In-gel Fluorescence Detection Research Articles

Detection and quantification of C-terminally tagged proteins by in-gel fluorescence

The analysis of recombinant proteins in complex solutions is often accomplished with tag-specific antibodies in western blots. Recently, I introduced an antibody-free alternative wherein tagged proteins are visualized directly within polyacrylamide gels. For this, I used the protein ligase Connectase to selectively attach fluorophores to target proteins possessing an N-terminal recognition sequence. In this study, I extend this methodology to encompass the detection and quantification of C-terminally tagged proteins. Similar to the N-terminal labeling method, this adapted procedure offers increased speed, heightened sensitivity, and an improved signal-to-noise ratio when compared to western blots. It also eliminates the need for sample-specific optimization, enables more consistent and precise quantifications, and uses freely available reagents. This study broadens the applicability of in-gel fluorescence detection methods and thereby facilitates research on recombinant proteins.

Efficient flavinylation of glycosomal fumarate reductase by its own ApbE domain in Trypanosoma brucei.

A subset of flavoproteins has a covalently attached flavin prosthetic group enzymatically attached via phosphoester bonding. In prokaryotes, this is catalysed by alternative pyrimidine biosynthesis E (ApbE) flavin transferases. ApbE-like domains are present in few eukaryotic taxa, for example the N-terminal domain of fumarate reductase (FRD) of Trypanosoma, a parasitic protist known as a tropical pathogen causing African sleeping sickness. We use the versatile reverse genetic tools available for Trypanosoma to investigate the flavinylation of glycosomal FRD (FRDg) invivo in the physiological and organellar context. Using direct in-gel fluorescence detection of covalently attached flavin as proxy for activity, we show that the ApbE-like domain of FRDg has flavin transferase activity invivo. The ApbE domain is preceded by a consensus flavinylation target motif at the extreme N terminus of FRDg, and serine 9 in this motif is essential as flavin acceptor. The preferred mode of flavinylation in the glycosome was addressed by stoichiometric expression and comparison of native and catalytically inactive ApbE domains. In addition to the trans-flavinylation activity, the ApbE domain catalyses the intramolecular cis-flavinylation with at least fivefold higher efficiency. We discuss how the higher efficiency due to unusual fusion of the ApbE domain to its substrate protein FRD may provide a selective advantage by faster FRD biogenesis during rapid metabolic adaptation of trypanosomes. The first 37 amino acids of FRDg, including the consensus motif, are sufficient as flavinylation target upon fusion to other proteins. We propose FRDg(1-37) as 4-kDa heat-stable, detergent-resistant fluorescent protein tag and suggest its use as a new tool to study glycosomal protein import.

Profiling protein expression in Klebsiella pneumoniae with a carbohydrate-based covalent probe

We report the application of a covalent probe based on a d-glucosamine scaffold for the profiling of the bacterial pathogen Klebsiella pneumoniae. Incubation of K. pneumoniae lysates with the probe followed by electrophoretic separation and in-gel fluorescence detection allowed the generation of strain-specific signatures and the differentiation of a carbapenem-resistant strain. The labelling profile of the probe was independent of its anomeric configuration and included several low-abundance proteins not readily detectable by conventional protein staining. Initial target identification experiments by mass spectrometry suggest that target proteins include several carbohydrate-recognising proteins, which indicates that the sugar scaffold may have a role for target recognition.

In-gel fluorescence detection by DNA polymerase elongation.

Fluorescence-based DNA readouts are increasingly important in biological research, owing to enhanced analytical sensitivity and multiplexing capability. In this study, we characterize an in-gel polymerase elongation process to understand the reaction kinetics and transport limitations, and we evaluate DNA sequence design to develop signal amplification strategies. Using fluorescently labeled nucleotides, we scrutinize polymerase elongation on single-stranded overhangs of DNA immobilized in polyacrylamide hydrogels. When polymerase elongation reactions were carried out with reactants diffused into the gels, we observed reaction completion after 2 h, indicating that the process was efficient but much slower than that predicted by models. Confocal microscopy revealed a nonuniform post-reaction fluorescence profile of the elongated DNA throughout the depth of the gel and that the time for complete fluorescence penetration was proportional to the immobilized DNA concentration. These observations suggest retarded diffusion of the polymerase, attributable to interactions between diffusing polymerase and immobilized DNA. This study will ultimately inform assay design by providing insight into the reaction completion time to ensure spatial uniformity of the fluorescence signal. In agreement with our hypothesis that incorporation of multiple labeled nucleotides per DNA strand results in an increased signal, incorporation of four labeled nucleotides resulted in a 2.3-fold increase in fluorescence intensity over one labeled nucleotide. Our results further suggest that the fluorescence signal increases with spacing between labeled nucleotides, validating the number of and spacing between labeled nucleotides as tunable parameters for signal amplification. In-gel polymerase-based fluorescence readout is promising for signal amplification when considering both transport limitations and DNA sequence design.

Membrane Protein Production and Purification from Escherichia coli and Sf9 Insect Cells.

A major obstacle to studying membrane proteins by biophysical techniques is the difficulty in producing sufficient amounts of materials for functional and structural studies. To overexpress the target membrane protein heterologously, especially an eukaryotic protein, a key step is to find the optimal host expression system and perform subsequent expression optimization. In this chapter, we describe protocols for screening membrane protein production using bacterial and insect cells, solubilization screening, large-scale production, and commonly used affinity chromatography purification methods. We discuss general optimization conditions, such as promoters and tags, and describe current techniques that can be used in any laboratory without specialized expensive equipment. Especially for insect cells, GFP fusions are particularly useful for localization and in-gel fluorescence detection of the proteins on SDS-PAGE. We give detailed protocols that can be used to screen the best expression and purification conditions for membrane protein study.

An efficient screening method for purifying and crystallizing membrane proteins using modified clear-native PAGE

Membrane proteins, such as G-protein coupled receptors, control communication between cells and their environments and are indispensable for many cellular functions. Nevertheless, structural studies on membrane proteins lag behind those on water-soluble proteins, due to their low structural stability, making it difficult to obtain crystals for X-ray crystallography. Optimizing conditions to improve the stability of membrane proteins is essential for successful crystallization. However, the optimization usually requires large amounts of purified samples, and it is a time-consuming and trial-and-error process. Here, we report a rapid method for precrystallization screening of membrane proteins using Clear Native polyacrylamide gel electrophoresis (CN-PAGE) with the modified Coomassie Brilliant Blue G-250 (mCBB) stain that was reduced in sodium formate. A2A adenosine receptor (A2AAR) was selected as a target membrane protein, for which we previously obtained the crystal structure using an antibody, and was expressed as a red fluorescent protein fusion for in-gel fluorescence detection. The mCBB CN-PAGE method enabled the optimization of the solubilization, purification, and crystallization conditions of A2AAR using the solubilized membrane fraction expressing the protein without purification procedures. These data suggest the applicability of mCBB CN-PAGE technique to a wide variety of integral membrane proteins.

Design of an Activity-Based Probe for Human Neutrophil Elastase: Implementation of the Lossen Rearrangement To Induce Förster Resonance Energy Transfers.

Human neutrophil elastase is an important regulator of the immune response and plays a role in host defense mechanisms and further physiological processes. The uncontrolled activity of this serine protease may cause severe tissue alterations and impair inflammatory states. The design of an activity-based probe for human neutrophil elastase reported herein relies on a sulfonyloxyphthalimide moiety as a new type of warhead that is linker-connected to a coumarin fluorophore. The inhibitory potency of the activity-based probe was assessed against several serine and cysteine proteases, and the selectivity for human neutrophil elastase (Ki = 6.85 nM) was determined. The adequate fluorescent tag of the probe allowed for the in-gel fluorescence detection of human neutrophil elastase in the low nanomolar range. The coumarin moiety and the anthranilic acid function of the probe, produced in the course of a Lossen rearrangement, were part of two different Förster resonance energy transfers.

Validation, Identification, and Biological Consequences of the Site-specific O-GlcNAcylation Dynamics of Carbohydrate-responsive Element-binding Protein (ChREBP).

O-GlcNAcylation of carbohydrate-responsive element-binding protein (ChREBP) is believed as an important modulator of ChREBP activities, however little direct evidence of O-GlcNAcylation on ChREBP and no exact O-GlcNAcylation sites have been reported so far. Here, we validate O-GlcNAcylation on ChREBP in cell-free coupled transcription/translation system and in cells by chemoenzymatic and metabolic labeling, respectively. Moreover, for the first time, we identify O-GlcNAcylation on Ser614 in the C-terminus of ChREBP by mass spectrometry and validate two important sites, Thr517 and Ser839 for O-GlcNAcylation and their function via molecular and chemical biological method. Under high glucose conditions, Ser514 phosphorylation enhances ChREBP O-GlcNAcylation, maintaining the transcriptional activity of ChREBP; Ser839 O-GlcNAcylation is essential for Mlx-heterodimerization and DNA-binding activity enhancement, consequently inducing transcriptional activity. Ser839 O-GlcNAcylation is also crucial for ChREBP nuclear export partially by strengthening interactions with CRM1 and 14-3-3. This work is a detailed study of ChREBP O-GlcNAcylation and highlights the biological consequences of the site-specific O-GlcNAcylation dynamics of ChREBP.

Probing Protein-Protein Interactions with Genetically Encoded Photoactivatable Cross-Linkers.

Fundamental to all living organisms is the ability of proteins to interact with other biological molecules at the right time and location, with the proper affinity, and to do so reversibly. One well-established technique to study protein interactions is chemical cross-linking, a process in which proteins in close spatial proximity are covalently tethered together. An emerging technology that overcomes many limitations of traditional cross-linking methods is one in which photoactivatable cross-linking noncanonical amino acids are genetically encoded into a protein of interest using the cell's native translational machinery. These proteins can then be used to trap interacting biomolecules upon UV illumination. Here, we describe a method for the site-specific incorporation of photoactivatable cross-linking amino acids into fluorescently tagged proteins of interest in E. coli. Photo-cross-linking and analysis by SDS-PAGE using in-gel fluorescence detection, which provides rapid, highly sensitive, and specific detection of cross-linked adducts even in impure systems, are also described. An example expression and cross-linking experiment involving transmembrane signaling of a bacterial second messenger receptor system that controls biofilm formation is shown. All reagents needed to carry out these experiments are commercially available, and do not require special or unique technology to perform, making this method tractable to a broad community studying protein structure and function.

Phosphono Bisbenzguanidines as Irreversible Dipeptidomimetic Inhibitors and Activity-Based Probes of Matriptase-2.

Matriptase-2, a type II transmembrane serine protease, plays a key role in human iron homeostasis. Inhibition of matriptase-2 is considered as an attractive strategy for the treatment of iron-overload diseases, such as hemochromatosis and β-thalassemia. In the present study, synthetic routes to nine dipeptidomimetic inactivators were developed. Five active compounds (41-45) were identified and characterized kinetically as irreversible inhibitors of matriptase-2. In addition to a phosphonate warhead, these dipeptides possess two benzguanidine moieties as arginine mimetics to provide affinity for matriptase-2 by binding to the S1 and S3/S4 subpockets, respectively. This binding mode was strongly supported by covalent docking analysis. Compounds 41-45 were obtained as mixtures of two diastereomers and were therefore separated into the single epimers. Compound 45 A, with S configuration at the N-terminal amino acid and R configuration at the phosphonate carbon atom, was the most potent matriptase-2 inactivator with a rate constant of inactivation of 2790 m(-1) s(-1) and abolished the activity of membrane-bound matriptase-2 on the surface of intact cells. Based on the chemotyp of phosphono bisbenzguanidines, the design and synthesis of a fluorescent probe (51 A) by insertion of a coumarin label is described. The in-gel fluorescence detection of matriptase-2 was demonstrated by applying 51 A as the first activity-based probe for this enzyme.

Nonradioactive Analysis of Dynamic Protein Palmitoylation

Methods to study protein S-palmitoylation dynamics have previously relied on metabolic labeling with [(14)C]palmitate, which requires additional safety precautions and long exposures. Nonradioactive alkynyl palmitate analogs have been developed for in-gel fluorescence detection and affinity purification. Cells metabolically labeled with the commercially available analog 17-octadynoic acid are lysed and then combined with azide-linked reporter tags for efficient conjugation by copper-catalyzed click chemistry in phosphate buffer. This approach has been demonstrated to label hundreds of endogenous palmitoylated proteins and is compatible with traditional pulse-chase methods. This protocol describes the reagents and procedures for labeling and detection of dynamic palmitoylation in mammalian cells.

Mechanism of Bacterial Oligosaccharyltransferase

N-Linked glycosylation is an essential post-translational protein modification in the eukaryotic cell. The initial transfer of an oligosaccharide from a lipid carrier onto asparagine residues within a consensus sequon is catalyzed by oligosaccharyltransferase (OST). The first X-ray structure of a complete bacterial OST enzyme, Campylobacter lari PglB, was recently determined. To understand the mechanism of PglB, we have quantified sequon binding and glycosylation turnover in vitro using purified enzyme and fluorescently labeled, synthetic peptide substrates. Using fluorescence anisotropy, we determined a dissociation constant of 1.0 μm and a strict requirement for divalent metal ions for consensus (DQNAT) sequon binding. Using in-gel fluorescence detection, we quantified exceedingly low glycosylation rates that remained undetected using in vivo assays. We found that an alanine in the -2 sequon position, converting the bacterial sequon to a eukaryotic one, resulted in strongly lowered sequon binding, with in vitro turnover reduced 50,000-fold. A threonine is preferred over serine in the +2 sequon position, reflected by a 4-fold higher affinity and a 1.2-fold higher glycosylation rate. The interaction of the +2 sequon position with PglB is modulated by isoleucine 572. Our study demonstrates an intricate interplay of peptide and metal binding as the first step of protein N-glycosylation.

Development of clickable active site-directed photoaffinity probes for γ-secretase

We have developed clickable active site-directed photoaffinity probes for γ-secretase which incorporate a photoreactive benzophenone group and an alkyne handle for subsequent click chemistry mediated conjugation with azide-linked reporter tags for visualization (e.g., TAMRA-azide) or enrichment (e.g., biotin-azide) of labeled proteins. Specifically, we synthesized clickable analogs of L646 (2) and L505 (3) and validated specific labeling to presenilin–1N-terminal fragment (PS1-NTF), the active site aspartyl protease component within the γ-secretase complex. Additionally, we were able to identify signal peptide peptidase (SPP) by Western blot analysis. Furthermore, we analyzed the photo-labeled proteins in an unbiased fashion by click chemistry with TAMRA-azide followed by in-gel fluorescence detection. This approach expands the utility of γ-secretase inhibitor (GSI) photoaffinity probes in that labeled proteins can be tagged with any number of azide-linked reporters groups using a single clickable photoaffinity probe for target pull down and/or fluorescent imaging applications.

Chemical reporters for fluorescent detection and identification of O-GlcNAc-modified proteins reveal glycosylation of the ubiquitin ligase NEDD4-1

The dynamic modification of nuclear and cytoplasmic proteins by the monosaccharide N-acetyl-glucosamine (GlcNAc) continues to emerge as an important regulator of many biological processes. Herein we describe the development of an alkynyl-modified GlcNAc analog (GlcNAlk) as a new chemical reporter of O-GlcNAc modification in living cells. This strategy is based on metabolic incorporation of reactive functionality into the GlcNAc biosynthetic pathway. When combined with the Cu(I)-catalyzed [3 + 2] azide-alkyne cycloaddition, this chemical reporter allowed for the robust in-gel fluorescent visualization of O-GlcNAc and affinity enrichment and identification of O-GlcNAc-modified proteins. Using in-gel fluorescence detection, we characterized the metabolic fates of GlcNAlk and the previously reported azido analog, GlcNAz. We confirmed previous results that GlcNAz can be metabolically interconverted to GalNAz, whereas GlcNAlk does not, thereby yielding a more specific metabolic reporter of O-GlcNAc modification. We also used GlcNAlk, in combination with a biotin affinity tag, to identify 374 proteins, 279 of which were not previously reported, and we subsequently confirmed the enrichment of three previously uncharacterized proteins. Finally we confirmed the O-GlcNAc modification of the ubiquitin ligase NEDD4-1, the first reported glycosylation of this protein.

Robust in-gel fluorescence detection of mucin-type O-linked glycosylation

Mucin-type O-linked glycosylation is a common post translational modification of cell-surface and secretory pathway proteins and is implicated in vital biological processes as well as human disease. We report here the use of the metabolic chemical reporter GalNAz along with Cu(I)-catalyzed [3+2] azide-alkyne cycloaddition conditions for the robust, in-gel fluorescent visualization of mucin-type O-linked glycoproteins.

High-resolution Native-PAGE for membrane proteins capable of fluorescence detection and hydrodynamic state evaluation

An improved native polyacrylamide gel electrophoresis (PAGE) method capable of evaluating the hydrodynamic states of membrane proteins and allowing in-gel fluorescence detection was established. In this method, bis(alkyl) sulfosuccinate is used to provide negative charges for detergent-solubilized membrane proteins to facilitate proper electrophoretic migration without disturbing their native hydrodynamic states. The method achieved high-resolution electrophoretic separation, in good agreement with the elution profiles obtained by size exclusion chromatography. The applicability of in-gel fluorescence detection for tagged green fluorescent protein (GFP) facilitates the analysis of samples without any purification. This method might serve as a general analytical technique for assessing the folding, oligomerization, and protein complex formation of membrane proteins.

High-throughput construction of expression system using yeast Pichia pastoris, and its application to membrane proteins

The well-established method for high-throughput construction of an expression system of the yeast Saccharomyces cerevisiae uses homologous recombination between an expression plasmid and a target gene (with homologous regions of the plasmid on both ends added by PCR). This method has been widely used for membrane proteins using plasmids containing GFP, and has been successfully used to investigate the cellular localization and solubilization conditions of the proteins. Although the methanol-utilizing yeast Pichia pastoris is known as an excellent expression host, a method for high-throughput construction of an expression system like that in S. cerevisiae has not been reported. In this study, we have attempted to construct expression systems via homologous recombination in P. pastoris. The insertion of genes into a plasmid could be easily checked by colony-PCR. Expression systems for seven membrane proteins of medaka fish ( Oryzias latipes) and yeast ( S. cerevisiae) were constructed, and the expression of proteins was analyzed by fluorescence spectra, fluorescence microscopy, and SDS–PAGE (in-gel fluorescence detection).

Direct in-gel fluorescence detection and cellular imaging of O-GlcNAc-modified proteins.

We report an advanced chemoenzymatic strategy for the direct fluorescence detection, proteomic analysis, and cellular imaging of O-GlcNAc-modified proteins. O-GlcNAc residues are selectively labeled with fluorescent or biotin tags using an engineered galactosyltransferase enzyme and [3 + 2] azide-alkyne cycloaddition chemistry. We demonstrate that this approach can be used for direct in-gel detection and mass spectrometric identification of O-GlcNAc proteins, identifying 146 novel glycoproteins from the mammalian brain. Furthermore, we show that the method can be exploited to quantify dynamic changes in cellular O-GlcNAc levels and to image O-GlcNAc-glycosylated proteins within cells. As such, this strategy enables studies of O-GlcNAc glycosylation that were previously inaccessible and provides a new tool for uncovering the physiological functions of O-GlcNAc.

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.