increasing external ionic strength, the affinity relative to free DNA increases, which indicates a comparatively weak screening of electrostatic interactions inside the phage. The rate of binding increases with increasing ionic strength, reflecting an increase in effective pore size of the capsid as electrostatic interactions are screened and/or a faster diffusion of the dye through the DNA matrix inside the capsid as the DNA affinity is reduced. A combination of electron microscopy, light scattering, and linear dichroism show that the phages are intact after YO-PRO binding, whereas a small degree of capsid rupture cannot be excluded with BOXTO-PRO.     

High-sensitivity staining of proteins for one- and two-dimensional gel electrophoresis using post migration covalent staining with a ruthenium fluorophore

This paper describes the use of a ruthenium complex ((bis(2,2'-bipyridine)-4'-methyl-4-carboxybipyridine-ruthenium-N-succidimyl ester-bis(hexafluorophosphate), abbreviated below as ASCQ_Ru) commercially available and chemically pure. This new ruthenium complex ASCQ_Ru brings an activated ester, allowing the selective acylation of amino acid side chain amines for the post migration staining of proteins separated in 1-DE and 2-DE. The protocol used is a simple three-step protocol fixing the proteins in the gel, staining and then washing, as no lengthy destaining step is required. First the critical staining step was optimized. Although in solution the best described pH for acylating proteins with this reagent is phosphate buffer at pH 7.0, we found that best medium for in-gel staining is unbuffered ACN/water solution (20/80 v/v). The two other steps are less critical and classical conditions are satisfactory: fixing with 7% acetic acid/10% ethanol solution and washing four times for 10 min with water. Sensitivity tests were performed using 1-DE on protein molecular weight markers. We obtained a higher sensitivity than SYPRO Ruby with a detection limit of 80 pg of protein per well. However, contrary to SYPRO Ruby, ASCQ_Ru exhibits a logarithmic dependency on the amount of protein. The dynamic range is similar to SYPRO Ruby and is estimated between three and four orders of magnitude. Finally, the efficiency of the post migration ASCQ_Ru staining for 2-D gel separation is demonstrated on the whole protein extract from human colon carcinoma cells lines HCT 116. ASCQ_Ru gave the highest number of spot detected compared to other common stains Colloidal CBB, SYPRO Ruby and Deep Purple.     

Background-free,fast protein staining in sodium dodecyl sulfate polyacrylamide gel using counterion dyes,zincon and ethyl violet

A background-free, fast protein staining method in polyacrylamide gel electrophoresis using an acidic dye, zincon (ZC) and a basic dye, ethyl violet (EV) is described. It is based on the counterion dye staining technique that employs two oppositely charged dyes to form an ion-pair complex in staining solution. The selective binding of free dye molecules to proteins in acidic solution produces bluish violet-colored bands. It is a rapid and end-point staining procedure, involving only fixing and staining steps that are completed in 1-1.5 h. The detection limit of this method is 8-15 ng of protein that is comparable to the sensitivity of the colloidal Coomassie Brilliant Blue G (CBBG) stain. Due to its sensitivity and speed, this stain may be more practical than any other dye-based stains for routine laboratory purposes.     

Functional characterization of two-dimensional gel-separated proteins using sequential staining

Proteins separated by two-dimensional (2-D) gel electrophoresis can be visualized using various protein staining methods. This is followed by downstream procedures, such as image analysis, gel spot cutting, protein digestion, and mass spectrometry (MS), to characterize protein expression profiles within cells, tissues, organisms, or body fluids. Characterizing specific post-translational modifications on proteins using MS of peptide fragments is difficult and labor-intensive. Recently, specific staining methods have been developed and merged into the 2-D gel platform so that not only general protein patterns but also patterns of phosphorylated and glycosylated proteins can be obtained. We used the new Pro-Q Diamond phosphoprotein dye technology for the fluorescent detection of phosphoproteins directly in 2-D gels of mouse leukocyte proteins, and Pro-Q Emerald 488 glycoprotein dye to detect glycoproteins. These two fluorescent stains are compatible with general protein stains, such as SYPRO Ruby stain. We devised a sequential procedure using Pro-Q Diamond (phosphoprotein), followed by Pro-Q Emerald 488 (glycoprotein), followed by SYPRO Ruby stain (general protein stain), and finally silver stain for total protein profile. This multiple staining of the proteins in a single gel provided parallel determination of protein expression and preliminary characterization of post-translational modifications of proteins in individual spots on 2-D gels. Although this method does not provide the same degree of certainty as traditional MS methods of characterizing post-translational modifications, it is much simpler, faster, and does not require sophisticated equipment and expertise in MS.     

Effective staining of tumor cells by coumarin-6 depends on the stoichiometry of cyclodextrin complex formation

In a comprehensive picture of inclusion complex formation of the highly fluorescent dye coumarin-6 (C6) and betacyclodextrin (beta-CD), which was obtained using various fluorescence spectroscopic methods, it was demonstrated that up to three beta-CD rings can thread on the rod like dye molecule. Interaction of coumarins and modified coumarins with cellular organelles or proteins has been reported in several publications. Especially 7-amino-coumarins are characterized by unique properties like high fluorescence quantum yield and are thus already used successfully in different areas, like staining of fluorescent nanoparticles. We could show that Coumarin-6 made soluble by complexation with beta-cyclodextrin is able to stain eukaryotic cells specifically dependent on their origin and cellular behaviour. The staining reaction is independent from pH, is photo stable, and shows no cross talk with proteins in the cytoplasm and other staining procedures or erythrocytes. Staining with coumarin 6/cyclodextrin complexes can thus be used for fast discrimination of different cell types. Importantly, it could be shown that the ideal staining reaction is dependent on the stoichiometry of the complex-formation.     

Automated protein analysis by online detection of laser-induced fluorescence in slab gels and 3-D geometry gels

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) still remains the most reliable and comprehensive analytical method for the evaluation of protein extracts. However, conventional SDS-PAGE is time-consuming and, thus, unpractical if several tens or hundreds of samples must be examined. We show that SDS-PAGE protein analysis can be automated using slab gel DNA sequencers and compare the instrument's performance with conventional SDS-PAGE in terms of resolution, sensitivity and sample capacity. Labeled protein bands are detected online by laser-induced fluorescence (LIF) and the acquired signals are electronically stored for further processing, avoiding gel staining and scanning. Appropriate software allows immediate display of recorded data and convenient evaluation. The method provides a higher sensitivity and dynamic range than conventional Coomassie-stained gels and the resolution of proteins with different masses is independent of the polyacrylamide concentration. Internal markers can also be used for direct quantification and assignment of the molecular masses. Additionally, we present a novel electrophoresis instrument for the simultaneous separation and online LIF detection of all samples of a microtiterplate in parallel lanes in a 3-D geometry gel cylinder. The specific gel thermostatting concept prevents irregular sample migration (smiling) and improves the reproducibility and comparability of individual separation patterns. In combination with the expected large capacity of 384 or 1,536 samples, this makes the instrument a valuable tool for high-throughput comparative screening applications.     

Sensitive silver staining of protein in sodium dodecyl sulfate-polyacrylamide gels using an azo dye, calconcarboxylic acid, as a silver-ion sensitizer

A highly sensitive silver staining method for detecting proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was developed. It is based on the silver nitrate staining method but also employs an azo dye, calconcarboxylic acid (NN), as a silver-ion sensitizer. It increases silver binding on protein bands or spots by the formation of a silver-dye complex and also increases the reducing power of silver ions to metallic silver by NN itself with formaldehyde. After a 2 h gel fixing step, the protocol including sensitization, silver-ion impregnation, and reduction steps can be completed in 1 h. The sensitivity is superior to that of silver stain with glutardialdehyde as a silver-ion sensitizer. The detection limit of NN-silver stain is 0.05-0.2 ng protein. Considering the high sensitivity without using glutardialdehyde, the NN-silver stain would be useful for routine silver staining of proteins.     

Why do Congo Red,Evans Blue,and Trypan Blue differ in their complexation properties?

Congo Red, Evans Blue, and Trypan Blue dyes were evaluated in terms of their ability to form supramolecular systems in water solution. A geometric transformation set was defined to construct a supramolecular model system composed of eight dye molecules. The stability of the constructed multimolecular systems was estimated by molecular dynamics using AMBER 4.1 and DISCOVER force fields. The results essentially confirm the tendency toward self‐assembly in each case. However, Congo Red and Evans Blue were found to form stable, continuous, ribbon‐like supramolecular organizations, whereas Trypan Blue self‐assembly appeared defective; some additional deviations from planarity seem to be the main reason for the disturbance in self‐assembling. The extra rotation around the azo bonds in the Trypan Blue molecule is slightly extorted by the close proximity of sulfonic groups. This may also be the direct cause of the observed deviation from symmetry in the molecule of this dye. The results confirm that the self‐assembling capability of the compounds studied correlates with the capacity to complex proteins, supporting the idea that supramolecularity may create specific ligation properties. © 2000 John Wiley & Sons, Inc. J Comput Chem 21: 656–667, 2000     

Fast visible dye staining of proteins in one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gels compatible with matrix assisted laser desorption/ionization-mass spectrometry

A fast and matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) compatible protein staining method in one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1- and 2-D SDS-PAGE) is described. It is based on the counterion dye staining method that employs oppositely charged two dyes, zincon (ZC) and ethyl violet (EV) to form an ion-pair complex. The protocol, including fixing, staining and quick washing steps, can be completed in 1-1.5 h depending upon gel thickness. It has a sensitivity of 4-8 ng, comparable to that of colloidal Coomassie Brilliant Blue G (CBBG) staining with phosphoric acid in the staining solution. The counterion dye stain does not induce protein modifications that complicate interpretation of peptide mapping data from MS. Considering the speed, sensitivity and compatibility with MS, the counterion dye stain may be more practical than any other dye-based protein stains for routine proteomic researches.     

Promoted negative staining of proteins in SDS-PAGE using Eosin B compounded with magnesium chloride

In this study, we describe an effective visualizing technique for proteins in SDS-PAGE based on the organic dye, Eosin B, the sensitivity of which can be further strengthened by the addition of magnesium to the staining solution after electrophoresis. The newly developed protocol is low in cost and easily performed compared with the common methods for protein analysis in 1-D and 2-D gels. It provides a much better sensitivity (0.2 ng of single protein band) than that of imidazole-zinc negative stain for fixing and staining within 1 h, and an excellent performance in terms of compatibility with MALDI-TOF MS. The results show that similar identification scores and numbers of matched peptides were obtained by both methods. Furthermore, the effects of different metal salts on the quality of protein visualization by Eosin B were also investigated. Because of its sensitivity, stability, and safety, this stain may be a more practical method for protein determination in the routine laboratory.     

Electron transfer reaction in a single protein molecule observed by total internal reflection fluorescence microscopy

To observe an electron transfer (ET) process in a single protein molecule, we constructed a model system, Alexa-HCytb5, in which cytochrome b5 (Cytb5) is modified with a fluorescent probe, Alexa Fluor 647 dye. In this model system, intramolecular transfer of an electron from the Alexa dye to heme in Cytb5 is supposed to oxidize the probe and quench its fluorescence, and the ET reaction at the single-molecule level can be monitored as the intermittent change in the fluorescence intensity. Alexa-HCytb5 was fixed on the glass surface, and illumination of laser light by the total internal reflection resulted in blinking of the fluorescence from the single Alexa-HCytb5 molecule in the time scale of several hundred milliseconds. Each Alexa-HCytb5 molecule is characterized by its own rate constant of the blinking, corresponding to the ET rate constant at the single-molecule level, and its variation ranges between 1 and 10 s(-1). The current system thus enables us to visualize the ET reaction in the single protein molecule, and the protein ET reaction was found to be explained by the distribution of the rate constants. On the basis of the Marcus theory, we suggest that the origin of this rate distribution is the distance change associated with the structural fluctuation in the protein molecule.     

In-gel deglycosylation of sodiumdodecyl sulfate polyacrylamide gel electrophoresis-separated glycoproteins for carbohydrate estimation by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Mass determination by mass spectrometric methods (electrospray ionization mass spectrometry (ESI-MS), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS)) of sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)-separated proteins is a well known procedure and reliable protocols are available. In our efforts to use the established methods to determine the molecular mass of the disulfide bridged, heterodimeric glycoprotein GP3 and to determine the carbohydrate content of each protein subunit we developed an in-gel chemical deglycosylation method. For this purpose we established experimental conditions that allow maximum extraction of the high molecular mass protein subunits and developed a routine method to apply the HF-pyridine deglycosylation protocol to proteins isolated from polyacrylamide gel pieces. The novel protocol and extraction procedure described can be used to analyze O-glycosylated proteins up to 150 kDa after SDS-PAGE separation.     

Spectral imaging of single molecules by transmission grating-based epi-fluorescence microscopy

A spectral imaging method of single protein molecules labeled with a single fluorophore is presented. The method is based on a transmission grating and a routine fluorescence microscope. The bovine serum alubmin (BSA) and antiBSA molecules labeled with Alexa Fluor 488 and Alexa Fluor 594, respectively, are used as the model proteins. The fluorescence of single molecules is dispersed into zeroth-order spectrum and first-order spectrum by the transmission grating. Results show that the fluorescence emission spectrum of single molecule converted from the first-order spectral imaging is in good agreement with the bulk fluorescence spectrum. The spectral resolution of 2.4 nm/pixel is obtained, which is sufficient for identifying the molecular species in a multicomponent system.     

Direct Blue 71 staining of proteins bound to blotting membranes

A sensitive staining method for protein blots using Direct Blue 71 is described. It is based on the selective binding of dye molecules to proteins in acidic solution and produces bluish violet colored bands. It is a simple and rapid procedure, involving only staining and rinsing steps that occur within 7 min. The sensitivity of this method is 5-10 ng of protein on nitrocellulose (NC) and 10-20 ng on polyvinylidene difluoride (PVDF), which is tenfold better than that of the commonly used Ponceau S staining. Moreover, the staining is reversible for subsequent immunostaining, without impairing immunoreactivity. To remove the dye from the developed bands, changes in pH and hydrophobicity of the solvent are required. Due to its sensitivity, rapidity, simplicity, and low cost, this stain may be more practical than other dye-based stains or metal-based stains for routine laboratory purposes.     

A new photostable terrylene diimide dye for applications in single molecule studies and membrane labeling

A new terrylene diimide-based dye (WS-TDI) that is soluble in water has been synthesized, and its photophysical properties are characterized. WS-TDI forms nonfluorescing H-aggregates in water that show absorption bands being blue-shifted with respect to those of the fluorescing monomeric form. The ratio of monomeric WS-TDI to aggregated WS-TDI was determined to be 1 in 14 400 from fluorescence correlation spectroscopy (FCS) measurements, suggesting the presence of a large amount of soluble, nonfluorescent aggregates in water. The presence of a surfactant such as Pluronic P123 or CTAB leads to the disruption of the aggregates due to the formation of monomers in micelles. This is accompanied by a strong increase in fluorescence. A single molecule study of WS-TDI in polymeric films of PVA and PMMA reveals excellent photostability with respect to photobleaching, far above the photostability of other common water-soluble dyes, such as oxazine-1, sulforhodamine-B, and a water-soluble perylenediimide derivative. Furthermore, labeling of a single protein such as avidin is demonstrated by FCS and single molecule photostability measurements. The high tendency of WS-TDI to form nonfluorescent aggregates in water in connection with its high affinity to lipophilic environments is used for the fluorescence labeling of lipid membranes and membrane containing compartments such as artificial liposomes or endosomes in living HeLa cells. The superior fluorescence imaging quality of WS-TDI in such applications is demonstrated in comparison to other well-known membrane staining dyes such as Alexa647 conjugated with dextran and FM 4-64 lipophilic styryl dye.     

Determination of dye/protein ratios in a labeling reaction between a cyanine dye and bovine serum albumin by micellar electrokinetic chromatography using a diode laser-induced fluorescence detection

The degree of labeling, i.e., dye/protein ratio (D/P) is important for characterizing properties of dye labeling with proteins. A method for the determination of this ratio between a fluorescent cyanine dye and bovine serum albumin (BSA), based on the separation of the labeling mixture using micellar electrokinetic chromatography with diode laser-induced fluorescence detection, is described. Two methods for the determination of D/P were examined in this study. In these methods, a hydrolysis product and impurities, which are usually unfavorable compounds that are best excluded for protein analysis, were utilized to determine the amounts of dye bound to BSA. One is a direct method in which a ratio of the peak area of BSA to the total peak area of all the products produced in the labeling reaction was used for determining the average number of dye molecules bound to a single BSA molecule. The other is an indirect determination, which is based on diminution of all peak areas related to the products except for the labeled BSA. These methods were directly compared by means of a spectrophotometric method. The experimental results show that the indirect method is both reliable and sensitive. Therefore, D/P values can be determined at trace levels using the indirect method.     

A fluorescent derivatization method of proteins for the detection of low‐level impurities by microchip capillary gel electrophoresis

A novel pre‐chip fluorescent derivatization method is presented for protein sizing and quantification by microchip CGE. The derivatization reaction employed a water‐soluble and stable fluorescent dye and was performed under conditions that favored the formation of homogeneous reaction products. The method delivered in terms of protein sizing similar results as microchip CGE with on‐chip staining but showed an extended linear dynamic range for protein quantification encompassing four orders of magnitude. The sensitivity of the method was similar to standard silver‐stained planar gels. The characterization of derivatization reaction products by MS and preparative isoelectric focusing indicated that a constant degree of dye molecule tagging was obtained over a broad range of protein/dye ratios. The method allowed detecting and quantifying an impurity spiked into an antibody preparation down to a level of 0.05%. Advantages of this method compared with CGE approaches with pre‐column derivatization include a shorter analysis time and an increased robustness and ease of use.