YidC-driven membrane insertion of single fluorescent Pf3 coat proteins

The membrane insertion of single bacteriophage Pf3 coat proteins was observed by confocal fluorescence microscopy. Within seconds after addition of the purified and fluorescently labeled protein to liposomes or proteoliposomes containing the purified and reconstituted membrane insertase YidC of Escherichia coli, the translocation of the labeled residue was detected. The 50-amino-acid-long Pf3 coat protein was labeled with Atto520 and inserted into the proteoliposomes. Translocation of the dye into the proteoliposome was revealed by quenching the fluorescence outside of the vesicles. This allowed us to distinguish single Pf3 coat proteins that only bound to the surface of the liposomes from proteins that had inserted into the bilayer and translocated the dye into the lumen. The Pf3 coat protein required the presence of the YidC membrane insertase, whereas mutants that have a membrane-spanning region with an increased hydrophobicity were autonomously inserted into the liposomes without YidC.     

Controlled three-dimensional immobilization of biomolecules on chemically patterned surfaces

We used electron-beam lithography to fabricate chemical nanostructures, i.e. amino groups in aromatic self-assembled monolayers (SAMs) on gold surfaces. The amino groups are utilized as reactive species for mild covalent attachment of fluorescently labeled proteins. Since non-radiative energy transfer results in strong quenching of fluorescent dyes in the vicinity of the metal surfaces, different labeling strategies were investigated. Spacers of varying length were introduced between the gold surface and the fluorescently labeled proteins. First, streptavidin was directly coupled to the amino groups of the SAMs via a glutaraldehyde linker and fluorescently labeled biotin (X-Biotin) was added, resulting in a distance of approximately 2 nm between the dyes and the surface. Scanning confocal fluorescence images show that efficient energy transfer from the dye to the surface occurs, which is reflected in poor signal-to-background (S/B) ratios of approximately 1. Coupling of a second streptavidin layer increases the S/B-ratio only slightly to approximately 2. The S/B-ratio of the fluorescence signals could be further increased to approximately 4 by coupling of an additional fluorescently labeled antibody layer. Finally, we introduced tetraethylenepentamine as functional spacer molecule to diminish fluorescence quenching by the surface. We demonstrate that the use of this spacer in combination with multiple antibody layers enables the controlled fabrication of highly fluorescent three-dimensional nanostructures with S/B-ratios of >20. The presented technique might be used advantageously for the controlled three-dimensional immobilization of single protein or DNA molecules and the well-defined assembly of protein complexes.     

Directional surface plasmon-coupled emission: application for an immunoassay in whole blood

We present a new approach for performing fluorescence immunoassay in whole blood using fluorescently labeled anti-rabbit immunoglobulin G (IgG) on a silver surface. This approach, which is based on surface plasmon-coupled emission (SPCE), provides increased sensitivity and substantial background reduction due to exclusive selection of the signal from the fluorophores located near a bioaffinity surface. This article describes the effect of an optically dense sample matrix, namely human whole blood and serum, on the intensity of the SPCE. An antigen (rabbit IgG) was adsorbed to a slide covered with a thin silver metal layer, and the SPCE signal from the fluorophore-labeled anti-rabbit antibody, binding to the immobilized antigen, was detected. The effect of the sample matrix (buffer, human serum, or human whole blood) on the end-point immunoassay SPCE signal was studied. It was demonstrated that the kinetics of binding could be monitored directly in whole blood or serum. The results showed that human serum and human whole blood attenuate the SPCE end-point signal and the immunoassay kinetic signal only approximately two- and threefold, respectively, as compared with buffer, resulting in signals that are easily detectable even in whole blood. The high optical absorption of the hemoglobin can be tolerated because only fluorophores within a couple of hundred nanometers from the metallic film contribute to SPCE. Excited fluorophores outside the 200-nm layer do not contribute to SPCE, and their free space emission is not transmitted through the opaque metallic film into the glass substrate. We believe that SPCE has the potential of becoming a powerful approach for performing immunoassays based on surface-bound analytes or antibodies for many biomarkers directly in dense samples such as whole blood with no need for washing steps.     

Low-Cost Plasmonic Carbon Spacer for Surface Plasmon-Coupled Emission Enhancements and Ethanol Detection: a Smartphone Approach

Surface plasmon-coupled emission (SPCE) has led to significant advancements in analytical techniques on account of its unique characteristics that include highly polarized photon-sorting ability. In this study, we report the use of a low-cost activated carbon as a plasmonic spacer in the SPCE substrate for achieving 30-fold enhancement in fluorescence emission. We extend the use of this spacer in the presence of Rhodamine B Base, a lactone dye as the sensing material for smartphone-based ethanol detection on the SPCE platform. Ethanol detection from 1 to 6% concentration highlights the potential use of this technique in monitoring fermentation processes.     

Lateral mobility of integral proteins in red blood cell tethers.

The red blood cell membrane is a complex material that exhibits both solid- and liquidlike behavior. It is distinguished from a simple lipid bilayer capsule by its mechanical properties, particularly its shear viscoelastic behavior and by the long-range mobility of integral proteins on the membrane surface. Subject to sufficiently large extension, the membrane loses its shear rigidity and flows as a two-dimensional fluid. These experiments examine the change in integral protein mobility that accompanies the mechanical phenomenon of extensional failure and liquidlike flow. A flow channel apparatus is used to create red cell tethers, hollow cylinders of greatly deformed membrane, up to 36-microns long. The diffusion of proteins within the surface of the membrane is measured by the technique of fluorescence redistribution after photobleaching (FRAP). Integral membrane proteins are labeled directly with a fluorescein dye (DTAF). Mobility in normal membrane is measured by photobleaching half of the cell and measuring the rate of fluorescence recovery. Protein mobility in tether membrane is calculated from the fluorescence recovery rate after the entire tether has been bleached. Fluorescence recovery rates for normal membrane indicate that more than half the labeled proteins are mobile with a diffusion coefficient of approximately 4 x 10(-11) cm2/s, in agreement with results from other studies. The diffusion coefficient for proteins in tether membrane is greater than 1.5 x 10(-9) cm2/s. This dramatic increase in diffusion coefficient indicates that extensional failure involves the uncoupling of the lipid bilayer from the membrane skeleton.     

Directional surface plasmon-coupled emission from a 3 nm green fluorescent protein monolayer

High-sensitivity detection schemes are of great interest for a number of applications. Unfortunately, such schemes are usually high-cost. We demonstrate a low-cost approach to a high-sensitivity detection scheme based on surface plasmon-coupled emission (SPCE). The SPCE of a monomolecular layer of green fluorescent protein (GFP) is reported here. The protein was electrostatically attached to a thin, SiO(2)-protected silver film deposited on a quartz substrate. The visible, directional emission of GFP was observed at a sharp, well-defined angle of 47.5 degrees from the normal to the coupling prism, and the spectrum corresponded to that of GFP. The SPCE resulting from the reverse Kretschmann configuration showed a 12-fold enhancement over the free space fluorescence. The directional emission was 97% p-polarized. The directionality and high polarization can be coupled with the intrinsic spectral resolution of SPCE to be used in the design miniaturized spectrofluorometers. The observation of SPCE in the visible region of the spectrum from a monolayer of protein opens up new possibilities in protein-based sensing.     

Multi-wavelength immunoassays using surface plasmon-coupled emission

We describe a new method for multi-wavelength immunoassays using surface plasmon-coupled emission (SPCE). This phenomenon is coupling of excited fluorophores with a nearby thin metal film, in our case silver, resulting in strongly directional emission into the underlying glass substrate. The angle at which the radiation propagate through the prism depends on the surface plasmon angle for the relevant wavelength. These angles depend on emission wavelength, allowing measurement of multiple analytes using multiple emission wavelengths. We demonstrated this possibility using antibodies labeled with either Rhodamine Red-X or AlexaFluor 647. These antibodies were directed against an antigen protein bound to the silver surface. The emission from each labeled antibody occurred at a different angle on the glass prism, allowing independent measurement of surface binding of each antibody. This method of SPCE immunoassays can be readily extended to 4 or more wavelengths.     

Fluorescent labeling of proteins with amine-specific 1,3,2-(2H)-dioxaborine polymethine dye

A novel water-soluble amine-reactive dioxaborine trimethine dye was synthesized in a good yield and characterized. The potential of the dye as a specific reagent for protein labeling was demonstrated with bovine serum albumin and lysozyme. Its interaction with proteins was studied by fluorescence spectroscopy and gel electrophoresis. The covalent binding of this almost nonfluorescent dye to proteins results in a 75- to 78-fold increase of its emission intensity accompanied by a red shift of the fluorescence emission maximum by 27 to 45 nm, with fluorescence wavelengths of labeled biomolecules being more than 600 nm. The dye does not require activation for the labeling reaction and can be used in a variety of bioassay applications.     

In vivo fluorescence labeling of plasma membrane from plant tissue and cells

Eosin-5-maleimide (EMA) is a membrane impermeable thiol reagent and was used to label proteins of the outer surface of the plasma membrane from intact cells and plant tissue. The covalent reaction of the fluorescent dye with cysteine residues of proteins allowed the visualization of the labeled cell compartment by microscopy, as well as in membrane preparations of cell homogenates from corn, squash and carrots. Density gradients from a resuspended membrane pellet (1000–15 000 × g) showed a fluorescent band at 40–42% (w/w) sucrose in all specimens. This EMA-enriched zone was well separated from the protein maximum and from membranes, which could be classified as tonoplast, golgi apparatus and mitochondria by its marker enzymes. A conformity of the fluorescence maximum with maximum enzyme activity of the plasma membrane marker (UDPG)-steroyl-glucosyl transferase (SGT) could be observed in a few cases (e.g. corn), although SGT activity varied considerably (32–40% sucrose) according to the object and the tissue used.Microscopic analysis of the labeled cells detected a strong fluorescence in the region of the cell wall and the plasma membrane. The use of EMA as a tool for the labeling and identification of plant plasma membranes is discussed.     

Signal enhancement of surface plasmon-coupled emission (SPCE) with the evanescent field of surface plasmons on a bimetallic paraboloid biochip

We present a novel approach to the enhancement of surface plasmon-coupled emission (SPCE) using surface plasmon excitation in a bimetal (Ag/Au) layer and we validate the enhancement by presenting the results of a model human IgG immunoassay. Theoretical calculations using Fresnel's equations have been carried out to determine the optimum bimetallic composition and the resulting electric field enhancement. Signal enhancement of SPCE was confirmed using a range of bimetallic layers which were deposited on the surface of a high collection efficiency polymer array biochip and subsequently immobilized with Alexa Fluor 647 labeled anti-human IgG. The bimetallic film of Ag/Au (36/10nm) was determined as an optimum substrate for maximum SPCE signal which was a compromise between the long-term stability of the metal layer and the optimized evanescent field enhancement. An enhanced dose-dependent response was also demonstrated which was ～3 times greater than that detected with a pure gold layer. A human IgG immunoassay showed a dose-dependent response yielding a limit of detection of 1pg/ml by the 3σ rule. The improved performance of the bimetal layer compared to that of an assay carried out on a pure gold layer is attributed to the enhanced evanescent field intensity of surface plasmons in the bimetal combination which excites more fluorescence hence producing an enhanced SPCE signal. This result demonstrates the potential of the SPCE-based array biochips as a sensitive and high-throughput analysis platform for biomolecular interactions.     

Proximity of the protein moiety of a GPI-anchored protein to the membrane surface: a FRET study

GPI-anchored proteins are ubiquitous on the eukaryotic cell surface, where they are involved in a variety of functions ranging from adhesion to enzymatic catalysis. Indirect evidence suggests that the GPI anchor may hold the protein close to the plasma membrane; however, there is a lack of direct information on the proximity of the protein portion of GPI-anchored proteins to the bilayer surface. The present study uses fluorescence resonance energy transfer (FRET) to address this important problem. The GPI-anchored ectoenzyme placental alkaline phosphatase (PLAP) was purified from a plasma membrane extract of human placental microsomes without the use of butanol. The protein was fluorescently labeled at the N-terminus with 7-(dimethylamino)coumarin-4-acetic acid succinimidyl ester (DMACA-SE) or Oregon Green 488 succinimidyl ester (OG488-SE), and each was reconstituted by detergent dilution into defined lipid bilayer vesicles containing an increasing mole fraction of a fluorescent lipid probe. The fluorescence of the labeled PLAP donors was quenched in a concentration-dependent manner by the lipid acceptors. The energy transfer data were analyzed using an approach that describes FRET between a uniform distribution of donors and acceptors in an infinite plane. The distance of closest approach between the protein moiety of PLAP and the lipid-water interfacial region of the bilayer was estimated to be smaller than 10-14 A. This indicates that the protein portion of PLAP is located very close to the lipid bilayer, possibly resting on the surface. This contact may allow transmission of structural changes from the membrane surface to the protein, which could influence the behavior and catalytic properties of GPI-anchored proteins.     

Single molecular observation of the interaction of GroEL with substrate proteins.

To understand the mechanism of GroEL-assisted protein folding, we observed the interaction of fluorescence-labeled GroEL with fluorescence-labeled substrate proteins at the single molecule level by total internal reflection fluorescence microscopy. GroEL with a A133C mutation in the equatorial domain was labeled with a fluorescent dye, tetramethylrhodamine. As substrate proteins, we used the largely denatured and partly denatured forms of bovine beta-lactoglobulin, both labeled with another fluorescent dye, Cy5. The complexes formed by GroEL with these substrates were characterized by size-exclusion gel chromatography. The recovered complexes were then observed by fluorescence microscopy. For both substrates, agreement of the fluorescent spots for tetramethylrhodamine and Cy5 indicated formation of the complex at the single molecule level. Similar observation of macroscopic binding by size-exclusion chromatography and microscopic binding by the fluorescence microscopy was done for the folding intermediate of Cy5-labeled bovine rhodanese. The fluorescence microscopy opens a new avenue for studying the interaction of GroEL with substrate proteins.     

Radiative decay engineering 3. Surface plasmon-coupled directional emission

A new method of fluorescence detection that promises to increase sensitivity by 20- to 1000-fold is described. This method will also decrease the contribution of sample autofluorescence to the detected signal. The method depends on the coupling of excited fluorophores with the surface plasmon resonance present in thin metal films, typically silver and gold. The phenomenon of surface plasmon-coupled emission (SPCE) occurs for fluorophores 20-250 nm from the metal surface, allowing detection of fluorophores over substantial distances beyond the metal-sample interface. SPCE depends on interactions of the excited fluorophore with the metal surface. This interaction is independent of the mode of excitation; that is, it does not require evanescent wave or surface-plasmon excitation. In a sense, SPCE is the inverse process of the surface plasmon resonance absorption of thin metal films. Importantly, SPCE occurs over a narrow angular distribution, converting normally isotropic emission into easily collected directional emission. Up to 50% of the emission from unoriented samples can be collected, much larger than typical fluorescence collection efficiencies near 1% or less. SPCE is due only to fluorophores near the metal surface and may be regarded as emission from the induced surface plasmons. Autofluorescence from more distal parts of the sample is decreased due to decreased coupling. SPCE is highly polarized and autofluorescence can be further decreased by collecting only the polarized component or only the light propagating with the appropriate angle. Examples showing how simple optical configurations can be used in diagnostics, sensing, or biotechnology applications are presented. Surface plasmon-coupled emission is likely to find widespread applications throughout the biosciences.     

Use of fluorescence decay times of 8-ANS-protein complexes to study the conformational transitions in proteins which unfold through the molten globule state

The conformational transitions starting with the native protein, passing the molten globule state and finally approaching the unfolded state of proteins was investigated for bovine carbonic anhydrase B (BCAB) and human -lactalbumin (-HLA) by means of fluorescence decay time measurements of the dye 8-anilinonaphthalene-1-sulphonic acid (8-ANS). Stepwise denaturation was realized by using the denaturant guanidinium chloride (GdmCl). It was shown that 8-ANS bound with protein yields a double-exponential fluorescence decay, where both decay times considerably exceed the decay time of free 8-ANS in water. This finding reflects the hydrophobic environment of the dye molecules attached to the proteins.

The fluorescence lifetime of the short-time component is affected by protein association and can be effectively quenched by acrylamide, indicating that 8-ANS molecules preferentially bind at the protein surface. The fluorescence lifetime of the long-time component is independent of the protein and acrylamide concentration and may be related to protein-embedded dye molecules.

Changes of the long lifetime component upon GdmCl-induced denaturation and unfolding of BCAB and -HLA correlate well with overall changes of the protein conformation. The transition from native protein to the molten globule state is accompanied by an increase of the number of protein-embedded 8-ANS molecules, while the number of dye molecules located at the protein surface decreases. For the transition from the molten globule to the unfolded state was the opposite behaviour observed.     

Rapid purification of mammalian 70,000-dalton stress proteins: affinity of the proteins for nucleotides.

A new and rapid purification procedure has been developed for the mammalian 70,000-dalton (70-kDa) heat-shock (or stress) proteins. Both the constitutive 73-kDa protein and the stress-induced 72-kDa protein have been purified by a two-step procedure employing DE52 ion-exchange chromatography followed by affinity chromatography on ATP-agarose. The two proteins, present in approximately equal amounts in either the 12,000 X g supernatant or pellet of hypotonically lysed heat-shock-treated HeLa cells, were found to copurify in relatively homogenous form. The purified proteins were covalently labeled with the fluorescent dye tetramethylrhodamine isothiocyanate, and the fluorescently labeled proteins were introduced back into living rat embryo fibroblasts via microinjection. The microinjected cells maintained at 37 degrees C showed only diffuse nuclear and cytoplasmic fluorescence. After heat-shock treatment of the cells, fluorescence was observed throughout the nucleus and more prominently within the nucleolus. This result is consistent with our earlier indirect immunofluorescence studies which showed a nuclear and nucleolar distribution of the endogenous 72-kDa stress protein in heat-shock-treated mammalian cells. The result also indicates that, for at least the 72-kDa protein, (i) the protein has been purified in apparently "native" form and (ii) its nucleolar distribution is stress dependent.     

High resolution clear native electrophoresis for in-gel functional assays and fluorescence studies of membrane protein complexes

Clear native electrophoresis and blue native electrophoresis are microscale techniques for the isolation of membrane protein complexes. The Coomassie Blue G-250 dye, used in blue native electrophoresis, interferes with in-gel fluorescence detection and in-gel catalytic activity assays. This problem can be overcome by omitting the dye in clear native electrophoresis. However, clear native electrophoresis suffers from enhanced protein aggregation and broadening of protein bands during electrophoresis and therefore has been used rarely. To preserve the advantages of both electrophoresis techniques we substituted Coomassie dye in the cathode buffer of blue native electrophoresis by non-colored mixtures of anionic and neutral detergents. Like Coomassie dye, these mixed micelles imposed a charge shift on the membrane proteins to enhance their anodic migration and improved membrane protein solubility during electrophoresis. This improved clear native electrophoresis offers a high resolution of membrane protein complexes comparable to that of blue native electrophoresis. We demonstrate the superiority of high resolution clear native electrophoresis for in-gel catalytic activity assays of mitochondrial complexes I-V. We present the first in-gel histochemical staining protocol for respiratory complex III. Moreover we demonstrate the special advantages of high resolution clear native electrophoresis for in-gel detection of fluorescent labeled proteins labeled by reactive fluorescent dyes and tagged by fluorescent proteins. The advantages of high resolution clear native electrophoresis make this technique superior for functional proteomics analyses.     

Anomalous fluorescence enhancement of Cy3 and cy3.5 versus anomalous fluorescence loss of Cy5 and Cy7 upon covalent linking to IgG and noncovalent binding to avidin

This study provides a critical examination of protein labeling with Cy3, Cy5, and other Cy dyes. Two alternate situations were tested. (i) Antibodies were covalently labeled with Cy dye succinimidyl ester at various fluorophore/protein ratios and the fluorescence of the labeled antibodies was compared to that of free Cy dye. (ii) Fluorescent biotin derivatives were synthesized by derivatizing ethylenediamine with one biotin and one Cy3 (or Cy5) residue. The fluorescence properties of these biotin-Cy dye conjugates were examined at all ligand/(strept)avidin ratios (0 相似文献   

Dilution of protein‐surfactant complexes: A fluorescence study

Dilution of protein–surfactant complexes is an integrated step in microfluidic protein sizing, where the contribution of free micelles to the overall fluorescence is reduced by dilution. This process can be further improved by establishing an optimum surfactant concentration and quantifying the amount of protein based on the fluorescence intensity. To this end, we study the interaction of proteins with anionic sodium dodecyl sulfate (SDS) and cationic hexadecyl trimethyl ammonium bromide (CTAB) using a hydrophobic fluorescent dye (sypro orange). We analyze these interactions fluourometrically with bovine serum albumin, carbonic anhydrase, and beta‐galactosidase as model proteins. The fluorescent signature of protein–surfactant complexes at various dilution points shows three distinct regions, surfactant dominant, breakdown, and protein dominant region. Based on the dilution behavior of protein–surfactant complexes, we propose a fluorescence model to explain the contribution of free and bound micelles to the overall fluorescence. Our results show that protein peak is observed at 3 mM SDS as the optimum dilution concentration. Furthermore, we study the effect of protein concentration on fluorescence intensity. In a single protein model with a constant dye quantum yield, the peak height increases with protein concentration. Finally, addition of CTAB to the protein–SDS complex at mole fractions above 0.1 shifts the protein peak from 3 mM to 4 mM SDS. The knowledge of protein–surfactant interactions obtained from these studies provides significant insights for novel detection and quantification techniques in microfluidics.     

Analysis of allosteric signal transduction mechanisms in an engineered fluorescent maltose biosensor

We previously reported the construction of a family of reagentless fluorescent biosensor proteins by the structure-based design of conjugation sites for a single, environmentally sensitive small molecule dye, thus providing a mechanism for the transduction of ligand-induced conformational changes into a macroscopic fluorescence observable. Here we investigate the microscopic mechanisms that may be responsible for the macroscopic fluorescent changes in such Fluorescent Allosteric Signal Transduction (FAST) proteins. As case studies, we selected three individual cysteine mutations (F92C, D95C, and S233C) of Escherichia coli maltose binding protein (MBP) covalently labeled with a single small molecule fluorescent probe, N-((2-iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole (NBD), each giving rise to a robust FAST protein with a distinct maltose-dependent fluorescence response. The fluorescence emission intensity, anisotropy, lifetime, and iodide-dependent fluorescence quenching were determined for each conjugate in the presence and absence of maltose. Structure-derived solvent accessible surface areas of the three FAST proteins are consistent with experimentally observed quenching data. The D95C protein exhibits the largest fluorescence change upon maltose binding. This mutant was selected for further characterization, and residues surrounding the fluorophore coupling site were mutagenized. Analysis of the resulting mutant FAST proteins suggests that specific hydrogen-bonding interactions between the fluorophore molecule and two tyrosine side-chains, Tyr171 and Tyr176, in the open state but not the closed, are responsible for the dramatic fluorescence response of this construct. Taken together these results provide insights that can be used in future design cycles to construct fluorescent biosensors that optimize signaling by engineering specific hydrogen bonds between a fluorophore and protein.     

New donor-acceptor pair for fluorescent immunoassays by energy transfer

A novel F?rster donor-acceptor dye pair for an immunoassay based on resonance energy transfer (RET) is characterized with respect to its photophysical properties. As donor and acceptor, we chose the long-wavelength excitable cyanine dyes Cy5 and Cy5.5, respectively. Due to the perfect spectral overlap, an exceptionally high R(0) value of 68.7 A is obtained in solution. For biochemical applications, antibodies (IgG) are labeled with Cy5, while a tracer for competitive binding is synthesized by labeling bovine serum albumin (BSA) with an analyte derivative and Cy5.5. Binding the dyes to proteins at a low dye/protein ratio increases the fluorescence lifetimes and quantum yields, leading to an enhanced R(0) value of 85.2 A. At higher dye/protein ratios, the formation of nonfluorescent dimeric species causes a decrease in the fluorescence lifetime and quantum yield due to RET from monomeric dyes to dimers within one protein molecule. The F?rster distances could be calculated using the dimer absorption spectra to 83.9 and 83.6 A for Cy5 and Cy5.5, respectively. Upon binding of the Cy5-labeled IgG to the tracer, efficient quenching of Cy5 fluorescence is observed. Steady-state and time-resolved measurements reveal that approximately 50% of the quenching results in F?rster-type RET, while the residual quenching effect is caused by static quenching processes. The applicability of this dye pair is demonstrated in a homogeneous competitive immunoassay for the pesticide simazine.