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Selective Cross-Linking of Interacting Proteins Using Self-Labeling Tags
Abstract
We have designed molecules that permit the selective cross-linking (S-CROSS) of interacting proteins in cell lysates and the sensitive detection of the trapped complexes through in-gel fluorescence scanning. S-CROSS requires the expression of the putative interacting proteins as fusion to CLIP-tag or SNAP-tag, two protein tags that can be specifically labeled with synthetic probes. Bifunctional molecules that contain the substrates of the two tags connected via a fluorophore are used to selectively cross-link interacting proteins in cell lysate. The amount of trapped complex can be then quantified after SDS gel electrophoresis by in-gel fluorescence scanning. On the basis of a detailed kinetic analysis of the cross-linking reaction, we showed that the cross-linking efficiency can be used as an indicator of interaction between two proteins, allowing thereby the unambiguous identification of interacting protein pairs. We validated our approach by confirming a number of interactions through selective cross-linking and showed that it permits the quantitative and simultaneous analysis of multiple homotypic and heterotypic protein complexes and the differentiation between strong and weak protein-protein interactions.
... Gautier et al. developed a new AGT mutant, which can be used in combination with the SNAP-tag, but recognizes a different substrate for covalent coupling [6]. The newly developed tag, called CLIP-tag, was selected by phage display from a library containing randomly mutated SNAP mutants from a yeast library. ...
... to the original SNAP-tag, the new protein tag contains eight mutations and has a 100-fold increased reactivity towards benzylcytosine (BC) compared to BG-modified substrates. Gautier et al. also demonstrated the similar activities of the CLIP-tag compared to the SNAP-tag as well as their use to simultaneously and specifically label different proteins in living cells [6]. ...
... Additionally, the group applied the combination of both tags to a cross-linking technology which is also used to study proteinprotein interactions but has the limitation of low selectivity when using chemical cross linkers [6]. For a selective cross linking (S-CROSS) to detect protein-protein interactions in cell lysates, the proteins which should be analyzed were genetically fused to the SNAP-and CLIP-tag and a dye modified with BG and BC was developed. ...
Over the past few years, the SNAP-tag technology has become a methodology with great potential in a variety of applications, e.g. the (specific) visualization of individual proteins and studies of protein interaction in living cells. Furthermore, the tag can be used for immunopurification and detection of recombinant proteins or site-specific coupling of recombinant proteins to surfaces. Next to the in vitro applications, it also enables detection of tagged proteins in vivo. This review gives an overview of the SNAP-tag technology in different fields of research and its potential for future developments.
... This is peculiar, because the great majority of model organisms does not react with BC-, avoiding any endogenous activity [12]. The advantage of possessing two orthogonal SLPs to be genetically fused to two respective POIs is particularly suitable for in vivo and in vitro protein-protein interaction studies, by methodologies as the Selective crosslinking of interacting proteins (S-CROSS) [13], as well as by employing FRET fluorophore pairs conjugated to the BG-and BG-substrates. ...
... The expressed protein, the SsOGT-MC 8 (Fig. 2) was easily purified from the E. coli cell free extract by affinity chromatography. The catalytic activity (in terms of second-order rate constant; [12,13,15,16,26]) of this tag was determined at the relative physiological temperature (65°C): as the previously citated engineered variants, it exhibits activity on the BC-TMR substrate, whereas it was not possible to measure any activity on orthogonal BG-FL substrate, displaying a specular behaviour with respect to the TS SNAP (Table 1), thus impeding in both cases to determine a BC/BG ratio value. On the other hand, purified CLIP-tag reacts only 10 2 faster on BC-TMR than on BG-FL, partially in agreement with previous data [12,26], whereas SNAP-tag is generally more specific on BGderivatives (10 3 faster; Table 1) [12,26]. ...
Self-labelling protein tags (SLPs) are resourceful tools that revolutionized sensor imaging, having the versatile ability of being genetically fused with any protein of interest and undergoing activation with alternative probes specifically designed for each variant (namely, SNAP-tag, CLIP-tag and Halo-tag). Commercially available SLPs are highly useful in studying molecular aspects of mesophilic organisms, while they fail in characterizing model organisms that thrive in harsh conditions. By applying an integrated computational and structural approach, we designed a engineered variant of the alkylguanine-DNA-alkyl-transferase (OGT) from the hyper-thermophilic archaeon Saccharolobus solfataricus (SsOGT), with no DNA-binding activity, able to covalently react with O⁶-benzyl-cytosine (BC-) derivatives, obtaining the first thermostable CLIP-tag, named SsOGT-MC⁸.
The presented construct is able to recognize and to covalently bind BC- substrates with a marked specificity, displaying a very low activity on orthogonal benzyl-guanine (BG-) substrate and showing a remarkable thermal stability that broadens the applicability of SLPs. The rational mutagenesis that, starting from SsOGT, led to the production of SsOGT-MC⁸ was first evaluated by structural predictions to precisely design the chimeric construct, by mutating specific residues involved in protein stability and substrate recognition. The final construct was further validated by biochemical characterization and X-ray crystallography, allowing us to present here the first structural model of a CLIP-tag establishing the molecular determinants of its activity, as well as proposing a general approach for the rational engineering of any O⁶-alkylguanine-DNA-alkyl-transferase turning it into a SNAP- and a CLIP-tag variant.
... Unfortunately, production fell into several technical problems including extremely low yields, precipitation issues and failure to recover active intact proteins (data not shown). We therefore asked whether selective cross-linking (S-CROSS) (Gautier et al., 2009) could be employed to bind molecules of interest. S-CROSS is based on self-labeling tags (SNAP and CLIP), which covalently bind synthetic probes. ...
... S-CROSS is based on self-labeling tags (SNAP and CLIP), which covalently bind synthetic probes. S-CROSS has been already used to detect protein-protein interactions in living cells (Gautier et al., 2009) , (Lemercier et al., 2007). A recombinant version of the CRE recombinase fused to an N-terminal CLIP-tag (CLIP-Cre) was produced in E. Coli. ...
Blood pressure is one of the vital signs and its regulation is crucial for survival. Several mechanisms contribute to maintain it in a physiological range: renin-angiotensin-aldosterone system, the autonomous nervous system and specialized baroreceptors neurons. In this study, we demonstrate the existence of a new population of sensory neurons marked by TrkC and TH that innervate blood vessels and are important in the control of blood pressure, blood flow and heart rate. Using an inducible Cre line driven from the TrkC locus, we show that TrkC is expressed in 30% of DRG neurons and that a fourth of these neurons are TH+ and project to blood vessels. Activation of TrkC+ TH+ neurons leads to high blood pressure, decreased blood flow and increased heart rate variability. Loss of function experiments revealed that TrkC+ TH+ sensory neurons are crucial for life. Ablation of TrkC+ neurons results in low blood pressure, alteration of blood flow and increased heart rate variability. All these cardiovascular alterations lead ablate mice to death within 48 hours. We also demonstrate that TrkC+ neurons do not act directly on blood vessels, but they exert their functions through a circuit with the sympathetic nervous system. We thus identified a new population of sensory neurons involved in the regulation of blood pressure, blood flow and heart rate and we hope that this can lead to the development of new therapeutic strategies in the near future.
... To this end, we first attempted to generate fusion proteins of ligand and CRE recombinase but were unable to recover active proteins at sufficient yields. We therefore asked whether selective cross-linking (S-CROSS) 16 could be employed to bind molecules of interest. S-CROSS is based on self-labelling tags (SNAP and CLIP), which covalently bind synthetic probes and has been previously used to detect protein-protein interactions in living cells 16,19 . ...
... We therefore asked whether selective cross-linking (S-CROSS) 16 could be employed to bind molecules of interest. S-CROSS is based on self-labelling tags (SNAP and CLIP), which covalently bind synthetic probes and has been previously used to detect protein-protein interactions in living cells 16,19 . Recombinant CRE recombinase fused to a N-terminal CLIP-tag (CLIP-Cre) was produced in E. Coli and CLIP activity was confirmed by selective labelling with a BC-derivative fluorophore (BC 488 ) (Fig. S1E). ...
Gene delivery using vector or viral-based methods is often limited by technical and safety barriers. A promising alternative that circumvents these shortcomings is the direct delivery of proteins into cells. Here we introduce a non-viral, ligand-mediated protein delivery system capable of selectively targeting primary skin cells in-vivo. Using orthologous self-labelling tags and chemical cross-linkers, we conjugate large proteins to ligands that bind their natural receptors on the surface of keratinocytes. Targeted CRE-mediated recombination was achieved by delivery of ligand cross-linked CRE protein to the skin of transgenic reporter mice, but was absent in mice lacking the ligand’s cell surface receptor. We further show that ligands mediate the intracellular delivery of Cas9 allowing for CRISPR-mediated gene editing in the skin more efficiently than adeno-associated viral gene delivery. Thus, a ligand-based system enables the effective and receptor-specific delivery of large proteins and may be applied to the treatment of skin-related genetic diseases.
... Thus, sulfonation and/or disubstitutions are two means to prevent cellular entry and, thereby, to maintain target specificity. Of note, although disubstituted sulfonated Cy3 and Cy5 have been previously reported [29] , their cell permeation properties were not examined. Comparison between the mono vs. disubstituted sulfonated Cy5 showed comparable cell surface fluorescence (Figure S4A), and maximal labelling following as short as 5 minutes of incubation ( Figure S4B). ...
Magnetic resonance imaging (MRI) is a powerful imaging modality, widely employed in research and clinical settings. However, MRI images suffer from low signals and a lack of target specificity. We aimed to develop a multimodal imaging probe to detect targeted cells by MRI and fluorescence microscopy. We synthesized a trifunctional imaging probe consisting of a SNAP‐tag substrate for irreversible and specific labelling of cells, cyanine dyes for bright fluorescence, and a chelated GdIII molecule for enhancing MRI contrast. Our probes exhibit specific and efficient labelling of genetically defined cells (expressing SNAP‐tag at their membrane), bright fluorescence and MRI signal. Our synthetic approach provides a versatile platform for the production of multimodal imaging probes, particularly for light microscopy and MRI.
... This feature of the enzyme has been exploited to attach various functional molecules such as fluorescent dyes, DNA oligonucleotides, and other ligands. [60][61][62] The SNAP-tag was also used for live-cell imaging using dSTORM. 63 Similarly, HALO-tag was derived from the enzyme haloalkane dehalogenase from Rhodococcus sp. ...
Super-resolution imaging is becoming a commonly employed tool to visualize biological targets in unprecedented detail. DNA-PAINT is one of the single-molecule localization microscopy-based super-resolution imaging modalities allowing the ultra-high-resolution imaging with superior multiplexing capabilities. We discuss the importance of patterned DNA nanostructures in demonstrating the capabilities of DNA-PAINT and the design of various combinations of imager-docking strand pairs for imaging. Central to the implementation of DNA-PAINT imaging in a biological context is the generation of docking strand-conjugated binders against the target molecules. Several researchers have developed a variety of labelling probes for improving resolution while also providing multiplexing capabilities for the broader application of DNA-PAINT. This review provides a comprehensive summary of the repertoire of labelling probes used for DNA-PAINT in cells and the strategies implemented to chemically modify them with a docking strand.
... NIR uorescent probes, SC-Cy5 and SS-Cy5, were developed using a Cy5 scaffold that is connected with BG, BC, or both to conjugate SNAP-tag, CLIP-tag, or both ( Fig. 3B), allowing selective cross-linking of proteins in cell lysates for the investigation of protein-protein interactions. 39 This approach is more convenient than that by using commercially available bismaleimide cross-linker. Indeed, this system can detect weak protein-protein interactions, even with a K D value of 30 mM. ...
The development of near-infrared (NIR) fluorescent probes over the past few decades has changed the way that biomolecules are imaged, and thus represents one of the most rapidly progressing areas of research. Presently, NIR fluorescent probes are routinely used to visualize and understand intracellular activities. The ability to penetrate tissues deeply, reduced photodamage to living organisms, and a high signal-to-noise ratio characterize NIR fluorescent probes as efficient next-generation tools for elucidating various biological events. The coupling of self-labeling protein tags with synthetic fluorescent probes is one of the most promising research areas in chemical biology. Indeed, at present, protein-labeling techniques are not only used to monitor the dynamics and localization of proteins but also play a more diverse role in imaging applications. For instance, one of the dominant technologies employed in the visualization of protein activity and regulation is based on protein tags and their associated NIR fluorescent probes. In this mini-review, we will discuss the development of several NIR fluorescent probes used for various protein-tag systems.
... [22,25,26] We next tested the reversibility of split-frFAST using the ability of rapamycin to dissociate an AP1510-induced FKBP homodimer. [27,28] We co-expressed FKBP-N-frFAST and FKBP-C-frFAST in HEK 293T cells.C ells were pre-treated with AP1510 for 2hto form the FKBP homodimer and 10 mm HPAR-3OM was added to visualize the complemented split-frFAST.T he addition of rapamycin led to am edian fluorescence decrease of 5-fold, in agreement with ad isassembly of split-frFAST concomitant with the dissociation of the FKBP homodimer (Figure 2e-g). Time-lapse imaging showed that complementation was reversed within af ew minutes (Figure 2e,g), in agreement with the rapid disassembly of FKBP homodimer. ...
Far‐red emitting fluorescent labels are highly desirable for spectral multiplexing and deep tissue imaging. Here, we report a far‐red fluorescent chemogenetic reporter for the on‐demand imaging of fusion proteins in cells and in living multicellular organisms.
Abstract
Far‐red emitting fluorescent labels are highly desirable for spectral multiplexing and deep tissue imaging. Here, we describe the generation of frFAST (far‐red Fluorescence Activating and absorption Shifting Tag), a 14‐kDa monomeric protein that forms a bright far‐red fluorescent assembly with (4‐hydroxy‐3‐methoxy‐phenyl)allylidene rhodanine (HPAR‐3OM). As HPAR‐3OM is essentially non‐fluorescent in solution and in cells, frFAST can be imaged with high contrast in presence of free HPAR‐3OM, which allowed the rapid and efficient imaging of frFAST fusions in live cells, zebrafish embryo/larvae, and chicken embryos. Beyond enabling the genetic encoding of far‐red fluorescence, frFAST allowed the design of a far‐red chemogenetic reporter of protein–protein interactions, demonstrating its great potential for the design of innovative far‐red emitting biosensors.
... [22,25,26] We next tested the reversibility of split-frFAST using the ability of rapamycin to dissociate an AP1510-induced FKBP homodimer. [27,28] We co-expressed FKBP-N-frFAST and FKBP-C-frFAST in HEK 293T cells.C ells were pre-treated with AP1510 for 2hto form the FKBP homodimer and 10 mm HPAR-3OM was added to visualize the complemented split-frFAST.T he addition of rapamycin led to am edian fluorescence decrease of 5-fold, in agreement with ad isassembly of split-frFAST concomitant with the dissociation of the FKBP homodimer (Figure 2e-g). Time-lapse imaging showed that complementation was reversed within af ew minutes (Figure 2e,g), in agreement with the rapid disassembly of FKBP homodimer. ...
Far‐red emitting fluorescent labels are highly desirable for spectral multiplexing and deep tissue imaging. Here, we describe the generation of frFAST (far‐red Fluorescence Activating and absorption Shifting Tag), a 14‐kDa monomeric protein that forms a bright far‐red fluorescent assembly with (4‐hydroxy‐3‐methoxy‐phenyl)allylidene rhodanine (HPAR‐3OM). As HPAR‐3OM is essentially non‐fluorescent in solution and in cells, frFAST can be imaged with high contrast in presence of free HPAR‐3OM, which allowed the rapid and efficient imaging of frFAST fusions in live cells, zebrafish embryo/larvae, and chicken embryos. Beyond enabling the genetic encoding of far‐red fluorescence, frFAST allowed the design of a far‐red chemogenetic reporter of protein–protein interactions, demonstrating its great potential for the design of innovative far‐red emitting biosensors.
... Here we considered an alternative approach for hybrid voltage sensor design; localization of a synthetic voltage indicator to cells of interest using genetically encoded protein tags 19,20 . We focused on enzyme-based small protein tags such as the self-modifying enzyme SNAP 21,22 -tag, and transferase-mediated labeling of the acyl carrier protein (ACP) 23,24 -tag, as these technologies allow for rapid, irreversible labeling, and are compatible with in vivo imaging 25,26 . For the VSD component we found that derivatives of Nile Red, an environment-sensitive ('fluorogenic') dye that shows fluorescence enhancement upon transition from aqueous to hydrophobic solvent 27,28 , register membrane potential with high fidelity. ...
Optical monitoring of neuronal voltage using fluorescent indicators is a powerful approach for the interrogation of the cellular and molecular logic of the nervous system. Herein, a semisynthetic tethered voltage indicator (STeVI1) based upon nile red is described that displays voltage sensitivity when genetically targeted to neuronal membranes. This environmentally sensitive probe allows for wash‐free imaging and faithfully detects supra‐ and sub‐threshold activity in neurons.
... The newer methods of click chemistry, enzymatic or tag labelling, may be used to improve the conventional strategies of labelled toxins production [63]. Another possibility is development of new types of chimeric proteins bearing genetically encoded labels, for example, SNAP or CLIP tags [111,112]. Application of fluorescently labelled toxins may be extended to involve fluorescence/Förster resonance energy transfer (FRET), fluorescence lifetime imaging microscopy (FLIM), and single molecule tracking [113,114]. The arsenal of labelled toxins available already today suits the requirements of these advanced techniques and we await reports describing the results of such applications. ...
Animal toxins are traditional and indispensible molecular tools that find application in different fields of biochemistry, neurobiology and pharmacology. These compounds possess several outstanding properties such as high affinity and selectivity with respect to particular molecular targets, most importantly ion channels and neuroreceptors, and stability. In addition to using toxins per se, a wide variety of labelled modifications have been obtained including radioactive and fluorescent derivatives. Here, we discuss the major types of labelled toxins, methods of their production and principal possibilities of application ranging from receptor localization and visualization to development of screening systems and diagnostic tools, and drug discovery.
... 26 Because there is no additional cofactor necessary for the reaction between the BG-/BC-functionalized ligand and the respective tag, the labeling reaction will proceed in vitro (i.e., by using purified protein samples) and also in vivo (i.e., within living cells) by using membrane-permeable ligands ( Figure 1). To date, SNAP-and CLIP-tags have been widely used as fusion partners to label proteins of interest for cellular imaging, 26,28−31 super-resolution microscopy, 28,32 FRET-based biosensors on cell surfaces, 33 in vitro protein conformational studies, 34 functionalizing nanoparticles, 35 cross-linking proteins, 36 and immobilizing a protein of interest on a surface. 37 These chemical tags have also been employed for fluorescent labeling in smFRET measurements; 38 however, a systematic evaluation of this labeling strategy is still lacking. ...
Fluorescence resonance energy transfer (FRET) is a superb technique for measuring conformational changes of proteins on the single molecule level (smFRET) in real time. It requires introducing a donor and acceptor fluorophore pair at specific locations of the protein molecule of interest, which has often been a challenging task. By using two different self-labeling chemical tags, such as Halo-, TMP-, SNAP- and CLIP-tags, orthogonal labeling may be achieved rapidly and reliably. However, these comparatively large tags add extra distance and flexibility between the desired labeling location on the protein and the fluorophore position, which may affect the results. To systematically characterize chemical tags for smFRET measurement applications, we took the SNAP-tag/CLIP-tag combination as a model system and fused a flexible unstructured peptide, rigid polyproline peptides of various lengths and the calcium sensor protein calmodulin between the tags. We could reliably identify length variations as small as four residues in the polyproline peptide. In the calmodulin system, the added length introduced by these tags was even beneficial for revealing subtle conformational changes upon variation of the buffer conditions. This approach opens up new possibilities for studying conformational dynamics, especially in large protein systems that are difficult to specifically conjugate with fluorophores.
... This molecule therefore bridges the interacting partners with high selectivity. (Gautier et al., 2009). When linked together, SNAP-tag and CLIP tag can act as a FRET pair and be used to detect changes in the concentration of a metabolite (Brun et al., 2011; Brun et al., 2009). ...
The pursuit of quantitative biological information via imaging requires robust labeling approaches that can be used in multiple applications and with a variety of detectable colors and properties. In addition to conventional fluorescent proteins, chemists and biologists have come together to provide a range of approaches that combine dye chemistry with the convenience of genetic targeting. This hybrid-tagging approach amalgamates the rational design of properties available through synthetic dye chemistry with the robust biological targeting available with genetic encoding. In this review, we discuss the current range of approaches that have been exploited for dye targeting or for targeting and activation and some of the recent applications that are uniquely permitted by these hybrid-tagging approaches.
... The SNAP-tag has an active site cysteine residue that covalently binds O 6 -benzylguanine (BG) and its synthetic derivatives (29). BG can be conjugated to a variety of fluorophores and other labels (30), making it possible to tag and track the protein of interest directly by a variety of methods such as fluorescence microscopy, gel electrophoresis, and in vivo imaging (31)(32)(33). The SNAP-tag has been demonstrated not to affect the function of a large number of fusion proteins (34,35) and is an optimal approach for pulse-chase labeling experiments (34,36). ...
Proteins targeted to the plasma membrane (PM) of cells are degraded at different rates. Sorting motifs contained within the
cytoplasmic domains of transmembrane proteins, post-translational modifications (e.g. ubiquitination), and assembly into multiprotein or protein-lipid complexes all may affect the efficiency of endocytosis and
recycling and influence the delivery to degradative compartments. Using the SNAP-tag labeling system, we examined the turnover
of a model PM protein, the α chain of the interleukin-2 receptor (Tac). The surface lifetimes of SNAP-Tac fusions were influenced
by their mode of entry into cells (clathrin-dependent versus clathrin-independent), their orientation in the PM (transmembrane versus glycosylphosphatidylinositol-anchored), and ubiquitination in their cytosolic domains. In addition, shedding of SNAP-Tac
into the medium was greatly influenced by its O-linked glycosylation status. For a number of PM proteins, delivery to lysosomes and ectodomain shedding represent distinct
parallel mechanisms to determine protein half-life.
... Keep tetracycline or doxycycline concentrations as low as possible since they display certain toxicity [ 33 ]. 16. Hoechst 33342 counterstaining is recommended to include in most of the imaging experiments since it produces strong fl uorescence signal, which is convenient for fi nding the cells and focusing. ...
One of the most prominent self-labeling tags is SNAP-tag. It is an in vitro evolution product of the human DNA repair protein O
6-alkylguanine-DNA alkyltransferase (hAGT) that reacts specifically with benzylguanine (BG) and benzylchloropyrimidine (CP) derivatives, leading to covalent labeling of SNAP-tag with a synthetic probe (Gronemeyer et al., Protein Eng Des Sel 19:309–316, 2006; Curr Opin Biotechnol 16:453–458, 2005; Keppler et al., Nat Biotechnol 21:86–89, 2003; Proc Natl Acad Sci U S A 101:9955–9959, 2004). SNAP-tag is well suited for the analysis and quantification of fused target protein using fluorescence microscopy techniques. It provides a simple, robust, and versatile approach to the imaging of fusion proteins under a wide range of experimental conditions.
... Furthermore bi-functional cross linkers containing BG and BC can be used to generate bivalent and bi-functional antibody molecules. Such reagents have already been used for the selective crosslinking of protein in cell lysates [131]. Bispecific antibodies can be use to attract effector cells such as cytotoxic T-cells, NK-cells and macrophages [132][133][134] and in particular antibodies recruiting cytotoxic T cells to tumours by binding to CD3 have demonstrated impressive effects in clinical trials [135]. ...
... Thus we cannot exclude that we might have missed transient or low-affinity interactions. Future studies implementing fluorescence cross-correlation spectroscopy, BioID, and/or S-Cross may be necessary to identify such interactions (Baudendistel et al., 2005;Bacia et al., 2006;Bacia and Schwille, 2007;Gautier et al., 2009;Roux et al., 2012). The evidence presented here suggests that the C. elegans core PCM proteins SPD-2 and SPD-5 do not form stably associated complexes inside the cytoplasm of living C. elegans embryos. ...
Centrosomes are the main microtubule organizing centers in animal cells. Centrosomes consist of a pair of centrioles surrounded by a matrix of pericentriolar material (PCM) that assembles from cytoplasmic components. In C. elegans embryos, interactions between the coiled coil proteins SPD-5, SPD-2, and the kinase PLK-1 are critical for PCM assembly. However, it is not known if these interactions promote the formation of cytoplasmic complexes that are added to the PCM or if components only interact during incorporation into the PCM matrix. Here, we address this problem by using a combination of live-cell fluorescence correlation spectroscopy, mass spectrometry, and hydrodynamic techniques to investigate the native state of PCM components in the cytoplasm. We show that SPD-2 is monomeric and neither SPD-2 nor SPD-5 exist in complex with PLK-1. SPD-5 exists as a monomer but also forms complexes with the PP2A-regulatory proteins RSA-1 and RSA-2, which are required for microtubule organization at centrosomes. These results suggest that the interactions between SPD-2, SPD-5 and PLK-1 do not result in formation cytoplasmic complexes, but instead occur in the context of PCM assembly.
... 14,15 It is wellknown for its rapid reaction rate with BG derivatives and nontoxicity in cells. Covalent labeling of SNAP-tag with an affinity ligand or optical probe has been used for protein interaction studies, 16 drug discovery, 17 super-resolution imaging applications, 18 and the construction of fluorescent sensors. 19,20 Most of the SNAP-tag fluorogenic probes were created based on the FRET strategy by introducing a fluorescent quencher at either the C-8 or N-9 position of guanine. ...
One major limitation of labeling proteins with synthetic fluorophores is the high fluorescence background which necessitates extensive washing steps to remove unreacted fluorophores. In this paper, we describe a novel fluorogenic probe based on environment-sensitive fluorophore for the labeling with SNAP-tag proteins. The probe exhibits dramatic fluorescence turn-on of 280-folds upon being labeled to SNAP-tag. The major advantages of our fluorogenic probe are the dramatic fluorescence turn-on, ease of synthesis, high selectivity and rapid labeling with SNAP-tag. No-wash labeling of both intracellular and cell surface proteins were successfully achieved in living cells and the localization of these proteins was specifically visualized.
... S-CROSS utilizes a bifunctional crosslinker that contains the substrates of both tags linked by a chemical fluorophore ( Figure 1B); crosslinking efficiency depends critically on the proximity of the two proteins ( Figure 1C). The sensitivity of S-CROSS in detecting protein-protein interactions is comparable to affinity purification [15], but the experimental simplicity of the former makes it more suitable for mediumthroughput screens. We expressed the 31 centrosomal SNAP-tag and CLIP-tag fusion proteins in a pairwise fashion, as well as SNAP-tag fusions of GFP and mCherry as negative controls, and subjected the resulting 527 combinations to S-CROSS. ...
The centrosome functions as the main microtubule-organizing center of animal cells and is crucial for several fundamental cellular processes [1]. Abnormalities in centrosome number and composition correlate with tumor progression [2 and 3] and other diseases [4, 5 and 6]. Although proteomic studies have identified many centrosomal proteins, their interactions are incompletely characterized [7 and 8]. The lack of information on the precise localization and interaction partners for many centrosomal proteins precludes comprehensive understanding of centrosome biology. Here, we utilize a combination of selective chemical crosslinking and superresolution microscopy to reveal novel functional interactions among a set of 31 centrosomal proteins. We reveal that Cep57, Cep63, and Cep152 are parts of a ring-like complex localizing around the proximal end of centrioles. Furthermore, we identify that STIL, together with HsSAS-6, resides at the proximal end of the procentriole, where the cartwheel is located. Our studies also reveal that the known interactors Cep152 and Plk4 reside in two separable structures, suggesting that the kinase Plk4 contacts its substrate Cep152 only transiently, at the centrosome or within the cytoplasm. Our findings provide novel insights into protein interactions critical for centrosome biology and establish a toolbox for future studies of centrosomal proteins.
... Another limitation of the technique is the need for fairly extensive transfection protocols and/or stably transfected cell lines, as well as the lack of orthogonally acting chemical dimerizer systems, although the technique has been expanded to a broad variety of signaling events. [160] Hope might come, in this respect, from the use of SNAP and CLIP tag fusions [161] or the FlAsH technique, [162] as these are amenable to small molecule manipulation for cross-linking proteins. ...
Since its discovery in the late 1980s, phosphoinositide 3-kinase (PI3K), and its isoforms have arguably reached the forefront of signal transduction research. Regulation of this lipid kinase, its functions, its effectors, in short its entire signaling network, has been extensively studied. PI3K inhibitors are frequently used in biochemistry and cell biology. In addition, many pharmaceutical companies have launched drug-discovery programs to identify modulators of PI3Ks. Despite these efforts and a fairly good knowledge of the PI3K signaling network, we still have only a rudimentary picture of the signaling dynamics of PI3K and its lipid products in space and time. It is therefore essential to create and use novel biological and chemical tools to manipulate the phosphoinositide signaling network with spatial and temporal resolution. In this review, we discuss the current and potential future tools that are available and necessary to unravel the various functions of PI3K and its isoforms.
... It is noteworthy that until now lanthanide derived fluorescent substrates are not permeant, therefore only receptors targeted to the cell surface, presenting an extracellular SNAP-tag will be labeled. By contrast other substrates such as tetramethyl rhodamine derivatives are permeant, allowing intracellular protein labeling ( Gautier et al., 2009). Other SLP-tags have been developed [e.g., CLIP-tag with benzyl cytosine ( Gautier et al., 2008), HaloTag (33 kDa) with HaloTag ligands ( Zhang et al., 2006)] allowing the labeling of different receptors with reduced cross-reactivity. ...
The concept of oligomerization of G protein-coupled receptor (GPCR) opens new perspectives regarding physiological function regulation. The capacity of one GPCR to modify its binding and coupling properties by interacting with a second one can be at the origin of regulations unsuspected two decades ago. Although the concept is interesting, its validation at a physiological level is challenging and probably explains why receptor oligomerization is still controversial. Demonstrating direct interactions between two proteins is not trivial since few techniques present a spatial resolution allowing this precision. Resonance energy transfer (RET) strategies are actually the most convenient ones. During the last two decades, bioluminescent resonance energy transfer and time-resolved fluorescence resonance energy transfer (TR-FRET) have been widely used since they exhibit high signal-to-noise ratio. Most of the experiments based on GPCR labeling have been performed in cell lines and it has been shown that all GPCRs have the propensity to form homo- or hetero-oligomers. However, whether these data can be extrapolated to GPCRs expressed in native tissues and explain receptor functioning in real life, remains an open question. Native tissues impose different constraints since GPCR sequences cannot be modified. Recently, a fluorescent ligand-based GPCR labeling strategy combined to a TR-FRET approach has been successfully used to prove the existence of GPCR oligomerization in native tissues. Although the RET-based strategies are generally quite simple to implement, precautions have to be taken before concluding to the absence or the existence of specific interactions between receptors. For example, one should exclude the possibility of collision of receptors diffusing throughout the membrane leading to a specific FRET signal. The advantages and the limits of different approaches will be reviewed and the consequent perspectives discussed.
Herein, we present a straightforward synthetic route for the design and synthesis of diverse heterobifunctional cyanine 5 dyes. We optimized the workup by harnessing the pH- and functional group-dependent solubility of the asymmetric cyanine 5 dyes. Therefore, purification through chromatography is deferred until the last synthesis step. Demonstrating successful large-scale synthesis, our modular approach prevents functional group degradation by introducing them in the last synthesis step. These modifiable heterobifunctional dyes offer significant utility in advancing biological studies.
Imaging proteins with high resolution is crucial for studying cellular physiology and pathology. Fluorescence imaging is a privileged method to visualize proteins with subcellular precision in live cells. In recent years, there has been a tremendous advance in the field of fluorescent dyes that are optically more sophisticated than genetically‐encodable fluorescent proteins. In this review, we aim to discuss modern bioconjugation methods to specifically incorporate these dyes into protein‐of‐interests. We focus on advances in live‐cell labeling strategies and fluorescent probes, especially the HaloTag, SNAP‐tag, TMP‐tag, and unnatural amino acid systems and their applications. These protein labeling methods, along with cutting‐edge dyes and novel microscopy methods, have become the infrastructure for biological research in the era of super‐resolution imaging.
Super-resolution fluorescence microscopy has emerged as a powerful tool for studying mitochondrial dynamics in living cells. However, the lack of photostable and chemstable probe makes long-term super-resolution imaging of mitochondria still a challenging work. Herein, we reported a 4-azetidinyl-naphthliamide derived SNAP-tag probe AN-BG exhibiting excellent fluorogenicity and photostability for long-term super-resolution imaging of mitochondrial dynamics. The azetidinyl group and naphthalimide fluorophore are in a flat conformation which can effectively suppress twisted intramolecular charge transfer and then effectively improve the brightness and photostability. This planarized molecular structure is conducive to the formation of fluorescence-quenched J-aggregates, and the protein labeling process will depolymerize the probes and restore fluorescence. Fluorescent labeling mitochondrial inner membrane proteins via SNAP tags overcomes the shortcomings that variations in mitochondrial inner membrane potential will release probes attached to mitochondria by electrostatic interactions. Therefore, AN-BG realized the stable labeling of mitochondria and the long-term imaging of mitochondrial dynamics under super-resolution microscopy.
Despite key roles in sister chromatid cohesion and chromosome organization, the mechanism by which cohesin rings are loaded onto DNA is still unknown. Here we combine biochemical approaches and cryoelectron microscopy (cryo-EM) to visualize a cohesin loading intermediate in which DNA is locked between two gates that lead into the cohesin ring. Building on this structural framework, we design experiments to establish the order of events during cohesin loading. In an initial step, DNA traverses an N-terminal kleisin gate that is first opened upon ATP binding and then closed as the cohesin loader locks the DNA against the ATPase gate. ATP hydrolysis will lead to ATPase gate opening to complete DNA entry. Whether DNA loading is successful or results in loop extrusion might be dictated by a conserved kleisin N-terminal tail that guides the DNA through the kleisin gate. Our results establish the molecular basis for cohesin loading onto DNA.
The development and function of tissues, blood and the immune system is dependent upon proximity for cellular recognition and communication. However, the detection of cell-to-cell contacts is limited due to a lack of reversible, quantitative probes that can function at these dynamic sites of irregular geometry. Described here is a novel chemo-genetic tool developed for fluorescent detection of protein-protein proximity and cell apposition that utilizes the Fluorogen Activating Protein (FAP) in combination with a Dye Activated by Proximal Anchoring (DAPA). The FAP-DAPA system has two protein components, the HaloTag and FAP, expressed on separate protein targets or in separate cells. The proteins function to bind and activate a compound that has the hexyl chloride (HexCl) ligand connected to malachite green (MG), the FAP fluorogen, via a poly(ethylene glycol) spacer spanning up to 28 nm. The dehalogenase protein, HaloTag, covalently binds the HexCl ligand, locally concentrating the attached MG. If the FAP is within range of the anchored fluorogen, it will bind and activate MG specifically when bath concentration is too low to saturate the FAP receptor. A new FAP variant was isolated with a 1000-fold reduced KD of ~10 – 100 nM so that the fluorogen activation reports proximity without artificially enhancing it. The system was characterized using purified FRB and FKBP fusion proteins and showed a doubling of fluorescence upon rapamycin induced complex formation. In co-cultured HEK293 cells (HaloTag and FAP-expressing) fluorescence increased at contact sites across a broad range of labeling conditions, more reliably providing contact-specific fluorescence activation with the lower-affinity FAP variant. When combined with suitable targeting and expression constructs, this labeling system may offer significant improvements in on-demand detection of intercellular contacts, potentially applicable in neurological and immunological synapse measurements and other transient, dynamic biological appositions that can be perturbed using other labeling methods that stabilize these interactions.
Herein we report a strategy to utilize the bioorthogonal reactivity and phosphorogenic property of iridium(III) polypyridine nitrone complexes and SNAP-tag protein for the modulation of emission and singlet oxygen (1O2) photosensitization in live cells.
Labeling a protein of interest (POI) with a fluorescent reporter is a powerful strategy for studying protein structures and dynamics in their native environments. Comparing to fluorescent proteins, synthetic dyes provide more choices in photophysical or photochemical attributes to microscopic characterizations. The specificity of bioorthogonal reactions in conjunction with the fidelity of subcellular destinations of genetically encoded protein tags can be employed to label POIs in live and fixed cells in a two-step process. In the present study the orthogonality of the strain-promoted azide-alkyne cycloaddition (SPAAC) and the inverse electron demand Diels-Alder (IEDDA) reaction is corroborated in concurrent labeling of two different intracellular targets. An azido group and a strained alkene are first installed at specific subcellular locations via orthogonal enzymatic reactions of the genetically incorporated SNAP- and CLIP-tags. The subsequent bioorthogonal reactions with fluorophores carrying matching reactive functionalities result in the simultaneous dual labeling. The two-step “orthogonal-bioorthogonal” labeling process would increase the utilities of SNAP/CLIP-tags, and as a consequence would expand the capability of decorating biological specimen with functionalities beyond fluorophores to potentially include spin labels, radioactive tracers, or catalysts.
Self-interacting proteins (SIPs) play a crucial role in investigation of various biochemical developments. In this work, a novel computational method was proposed for accelerating SIPs validation only using protein sequence. Firstly, the protein sequence was represented as Position-Specific Weight Matrix (PSWM) containing protein evolutionary information. Then, we incorporated the Legendre Moment (LM) and Sparse Principal Component Analysis (SPCA) to extract essential and anti-noise evolutionary feature from the PSWM. Finally, we utilized robust Probabilistic Classification Vector Machine (PCVM) classifier to carry out prediction. In the cross-validated experiment, the proposed method exhibits high accuracy performance with 95.54% accuracy on S.erevisiae dataset, which is a significant improvement compared to several competing SIPs predictors. The empirical test reveal that the proposed method can efficiently extracts salient features from protein sequences and accurately predict potential SIPs.
Optical monitoring of neuronal voltage using fluorescent indicators is a powerful approach for interrogation of the cellular and molecular logic of the nervous system. Here we describe Hybrid, Anchored to Protein tag, voltage Indicator based on Nile Red (HAPI-Nile Red) that displays voltage sensitivity when genetically targeted to neuronal membranes. This environmentally sensitive probe allows for wash-free imaging and faithfully detects supra- and subthreshold activity in neurons.
The self-labeling protein tags are robust and versatile tools for studying different molecular aspects of cell biology. In order to be suitable for a wide spectrum of experimental conditions, it is mandatory that these systems are stable after the fluorescent labeling reaction and do not alter the properties of the fusion partner. SsOGT-H5 is an engineered variant alkylguanine-DNA-alkyl-transferase (OGT) of the hyperthermophilic archaeon Sulfolobus solfataricus, and it represents an alternative solution to the SNAP-tag® technology under harsh reaction conditions. Here we present the crystal structure of SsOGT-H5 in complex with the fluorescent probe SNAP-Vista Green® (SsOGT-H5-SVG) that reveals the conformation adopted by the protein upon the trans-alkylation reaction with the substrate, which is observed covalently bound to the catalytic cysteine residue. Moreover, we identify the amino acids that contribute to both the overall protein stability in the post-reaction state and the coordination of the fluorescent moiety stretching-out from the protein active site. We gained new insights in the conformational changes possibly occurring to the OGT proteins upon reaction with modified guanine base bearing bulky adducts; indeed, our structural analysis reveals an unprecedented conformation of the active site loop that is likely to trigger protein destabilization and consequent degradation. Interestingly, the SVG moiety plays a key role in restoring the interaction between the N- and C-terminal domains of the protein that is lost following the new conformation adopted by the active site loop in the SsOGT-H5-SVG structure. Molecular dynamics simulations provide further information into the dynamics of SsOGT-H5-SVG structure, highlighting the role of the fluorescent ligand in keeping the protein stable after the trans-alkylation reaction.
In this paper, we present a novel charge-free fluorescence switchable near-infrared (IR) dye based on merocyanine for target specific imaging. In contrast to the typical bathochromic shift approach by extending π-conjugation, the bathochromic shift of our merocyanine dye to the near-IR region is due to an unusual S-cis diene conformer. This is the first example where a fluorescent dye adopts the stable S-cis conformation. In addition to the novel bathochromic shift mechanism, the dye exhibits fluorescence-switchable properties in response to polarity and viscosity. By incorporating a protein specific ligand to the dye, the probes (for SNAP-tag and hCAII proteins) exhibited dramatic fluorescence increase (up to 300-fold) upon binding with its target protein. The large fluorescence enhancement, near-IR absorption/emission and charge-free scaffold enabled no-wash and site-specific imaging of target proteins in living cells and in vivo with minimum background fluorescence. We believe that our unconventional approach for a near-IR dye with the S-cis diene conformation can lead to new strategies for the design of near-IR dyes.
Protein labeling by using a protein tag and tag-specific fluorescent probes is increasingly becoming a useful technique for the real-time imaging of proteins in living cells. SNAP-tag as one of the most prominent fusion tags has been widely used and already commercially available. Recently, various fluorogenic probes for SNAP-tag based protein labeling were reported. Owing to turn-on fluorescence response, fluorogenic probes for SNAP-tag minimize the fluorescence background caused by unreacted or nonspecifically bound probes and allow for direct imaging in living cells without wash-out steps. Thus, real-time analysis of protein localization, dynamics and interactions has been made possible by SNAP-tag fluorogenic probes. In this mini-review, we describe the design strategies of fluorogenic probes for SNAP-tag and their applications in cellular protein labeling.
When studying the underlying functionality of proteins, dual labeling is a common key strategy. The ability to monitor biomolecular conformation dynamics is proving essential to our understanding of protein structure and how it can influence subsequent functionality. Of the methodologies available to probe protein conformation, transfer mechanisms, such as photoinduced electron transfer (PET), and especially single-molecule ensemble Forster resonance ET (smFRET), have proven particularly useful. As with the intramolecular studies, FRET and the use of the biarsenical dyes appear to be the most popular methods of monitoring intermolecular interactions. Some of the scientific innovations in protein and antibody labeling have led to progress in the medical arena, where tumor detection and therapeutic applications have become a leading area of research. This chapter summarizes the current methods used in cell membrane staining for eventual use in tumor detection. Specifically, the focus is pretargeting schemes that allow for in vitro and in vivo staining and optical imaging.
In vivo protein ligation is of emerging interest as a means of endowing proteins with new properties in a controlled fashion. Tools to site-specifically and covalently modify proteins with small molecules, peptides, or other proteins in living cells are few and far between. Here, we describe the development of a Staphylococcus aureus sortase (SrtA)-based protein ligation approach for site-specific conjugation of fluorescent dyes and ubiquitin (Ub) to modify proteins in Caenorhabditis elegans. Hepta-mutant SrtA (SrtA7m) expressed in C. elegans is functional and supports in vitro sortase reactions in a low-Ca(2+) environment. Feeding SrtA7m-expressing C. elegans with small peptide-based probes such as (Gly)3- biotin or (Gly)3-fluorophores enables in vivo target protein modification. SrtA7m also catalyzes the circularization of suitably modified linear target proteins in vivo and allows the installation of F-box domains on targets to induce their degradation in a ubiquitin-dependent manner. This is a noninvasive method to achieve in vivo protein labeling, protein circularization, and targeted degradation in C. elegans. This technique should improve our ability to monitor and alter the function of intracellular proteins in vivo.
Putting fluorescent and other labels on proteins and small molecules in living cells is an important tool for studying cell biology and developing drugs. The required chemical reactions need to be bioorthogonal to produce selectivity. Here, we summarize currently used bioorthogonal reactions that have been successfully applied for labeling biomolecules in cells. Further, we discuss the various methods available to include orthogonally reactive groups into proteins.
A versatile chemical labeling approach was developed, where intracellular proteins were first incorporated with a bioorthogonal group via affinity conjugation, and subsequently labeled via strain-promoted cycloaddition reactions in live cells.
Currently most of the fluorogenic probes are designed for the detection of enzymes which work by converting the non-fluorescence substrate into the fluorescence product via an enzymatic reaction. On the other hand, the design of fluorogenic probes for non-enzymatic proteins remains a great challenge. Herein, we report a general strategy to create near-IR fluorogenic probes, where a small molecule ligand is conjugated to a novel γ-phenyl-substituted Cy5 fluorophore, for the selective detection of proteins through a non-enzymatic process. Detail mechanistic studies reveal that the probes self-assemble to form fluorescence-quenched J-type aggregate. In the presence of target analyte, bright fluorescence in the near-IR region is emitted through the recognition-induced disassembly of the probe aggregate. This Cy5 fluorophore is a unique self-assembly/disassembly dye as it gives remarkable fluorescence enhancement. Based on the same design, three different fluorogenic probes were constructed and one of them was applied for the no-wash imaging of tumor cells for the detection of hypoxia-induced cancer-specific biomarker, transmembrane-type carbonic anhydrase IX.
Chemical dimerizers are powerful non-invasive tools for bringing molecules together inside intact cells. We recently introduced a rapidly reversible chemical dimerizer system which enables transient translocation of enzymes to and from the plasma membrane (PM). Here we have applied this system to transiently activate phosphatidylinositol 4,5-bisphosphate (PIP2) breakdown at the PM via translocation of phosphoinositide 5-phosphatase (5Ptase). We found that the PIP2 sensor phospholipase C-δ PH domain (PLCδ-PH) is released from the PM upon addition of the reversible chemical dimerizer rCD1. By outcompeting rCD1, rapid release of the 5Ptase from the PM is followed by PIP2 recovery. This permits the observation of the PIP2-dependent clathrin assembly at the PM.
Copyright © 2015. Published by Elsevier Ltd.
Chemists and biologists have long recognized small molecule probes as powerful tools for functional genomics and proteomics studies. The possibility of specifically attaching chemical probes to individual proteins with spatial and temporal resolution has greatly improved our ability to visualize and characterize proteins in their native environment. The continued development of novel molecular probes for protein labeling is, therefore, of fundamental importance to gain new insights into biological processes in living cells and organisms. Several excellent approaches for the site-specific labeling of fusion proteins with chemical probes exist. Herein I discuss the design and generation of chemical probes for the SNAP-tag and CLIP-tag systems. The first part of this chapter is dedicated to reviewing the principles of the SNAP-tag technology, followed by a section dedicated to the development of chemical probes for unique applications, such as super-resolution imaging, protein trafficking and recycling, protein-protein interactions, and biomolecular sensing. The last part of the chapter contains experimental protocols and technical notes for the synthesis of selected SNAP-tag substrates and labeling of SNAP-tag fusion proteins in vitro and in living cells.
In this protocol we describe the incorporation of bio-orthogonal amino acids as a versatile method for visualizing and identifying de novo-synthesized proteins in the roundworm Caenorhabditis elegans. This protocol contains directions on implementing three complementary types of analysis: 'click chemistry' followed by western blotting, click chemistry followed by immunofluorescence, and isobaric tags for relative and absolute quantification (iTRAQ) quantitative mass spectrometry. The detailed instructions provided herein enable researchers to investigate the de novo proteome, an analysis that is complicated by the fact that protein molecules are chemically identical to each other, regardless of the timing of their synthesis. Our protocol circumvents this limitation by identifying de novo-synthesized proteins via the incorporation of the chemically modifiable azidohomoalanine instead of the natural amino acid methionine in the nascent protein, followed by facilitating the visualization of the resulting labeled proteins in situ. It will therefore be an ideal tool for studying de novo protein synthesis in physiological and pathological processes including learning and memory. The protocol requires 10 d for worm growth, liquid culture and synchronization; 1-2 d for bio-orthogonal labeling; and, with regard to analysis, 3-4 d for western blotting, 5-6 d for immunofluorescence or ∼3 weeks for mass spectrometry.
The ability to introduce any chemical probe to any endogenous target protein in its native environment, that is in cells and in vivo, is anticipated to provide various new exciting tools for biological and biomedical research. Although still at the prototype stage, the ligand-directed tosyl (LDT) chemistry is a novel type of affinity labeling technique that we developed for such a dream. This chemistry allows for modifying native proteins by various chemical probes with high specificity in various biological settings ranging from in vitro (in test tubes) to in living cells and in vivo. Since the first report, the list of proteins that are successfully labeled by the LDT chemistry has been increasing. A growing number of studies have demonstrated its utility to create semisynthetic proteins directly in cellular contexts. The in situ generated semisynthetic proteins are applicable for various types of analysis and imaging of intracellular biological processes. In this review, we summarize the basic properties of the LDT chemistry and its applications toward in situ engineering and analysis of native proteins in living systems. Current limitations and future challenges of this area are also described.
Ligand binding promotes conformational rearrangement of the epidermal growth factor receptor (EGFR) leading to receptor autophosphorylation and downstream signaling. However, transient interactions between unstimulated EGFR molecules on the cell surface are not fully understood. In this report, we describe the investigation of homodimer formation of EGFR by means of an SNAP-tag based selective crosslinking approach (S-CROSS). EGFR homodimers were selectively captured in living cells and utilized for analysis of protein receptor interactions on the plasma membrane and ligand-induced activation. We showed that EGFR forms homodimers in unstimulated cells with efficiencies similar to those seen in cells treated with the epidermal growth factor ligand (EGF) supporting the existence of constitutive transient receptor-receptor interactions. EGFR crosslinked homodimers displayed a substantially increase in kinase activation upon ligand stimulation. Interestingly, in unstimulated cells the levels of spontaneous phosphorylation were found to correlate with the yields of the crosslinked homodimers species. In addition, we demonstrated that this crosslinking approach can be applied to interrogate the effect of small molecule inhibitors on receptor dimerization and kinase activity. Our crosslinking assay provides a new tool to dissect ligand-independent dimerization and activation mechanisms of receptor tyrosine kinases, many of which are important anticancer drug targets.
Die Bestimmung von Proteinfunktionen in lebenden Zellen ist essenziell, um Zellen und Organismen zu verstehen. Von besonderer Wichtigkeit ist dabei die Ermittlung und Beeinflussung von Protein‐Protein‐Interaktionen. In den letzten Jahren wurden mehrere Methoden entwickelt, um Protein‐Protein‐Interaktionen in lebenden Zellen zu messen bzw. zu induzieren. Daher sind jetzt Werkzeuge verfügbar, um intrazelluläre Abläufe spezifisch durch reversibles oder irreversibles Verknüpfen zu manipulieren. Dies kann neue Türen öffnen, um komplizierte Proteinnetzwerke im Detail zu analysieren. Hier beschreiben wir die verfügbaren Verknüpfungs‐ und Dimerisierungsreagentien und ihre Anwendungen.
Advances in the development of new fluorescent reporters and imaging techniques have revolutionized our ability to directly visualize biological processes in living systems. Real-time analysis of protein localization, dynamics, and interactions has been made possible by site-specific protein labeling with custom designed probes. This review outlines some of the most recent advances in the design and application of live-cell imaging probes, with a particular focus on SNAP-tag technology. Specific examples illustrating applications in superresolution and single-molecule imaging, protein trafficking and recycling, and protein-protein interactions are presented.
Recent advancements in analytical methods relating organic chemistry, molecular biology, spectroscopy, engineering, and chemical biology have provided powerful tools to explore different selective fluorescent labeling techniques for analyzing molecular events in cells. New fluorescent labeling techniques have been developed and improvised depending on requirements like non-specificity, selectivity, small size, and of course, high yield. Along with synthesized small fluorophores that are used mostly for chemical labeling, fluorescent proteins such as green fluorescent protein (GFP) and its variants are used to label living cells genetically, both in vivo and in vitro. Fluorescent labeling has been extended over a large area of interdisciplinary research to visualize the functions and conditions of molecules using lower concentrations of the fluorescent material. Considering the wide applications of fluorescent techniques in various fields, the basic principles of different fluorescent labeling techniques relevant to chemistry and biology are discussed in this review.
Protein microarrays are valuable tools for protein assays. Reducing spot sizes from micro- to nano-scale facilitates miniaturization of platforms and consequently decreased material consumption, but faces inherent challenges in the reduction of fluorescent signals and compatibility with complex solutions. Here we show that vertical arrays of nanowires (NWs) can overcome several bottlenecks of using nanoarrays for extraction and analysis of proteins. The high aspect ratio of the NWs results in a large surface area available for protein immobilization and renders passivation of the surface between the NWs unnecessary. Fluorescence detection of proteins allows quantitative measurements and spatial resolution, enabling us to track individual NWs through several analytical steps, thereby allowing multiplexed detection of different proteins immobilized on different regions of the NW array. We use NW arrays for on-chip extraction, detection and functional analysis of proteins on a nano-scale platform that holds great promise for performing protein analysis on minute amounts of material. The demonstration made here on highly ordered arrays of indium arsenide (InAs) NWs is generic and can be extended to many high aspect ratio nanostructures.
Cell activation initiated by receptor ligands or oncogenes triggers complex and convoluted intracellular signaling. Techniques initiating signals at defined starting points and cellular locations are attractive to elucidate the output of selected pathways. Here, we present the development and validation of a protein heterodimerization system based on small molecules cross-linking fusion proteins derived from HaloTags and SNAP-tags. Chemical dimerizers of HaloTag and SNAP-tag (HaXS) show excellent selectivity and have been optimized for intracellular reactivity. HaXS force protein-protein interactions and can translocate proteins to various cellular compartments. Due to the covalent nature of the HaloTag-HaXS-SNAP-tag complex, intracellular dimerization can be easily monitored. First applications include protein targeting to cytoskeleton, to the plasma membrane, to lysosomes, the initiation of the PI3K/mTOR pathway, and multiplexed protein complex formation in combination with the rapamycin dimerization system.
The tumor suppressor p53 is in equilibrium at cellular concentrations between dimers and tetramers. Oncogenic mutant p53 (mut) exerts a dominant-negative effect on co-expression of p53 wild-type (wt) and mut alleles in cancer cells. It is believed that wt and mut form hetero-tetramers of attenuated activity, via their tetramerization domains. Using electrospray mass spectrometry on isotopically labeled samples, we measured directly the composition and rates of formation of p53 complexes in the presence and absence of response element DNA. The dissociation of tetramers was unexpectedly very slow (t(1/2) = 40 min) at 37 degrees C, matched by slow association of dimers, which is approximately four times longer than the half-life of spontaneous denaturation of wt p53. On mixing wt tetramers with the oncogenic contact mutant R273H of low DNA affinity, we observed the same slow formation of only wt(4), wt(2)mut(2), and mut(4), in the ratio 1:2:1, on a cellular time scale. On mixing wt and mut with response element DNAs P21 and BAX, we observed only the complexes wt(4)xDNA, wt(2)mut(2)xDNA, and mut(4)xDNA, with relative dissociation constants 1:4:71 and 1:13:85, respectively, accounting for the dominant-negative effect by weakened affinity. p53 dimers assemble rapidly to tetramers on binding to response element DNA, initiated by the p53 DNA binding domains. The slow oligomerization of free p53, competing with spontaneous denaturation, has implications for the possible regulation of p53 by binding proteins and DNA that affect tetramerization kinetics as well as equilibria.
(Chemical Equation Presented) Labeled and linked: The small-molecule binding site of Escherichia coli lipoic acid ligase was re-engineered to accept a fluorinated aryl azide probe in place of lipoic acid. Labeling with this mutant is highly specific for LAP fusion proteins. In cell lysate, FK506 binding protein was labeled and rapamycin-dependent photocross-linking to its interaction partner was demonstrated.
Complexed with its intracellular receptor, FKBP12, the natural product rapamycin inhibits G1 progression of the cell cycle in a variety of mammalian cell lines and in the yeast Saccharomyces cerevisae. Previously, a mammalian protein that directly associates with FKBP12-rapamycin has been identified and its encoding gene has been cloned from both human (designated FRAP) [Brown, E.J., Albers, M.W., Shin, T.B., Ichikawa, K., Keith, C.T., Lane, W.S. & Schreiber, S.L. (1994) Nature (London) 369, 756-758] and rat (designated RAFT) [Sabatini, D.M., Erdjument-Bromage, H., Lui, M., Tempst, P. & Snyder, S.H. (1994) Cell 78, 35-43]. The full-length FRAP is a 289-kDa protein containing a putative phosphatidylinositol kinase domain. Using an in vitro transcription/translation assay method coupled with proteolysis studies, we have identified an 11-kDa FKBP12-rapamycin-binding domain within FRAP. This minimal binding domain lies N-terminal to the kinase domain and spans residues 2025-2114. In addition, we have carried out mutagenesis studies to investigate the role of Ser2035, a potential phosphorylation site for protein kinase C within this domain. We now show that the FRAP Ser2035-->Ala mutant displays similar binding affinity when compared with the wild-type protein, whereas all other mutations at this site, including mimics of phosphoserine, abolish binding, presumably due to either unfavorable steric interactions or induced conformational changes.
A computer program with the code name DYNAFIT was developed for fitting either the initial velocities or the time course of enzyme reactions to an arbitrary molecular mechanism represented symbolically by a set of chemical equations. Seven numerical tests and five graphical tests are applied to judge the goodness of fit. Experimental data on the inhibition of the dissociative dimeric proteinase from HIV were used in four test examples. A set of initial velocities was analyzed to see if a tight-binding inhibitor could bind to the HIV proteinase monomer. Three different sets of progress curves were analyzed (i) to determine the kinetic properties of an irreversible inhibitor, (ii) to investigate the dissociation and denaturation mechanism for the protease dimer, and (iii) to investigate the inhibition mechanism for a transient inhibitor. The program is available by anonymous ftp via uwmml.pharmacy.wisc.edu and on the World Wide Web via http://uwmml.pharmacy.wisc.edu.
The use of low molecular weight organic compounds to induce dimerization or oligomerization of engineered proteins has wide-ranging utility in biological research as well as in gene and cell therapies. Chemically induced dimerization can be used to activate intracellular signal transduction pathways or to control the activity of a bipartite transcription factor. Dimerizer systems based on the natural products cyclosporin, FK506, rapamycin, and coumermycin have been described. However, owing to the complexity of these compounds, adjusting their binding or pharmacological properties by chemical modification is difficult. We have investigated several families of readily prepared, totally synthetic, cell-permeable dimerizers composed of ligands for human FKBP12. These molecules have significantly reduced complexity and greater adaptability than natural product dimers. We report here the efficacies of several of these new synthetic compounds in regulating two types of protein dimerization events inside engineered cells--induction of apoptosis through dimerization of engineered Fas proteins and regulation of transcription through dimerization of transcription factor fusion proteins. One dimerizer in particular, AP1510, proved to be exceptionally potent and versatile in all experimental contexts tested.
Chemically induced dimerization provides a general way to gain control over intracellular processes. Typically, FK506-binding protein (FKBP) domains are fused to a signaling domain of interest, allowing crosslinking to be initiated by addition of a bivalent FKBP ligand. In the course of protein engineering studies on human FKBP, we discovered that a single point mutation in the ligand-binding site (Phe-36 --> Met) converts the normally monomeric protein into a ligand-reversible dimer. Two-hybrid, gel filtration, analytical ultracentrifugation, and x-ray crystallographic studies show that the mutant (F(M)) forms discrete homodimers with micromolar affinity that can be completely dissociated within minutes by addition of monomeric synthetic ligands. These unexpected properties form the basis for a "reverse dimerization" regulatory system involving F(M) fusion proteins, in which association is the ground state and addition of ligand abolishes interactions. We have used this strategy to rapidly and reversibly aggregate fusion proteins in different cellular compartments, and to provide an off switch for transcription. Reiterated F(M) domains should be generally useful as conditional aggregation domains (CADs) to control intracellular events where rapid, reversible dissolution of interactions is required. Our results also suggest that dimerization is a latent property of the FKBP fold: the crystal structure reveals a remarkably complementary interaction between the monomer binding sites, with only subtle changes in side-chain disposition accounting for the dramatic change in quaternary structure.
Characterizing the movement, interactions, and chemical microenvironment of a protein inside the living cell is crucial to a detailed understanding of its function. Most strategies aimed at realizing this objective are based on genetically fusing the protein of interest to a reporter protein that monitors changes in the environment of the coupled protein. Examples include fusions with fluorescent proteins, the yeast two-hybrid system, and split ubiquitin. However, these techniques have various limitations, and considerable effort is being devoted to specific labeling of proteins in vivo with small synthetic molecules capable of probing and modulating their function. These approaches are currently based on the noncovalent binding of a small molecule to a protein, the formation of stable complexes between biarsenical compounds and peptides containing cysteines, or the use of biotin acceptor domains. Here we describe a general method for the covalent labeling of fusion proteins in vivo that complements existing methods for noncovalent labeling of proteins and that may open up new ways of studying proteins in living cells.
MDM2 binds the p53 tumor suppressor protein with high affinity and negatively modulates its transcriptional activity and stability. Overexpression of MDM2, found in many human tumors, effectively impairs p53 function. Inhibition of MDM2-p53 interaction can stabilize p53 and may offer a novel strategy for cancer therapy. Here, we identify potent and selective small-molecule antagonists of MDM2 and confirm their mode of action through the crystal structures of complexes. These compounds bind MDM2 in the p53-binding pocket and activate the p53 pathway in cancer cells, leading to cell cycle arrest, apoptosis, and growth inhibition of human tumor xenografts in nude mice.
In response to epidermal growth factor (EGF), the mitogen-activated protein kinase ERK2 translocates into the nucleus. To probe the mechanisms regulating the subcellular localization of ERK2, we used live cell imaging to examine the interaction between MEK1 and ERK2. Fluorescence resonance energy transfer (FRET) studies show that MEK1 and ERK2 directly interact and demonstrate that this interaction in the cytoplasm is largely responsible for cytoplasmic retention of ERK2. Stimulation with EGF caused loss of FRET as ERK separated from MEK and moved into the nucleus. FRET was recovered as ERK returned to the cytosol, indicating ERK reassociation with MEK in the cytoplasm. The EGF-induced transit of ERK through the nucleus was complete within 20 min, and there was no significant movement of MEK into the nucleus. Fluorescence recovery after photobleaching experiments was used to assess the rate of movement of MEK and ERK. The steady-state rate of ERK entry into the nucleus in resting cells was energy-independent and greater than the rate of ERK entry upon EGF stimulation. This suggests that the rate constant for ERK transport across the nuclear membrane is not limiting nuclear entry. Thus, we suggest that the movement of ERK into and out of the nucleus in response to agonist occurs primarily by diffusion and is controlled by interactions with binding partners in the cytosol and nucleus. No evidence of ERK dimerization was detected by FRET methods; the kinetics for nucleocytoplasmic transport were unaffected by mutations in the ERK putative dimerization domain.
A major challenge in understanding the networks of interactions that control cell and organism function is the definition of protein interactions. Solid-phase peptide synthesis has allowed the photo-cross-linkable amino acid p-benzoyl-L-phenylalanine (pBpa; Fig. 1a) to be site-specifically incorporated into peptide chains, to facilitate the definition of peptide-ligand complexes. This method, however, is limited to the in vitro study of peptides and small proteins. An innovative development allows the incorporation of a site-specific photo-cross-linker into virtually any protein that can be expressed in Escherichia coli, thereby promoting in vivo or in vitro cross-linking of proteins. The method relies on an orthogonal aminoacyl tRNA synthetase-tRNACUA pair that incorporates pBpa at the position encoded by the amber codon (UAG) in any gene transformed into E. coli (Fig. 1b). The system described in this protocol uses two plasmids: a p15A-based plasmid to express the orthogonal tRNA and synthetase pair (pDULE) and a second plasmid containing an amber mutant of the gene of interest. To produce the photo-cross-linker-containing protein, cultures of E. coli carrying both plasmids are grown in the presence of the unnatural amino acid. To photo-cross-link the protein to its binding partner in vivo or in vitro, cells or purified proteins, respectively, are exposed to UV light (Fig. 2).
To comprehend the Ras/ERK MAPK cascade, which comprises Ras, Raf, MEK, and ERK, several kinetic simulation models have been developed. However, a large number of parameters that are essential for the development of these models are still missing and need to be set arbitrarily. Here, we aimed at collecting these missing parameters using fluorescent probes. First, the levels of the signaling molecules were quantitated. Second, to monitor both the activation and nuclear translocation of ERK, we developed probes based on the principle of fluorescence resonance energy transfer. Third, the dissociation constants of Ras.Raf, Raf.MEK, and MEK.ERK complexes were estimated using a fluorescent tag that can be highlighted very rapidly. Finally, the same fluorescent tag was used to measure the nucleocytoplasmic shuttling rates of ERK and MEK. Using these parameters, we developed a kinetic simulation model consisting of the minimum essential members of the Ras/ERK MAPK cascade. This simple model reproduced essential features of the observed activation and nuclear translocation of ERK. In this model, the concentration of Raf significantly affected the levels of phospho-MEK and phospho-ERK upon stimulation. This prediction was confirmed experimentally by decreasing the level of Raf using the small interfering RNA technique. This observation verified the usefulness of the parameters collected in this study.
Protein-fragment complementation assays (PCAs) provide a general strategy to study the dynamics of protein-protein interactions in vivo and in vitro. The full potential of PCA requires assays that are fully reversible and sensitive at subendogenous protein expression levels. We describe a new assay that meets these criteria, based on the Gaussia princeps luciferase enzyme, demonstrating chemical reversal, and induction and inhibition of a key interaction linking insulin and TGFbeta signaling.
Mdm2, a key negative regulator of the p53 tumor suppressor, is a RING-type E3 ubiquitin ligase. The Mdm2 RING domain can be biochemically fractionated into two discrete species, one of which exists as higher order oligomers that are visible by electron microscopy, whereas the other is a monomer. Both fractions are ATP binding and E3 ligase activity competent, although the oligomeric fraction exhibits lower dependence on the E2 component of ubiquitin polymerization reactions. The extreme C-terminal five amino acids of Mdm2 are essential for E3 ligase activity in vivo and in vitro, as well as for oligomeric assembly of the protein. A single residue (phenylalanine 490) in that sequence is critical for both properties. Interestingly, the C-terminus of the Mdm2 homologue, MdmX (itself inert as an E3 ligase), can fully substitute for the equivalent segment of Mdm2 and restore its E3 activity. We further show that the Mdm2 C-terminus is involved in intramolecular interactions and can set up a platform for direct protein-protein interactions with the E2.
Mapping protein-protein interactions is an invaluable tool for understanding protein function. Here, we report the first large-scale study of protein-protein interactions in human cells using a mass spectrometry-based approach. The study maps protein interactions for 338 bait proteins that were selected based on known or suspected disease and functional associations. Large-scale immunoprecipitation of Flag-tagged versions of these proteins followed by LC-ESI-MS/MS analysis resulted in the identification of 24,540 potential protein interactions. False positives and redundant hits were filtered out using empirical criteria and a calculated interaction confidence score, producing a data set of 6463 interactions between 2235 distinct proteins. This data set was further cross-validated using previously published and predicted human protein interactions. In-depth mining of the data set shows that it represents a valuable source of novel protein-protein interactions with relevance to human diseases. In addition, via our preliminary analysis, we report many novel protein interactions and pathway associations.
The tumor suppressor protein p53 induces or represses the expression of a variety of target genes involved in cell cycle control, senescence, and apoptosis in response to oncogenic or other cellular stress signals. It exerts its function as guardian of the genome through an intricate interplay of independently folded and intrinsically disordered functional domains. In this review, we provide insights into the structural complexity of p53, the molecular mechanisms of its inactivation in cancer, and therapeutic strategies for the pharmacological rescue of p53 function in tumors. p53 emerges as a paradigm for a more general understanding of the structural organization of modular proteins and the effects of disease-causing mutations.
Cell-surface proteins are important in cell-cell communication. They assemble into heterocomplexes that include different receptors and effectors. Elucidation and manipulation of such protein complexes offers new therapeutic possibilities. We describe a methodology combining time-resolved fluorescence resonance energy transfer (FRET) with snap-tag technology to quantitatively analyze protein-protein interactions at the surface of living cells, in a high throughput-compatible format. Using this approach, we examined whether G protein-coupled receptors (GPCRs) are monomers or assemble into dimers or larger oligomers--a matter of intense debate. We obtained evidence for the oligomeric state of both class A and class C GPCRs. We also observed different quaternary structure of GPCRs for the neurotransmitters glutamate and gamma-aminobutyric acid (GABA): whereas metabotropic glutamate receptors assembled into strict dimers, the GABA(B) receptors spontaneously formed dimers of heterodimers, offering a way to modulate G-protein coupling efficacy. This approach will be useful in systematic analysis of cell-surface protein interaction in living cells.
Genetically encoded fluorescent sensor proteins offer the possibility to probe the concentration of key metabolites in living cells. The approaches currently used to generate Such fluorescent sensor proteins lack. generality, as they require a protein that undergoes a conformational change upon metabolite binding. Here we present an approach that overcomes this limitation. Our biosensors consist of SNAP-tag, a fluorescent protein and a metabolite-binding protein. SNAP-tag is specifically labeled with a synthetic molecule containing a ligand of the metabolite-binding protein and a fluorophore. In the labeled sensor, the metabolite of interest displaces the intramolecular ligand from the binding protein, thereby shifting the sensor protein from a closed to an open conformation. The readout is a concomitant ratiometric change in the fluorescence intensities of the fluorescent protein and the tethered fluorophore. The observed ratiometric changes compare favorably with those achieved in genetically encoded fluorescent sensor proteins. Furthermore, the modular design of our sensors permits the facile generation of ratiometric fluorescent sensors at wavelengths not covered by autofluorescent proteins. These features should allow semisynthetic fluorescent sensor proteins based on SNAP-tag to become important tools for probing previously inaccessible metabolites.
Split-protein sensors are increasingly used as analytical tools to identify, measure, and manipulate protein–protein interactions in living cells. By using split-ubiquitin as a representative example, their general and their specific properties as well as their diverse applications in proteomics, cell biology, and synthetic biology are discussed.
Transforming growth factor-beta (TGF-beta) family members bind to receptors that consist of heteromeric serine-threonine kinase subunits (type I and type II). In a yeast genetic screen, the immunophilin FKBP-12, a target of the macrolides FK506 and rapamycin, interacted with the type I receptor for TGF-beta and with other type I receptors. Deletion, point mutation, and co-immunoprecipitation studies further demonstrated the specificity of the interaction. Excess FK506 competed with type I receptors for binding to FKBP-12, which suggests that these receptors share or overlap the macrolide binding site on FKBP-12, and therefore they may represent its natural ligand. The specific interaction between the type I receptors and FKBP-12 suggests that FKBP-12 may play a role in type I receptor-mediated signaling.
Intermolecular and intramolecular FRET between two spectrally overlapping green fluorescent protein variants fused to two different host proteins or at two different sites within the same protein offers a unique opportunity to monitor real-time protein-protein interactions or protein conformational changes. By using fluorescence digital imaging microscopy, one can visualize the location of green fluorescent proteins within a living cell and follow the time course of the changes in FRET corresponding to cellular events at a millisecond time resolution. The observation of such dynamic molecular events in vivo provides vital insight into the action of biological molecules.
In the cell, homo- and heteroassociations of polypeptide chains evolve and take place within subcellular compartments that are crowded with many other cellular macromolecules. In vivo chemical cross-linking of proteins is a powerful method to examine changes in protein oligomerization and protein-protein interactions upon cellular events such as signal transduction. This chapter is intended to provide a guide to the selection of the cell-membrane-permeable cross-linkers, the optimization of in vivo cross-linking conditions, and the identification of specific cross-links in a cellular context where the frequency of random collisions is high. By combining the chemoselectivity of the homo-bifunctional cross-linker and the length of its spacer arm with knowledge on the protein structure, we show that selective cross-links can be introduced specifically on either the dimer or the hexamer form of the same polypeptide in vitro as well as in vivo, using the human type B nucleoside diphosphate kinase as a protein model.
Cellular biochemical machineries, what we call pathways, consist of dynamically assembling and disassembling macromolecular complexes. Although our models for the organization of biochemical machines are derived largely from in vitro experiments, do they reflect their organization in intact, living cells? We have developed a general experimental strategy that addresses this question by allowing the quantitative probing of molecular interactions in intact, living cells. The experimental strategy is based on protein-fragment complementation assays (PCA), a method whereby protein interactions are coupled to refolding of enzymes from cognate fragments where reconstitution of enzyme activity acts as the detector of a protein interaction. A biochemical machine or pathway is defined by grouping interacting proteins into those that are perturbed in the same way by common factors (hormones, metabolites, enzyme inhibitors, and so on). In this chapter we review some of the essential principles of PCA and provide details and protocols for applications of PCA, particularly in mammalian cells, based on three PCA reporters, dihydrofolate reductase, green fluorescent protein, and beta-lactamase.
We report a method of photo-cross-linking proteins in mammalian cells, which is based on site-specific incorporation of a photoreactive amino acid, p-benzoyl-L-phenylalanine (pBpa), through the use of an expanded genetic code. To analyze the cell signaling interactions involving the adaptor protein Grb2, pBpa was incorporated in its Src homology 2 (SH2) domain. The human GRB2 gene with an amber codon was introduced into Chinese hamster ovary (CHO) cells, together with the genes for the Bacillus stearothermophilus suppressor tRNA(Tyr) and a pBpa-specific variant of Escherichia coli tyrosyl-tRNA synthetase (TyrRS). The Grb2 variant with pBpa in the amber position was synthesized when pBpa was included in the growth medium. Upon exposure of cells to 365-nm light, protein variants containing pBpa in the positions proximal to the ligand-binding pocket were cross-linked with the transiently expressed epidermal growth factor (EGF) receptor in the presence of an EGF stimulus. Cross-linked complexes with endogenous proteins were also detected. In vivo photo-cross-linking with pBpa incorporated in proteins will be useful for studying protein-protein interactions in mammalian cells.
Rapamycin is an important immunosuppressant, a possible anticancer therapeutic, and a widely used research tool. Essential to its various functions is its ability to bind simultaneously to two different proteins, FKBP and mTOR. Despite its widespread use, a thorough analysis of the interactions between FKBP, rapamycin, and the rapamycin-binding domain of mTOR, FRB, is lacking. To probe the affinities involved in the formation of the FKBP.rapamycin.FRB complex, we used fluorescence polarization, surface plasmon resonance, and NMR spectroscopy. Analysis of the data shows that rapamycin binds to FRB with moderate affinity (K(d) = 26 +/- 0.8 microM). The FKBP12.rapamycin complex, however, binds to FRB 2000-fold more tightly (K(d) = 12 +/- 0.8 nM) than rapamycin alone. No interaction between FKBP and FRB was detected in the absence of rapamycin. These studies suggest that rapamycin's ability to bind to FRB, and by extension to mTOR, in the absence of FKBP is of little consequence under physiological conditions. Furthermore, protein-protein interactions at the FKBP12-FRB interface play a role in the stability of the ternary complex.
Systematic mapping of protein-protein interactions, or 'interactome' mapping, was initiated in model organisms, starting with defined biological processes and then expanding to the scale of the proteome. Although far from complete, such maps have revealed global topological and dynamic features of interactome networks that relate to known biological properties, suggesting that a human interactome map will provide insight into development and disease mechanisms at a systems level. Here we describe an initial version of a proteome-scale map of human binary protein-protein interactions. Using a stringent, high-throughput yeast two-hybrid system, we tested pairwise interactions among the products of approximately 8,100 currently available Gateway-cloned open reading frames and detected approximately 2,800 interactions. This data set, called CCSB-HI1, has a verification rate of approximately 78% as revealed by an independent co-affinity purification assay, and correlates significantly with other biological attributes. The CCSB-HI1 data set increases by approximately 70% the set of available binary interactions within the tested space and reveals more than 300 new connections to over 100 disease-associated proteins. This work represents an important step towards a systematic and comprehensive human interactome project.
Although epidermal growth factor receptor (EGFR; also called ErbB1) and its relatives initiate one of the most well-studied signalling networks, there is not yet a genome-wide view of even the earliest step in this pathway: recruitment of proteins to the activated receptors. Here we use protein microarrays comprising virtually every Src homology 2 (SH2) and phosphotyrosine binding (PTB) domain encoded in the human genome to measure the equilibrium dissociation constant of each domain for 61 peptides representing physiological sites of tyrosine phosphorylation on the four ErbB receptors. This involved 77,592 independent biochemical measurements and provided a quantitative protein interaction network that reveals many new interactions, including ones that fall outside of our current view of domain selectivity. By slicing through the network at different affinity thresholds, we found surprising differences between the receptors. Most notably, EGFR and ErbB2 become markedly more promiscuous as the threshold is lowered, whereas ErbB3 does not. Because EGFR and ErbB2 are overexpressed in many human cancers, our results suggest that the extent to which promiscuity changes with protein concentration may contribute to the oncogenic potential of receptor tyrosine kinases, and perhaps other signalling proteins as well.
Protein microarrays are an attractive approach for the high-throughput analysis of protein function, but their impact on proteomics has been limited by the technical difficulties associated with their generation. Here we demonstrate that fusion proteins of O6-alkylguanine-DNA alkyltransferase (AGT) can be used for the simple and reliable generation of protein microarrays for the analysis of protein function. Important features of the approach are the selectivity of the covalent immobilization; this allows for direct immobilization of proteins out of cell extracts, and the option both to label and to immobilize AGT fusion proteins, which allows for direct screening for protein-protein interactions between different AGT fusion proteins. In addition to the identification of protein-protein interactions, AGT-based protein microarrays can be used for the characterization of small molecule-protein interactions or post-translational modifications. The potential of the approach was demonstrated by investigating the post-translational modification of acyl carrier protein (ACP) from E. coli by different phosphopantetheine transferases (PPTases), yielding insights into the role of selected ACP amino acids in the ACP-PPTase interaction.
The specific reaction of O6-alkylguanine-DNA alkyltransferase (AGT) with O6-benzylguanine (BG) derivatives allows for a specific labeling of AGT fusion proteins with chemically diverse compounds in living cells and in vitro. The efficiency of the labeling depends on a number of factors, most importantly on the reactivity, selectivity and stability of AGT. Here, we report the use of directed evolution and two different selection systems to further increase the activity of AGT towards BG derivatives by a factor of 17 and demonstrate the advantages of this mutant for the specific labeling of AGT fusion proteins displayed on the surface of mammalian cells. The results furthermore identify two regions of the protein outside the active site that influence the activity of the protein towards BG derivatives.
Cells technique: Small molecules have been synthesized that enable the covalent and irreversible dimerization of fusion proteins of O6-alkylguanine-DNA alkyltransferase (AGT or SNAP-Tag) in vitro and in living cells. The cross-linking efficiency of AGT fusion proteins provides a measure to characterize the proximity and interactions of protein pairs in living cells (see scheme).
Changes in the interactions among proteins that participate in a biochemical pathway can reflect the immediate regulatory responses to intrinsic or extrinsic perturbations of the pathway. Thus, methods that allow for the direct detection of the dynamics of protein-protein interactions can be used to probe the effects of any perturbation on any pathway of interest. Here we describe experimental strategies - based on protein-fragment complementation assays (PCAs) - that can achieve this. PCA-based strategies can be used with or instead of traditional target-based drug discovery strategies to identify novel pathway-component proteins of therapeutic interest, to increase the quantity and quality of information about the actions of potential drugs, and to gain insight into the intricate networks that make up the molecular machinery of living cells.
A large number of methods have been developed over the years to study protein-protein interactions. Many of these techniques are now available to the nonspecialist researcher thanks to new affordable instruments and/or resource centres. A typical protein-protein interaction study usually starts with an initial screen for novel binding partners. We start this review by describing three techniques that can be used for this purpose: (i) affinity-tagged proteins (ii) the two-hybrid system and (iii) some quantitative proteomic techniques that can be used in combination with, e.g., affinity chromatography and coimmunoprecipitation for screening of protein-protein interactions. We then describe some public protein-protein interaction databases that can be searched to identify previously reported interactions for a given bait protein. Four strategies for validation of protein-protein interactions are presented: confocal microscopy for intracellular colocalization of proteins, coimmunoprecipitation, surface plasmon resonance (SPR) and spectroscopic studies. Throughout the review we focus particularly on the advantages and limitations of each method.
We have observed differences in the infrared spectra of viable fibroblast cells depending on whether the cells were in the exponential (proliferating) or plateau (nonproliferating) phase of growth. The spectral changes were observed even after correcting for cell number and volume, ruling out these trivial explanations. Several of the changes occurred for both transformed and normal cell lines and were greater for the normal cell line. The biochemical basis of the spectral changes was estimated by fitting the cell spectra to a linear superposition of spectra for the major biochemical components of mammalian cells (DNA, RNA, protein, lipids, and glycogen). The ratios of RNA/lipid and protein/lipid increased when the cells were in the exponential phase compared to the plateau phase of growth. The fits of cell spectra to individual biochemical components also demonstrated that DNA is a relatively minor spectroscopic component as would be expected biochemically. Contrary to other reports in the literature, our data demonstrate that determining DNA content or structure using Fourier transform infrared spectroscopy data is difficult due to the relatively small amount of DNA and the overlap of DNA bands with the absorption bands of other biochemical components.
Dynamic protein interactions play a significant part in many cellular processes. A technique that shows considerable promise in elucidating such interactions is Förster resonance energy transfer (FRET). When combined with multiple, colored fluorescent proteins, FRET permits high spatial resolution assays of protein-protein interactions in living cells. Because FRET signals are usually small, however, their measurement requires careful interpretation and several control experiments. Nevertheless, the use of FRET in cell biological experiments has exploded over the past few years. Here we describe the physical basis of FRET and the fluorescent proteins appropriate for these experiments. We also review the approaches that can be used to measure FRET, with particular emphasis on the potential artifacts associated with each approach.
A new label transfer method is presented that overcomes most of the limitations of current systems. A protein of interest is tagged with a tetracysteine sequence (FlAsH receptor peptide (FRP)) that binds tightly and specifically to a chimeric molecule 3,4dihydroxyphenylalanine-biotin-4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein (DOPA-biotin-FlAsH). Upon brief periodate oxidation, the DOPA moiety is cross-linked to nearby surface-exposed nucleophiles. Boiling the products in excess dithiol dissolves the FlAsH-FRP interaction, resulting in transfer of the biotin tag to the partner proteins, allowing them to be identified by standard methods.
Homotypic and heterotypic protein interactions are crucial for all levels of cellular function, including architecture, regulation, metabolism, and signaling. Therefore, protein interaction maps represent essential components of post-genomic toolkits needed for understanding biological processes at a systems level. Over the past decade, a wide variety of methods have been developed to detect, analyze, and quantify protein interactions, including surface plasmon resonance spectroscopy, NMR, yeast two-hybrid screens, peptide tagging combined with mass spectrometry and fluorescence-based technologies. Fluorescence techniques range from co-localization of tags, which may be limited by the optical resolution of the microscope, to fluorescence resonance energy transfer-based methods that have molecular resolution and can also report on the dynamics and localization of the interactions within a cell. Proteins interact via highly evolved complementary surfaces with affinities that can vary over many orders of magnitude. Some of the techniques described in this review, such as surface plasmon resonance, provide detailed information on physical properties of these interactions, while others, such as two-hybrid techniques and mass spectrometry, are amenable to high-throughput analysis using robotics. In addition to providing an overview of these methods, this review emphasizes techniques that can be applied to determine interactions involving membrane proteins, including the split ubiquitin system and fluorescence-based technologies for characterizing hits obtained with high-throughput approaches. Mass spectrometry-based methods are covered by a review by Miernyk and Thelen (2008; this issue, pp. 597-609). In addition, we discuss the use of interaction data to construct interaction networks and as the basis for the exciting possibility of using to predict interaction surfaces.
The visualization of complex cellular processes involving multiple proteins requires the use of spectroscopically distinguishable fluorescent reporters. We have previously introduced the SNAP-tag as a general tool for the specific labeling of SNAP-tag fusion proteins in living cells. The SNAP-tag is derived from the human DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT) and can be covalently labeled in living cells using O6-benzylguanine derivatives bearing a chemical probe. Here we report the generation of an AGT-based tag, named CLIP-tag, which reacts specifically with O2-benzylcytosine derivatives. Because SNAP-tag and CLIP-tag possess orthogonal substrate specificities, SNAP and CLIP fusion proteins can be labeled simultaneously and specifically with different molecular probes in living cells. We furthermore show simultaneous pulse-chase experiments to visualize different generations of two different proteins in one sample.
Immunoprecipitation (IP) uses the specificity of antibodies to isolate target proteins (antigens) out of complex sample mixtures. Three different approaches for performing IP will be discussed; traditional (classical) method, oriented affinity method and direct affinity method. The traditional method of incubating the IP antibody with the sample and sequentially binding to Protein A or G agarose beads (resin) facilitates the most efficient target antigen recovery. However, this approach results in the target protein becoming contaminated with the IP antibody that can interfere with downstream analyses. The orientated affinity method uses Protein A or G beads to serve as an anchor to which the IP antibody is crosslinked thereby preventing the antibody from co-eluting with the target protein. Similarly, the direct affinity method also immobilizes the IP antibody except in this case it is directly attached to a chemically activated support. Both methods prevent co-elution of the IP antibody enabling reuse of the immunomatrix. All three approaches have unique advantages and can also be used for co-immunoprecipitation to study protein:protein interactions and investigate the functional proteome.
We report a new method for detection of protein-protein interactions in vitro and in cells. One protein partner is fused to Escherichia coli biotin ligase (BirA), while the other protein partner is fused to BirA's "acceptor peptide" (AP) substrate. If the two proteins interact, BirA will catalyze site-specific biotinylation of AP, which can be detected by streptavidin staining. To minimize nonspecific signals, we engineered the AP sequence to reduce its intrinsic affinity for BirA. The rapamycin-controlled interaction between FKBP and FRB proteins could be detected in vitro and in cells with a signal to background ratio as high as 28. We also extended the method to imaging of the phosphorylation-dependent interaction between Cdc25C phosphatase and 14-3-3epsilon phosphoserine/threonine binding protein. Protein-protein interaction detection by proximity biotinylation has the advantages of low background, high sensitivity, small AP tag size, and good spatial resolution in cells.
Multiprotein complexes partake in nearly all cell functions, thus the characterization and visualization of protein-protein interactions in living cells constitute an important step in the study of a large array of cellular mechanisms. Recently, noninvasive fluorescence-based methods using resonance energy transfer (RET), namely bioluminescence-RET (BRET) and fluorescence-RET (FRET), and those centered on protein fragment complementation, such as bimolecular fluorescence complementation (BiFC), have been successfully used in the study of protein interactions. These new technologies are nowadays the most powerful approaches for visualizing the interactions occurring within protein complexes in living cells, thus enabling the investigation of protein behavior in their normal milieu. Here we address the individual strengths and weaknesses of these methods when applied to the study of protein-protein interactions.
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