C-terminal modifications of a protein by UAG-encoded incorporation of puromycin during in vitro protein synthesis in the absence of release factor 1

Deactivation of release factor 1 by polyclonal antibodies in an in vitro translation system, which was used to express the esterase gene, led to the reversible elimination of naturally occurring termination. This technique allowed the antibiotic puromycin to be used as an acceptor substrate for the peptidyl residue in the peptidyl-transferase reaction. This resulted in more than 80 % yield of protein with C-terminally incorporated puromycin. pCpPuromycin that was either conjugated with the Cy3 fluorophor or biotin by N4 alkylation of cytosine, also acted as an acceptor substrate for the peptidyl-transferase reaction and was incorporated into the protein C terminus. The resulting conjugates possessed Cy3-specific fluorescence and affinity to streptavidin-coated surfaces, respectively. This left the enzymatic activity of the reporter protein unaffected. It was also shown that extension of puromycin on its 5'-hydroxyl end by up to ten deoxyoligonucleotides also allowed conjugation with the C terminus of in vitro translated protein when RF1-dependent termination was suppressed. However, the conjugation yield decreased upon addition of more than six nucleotides.     

Intein-mediated construction of a library of fluorescent Rab GTPase probes

Rab GTPases play a key role in the regulation of membrane trafficking. Post-translational geranylgeranylation is critical for their biological activity and is conferred by Rab geranylgeranyl transferease (RabGGTase), together with an accessory factor, Rab escort protein (REP). Mechanistic studies of Rab prenylation and identification of RabGGTase inhibitors require sensitive reporters of Rab prenylation. In the present work, a combination of protein engineering and expressed protein ligation was used to construct a library of semisynthetic Rab7 fluorescent conjugates. In order to avoid synthesis of a large number of fluorescently labeled peptides, we developed a strategy that combined thiol-reactive dye-labeling of cysteine with in vitro protein ligation. Application of this strategy required optimization of labeling and ligation conditions to promote thiol labeling and disfavor intramolecular cyclization. Using this approach, we constructed 46 fluorescent sensors with different spectral properties that reported on the interaction of Rab7 with RabGGTase, REP-1, and the overall prenylation reaction. Two constructs, Rab7Δ3CCK(NBD) and Rab7Δ2SCCC-dans, displayed 2.5- and 1.5-fold increase in fluorescence, respectively, upon prenylation. Moreover, dansyl-, NBD (4-nitro-benzofurazan)-, I-BA-, and I-SO-labeled Rab7 conjugates exhibited two- to tenfold change in fluorescence upon binding to REP or RabGGTase. These fluorescent sensors allowed us to monitor Rab prenylation in real time and to investigate the assembly of Rab-REP binary and Rab-REP-RabGGTase ternary complexes.     

Engineering substrate specificity of O6-alkylguanine-DNA alkyltransferase for specific protein labeling in living cells

Fusion proteins of human O(6)-alkylguanine-DNA alkyltransferase (AGT) can be specifically labeled with a wide variety of synthetic probes in mammalian cells; this makes them an attractive tool for studying protein function. However, to avoid undesired labeling of endogenous wild-type AGT (wtAGT), the specific labeling of AGT fusion proteins has been restricted to AGT-deficient mammalian cell lines. We present here the synthesis of an inhibitor of wtAGT and the generation of AGT mutants that are resistant to this inhibitor. This enabled the inactivation of wtAGT and specific labeling of fusion proteins of the AGT mutant in vitro and in living cells. The ability to specifically label AGT fusion proteins in the presence of endogenous AGT, after brief incubation of the cells with a small-molecule inhibitor, should significantly broaden the scope of application of AGT fusion proteins for studying protein function in living cells.     

Tunable photoactivation of a post-translationally modified signaling protein and its unmodified counterpart in live cells

An ideal technology for direct imaging of post-translationally modified proteins would be one in which the appearance of a fluorescent signal is linked to a modification dependent protein-activation event. Herein, we utilize the protein semisynthesis technique, expressed protein ligation (EPL), to prepare caged analogues of the signaling protein Smad2; the function and fluorescence of the analogues were then photocontrolled in a correlated fashion. We show that this strategy permits titration of the cellular levels of active phosphorylated Smad2 in its biologically relevant, full-length form. We also prepared a nonphosphorylated, caged full-length Smad2 analogue labeled with an orthogonal fluorophore, and simultaneously imaged the phosphorylated and nonphosphorylated forms of the protein in the same cell. This strategy should enable the dissection of the cellular consequences of post-translational modifications (PTMs) by direct comparison of the behavior of the modified and unmodified forms of the protein following uncaging.     

Ribosomal Synthesis of Cyclic Peptides with a Fluorogenic Oxidative Coupling Reaction

Ring around the peptides : We demonstrate a new method for the cyclization of peptides that involves the oxidative coupling of 5‐hydroxyindole and benzylamine. After two nonproteinogenic amino acids were incorporated into peptides by reprogramming the genetic code, cyclization took place rapidly upon the addition of K₃Fe(CN)₆ and generated a conjugated, fluorescent, heterocyclic structure.

     

Optimized fluorescent trimethoprim derivatives for in vivo protein labeling

The combined technologies of optical microscopy and selective probes allow for real-time analysis of protein function in living cells. Synthetic chemistry offers a means to develop specific, protein-targeted probes that exhibit greater optical and chemical functionality than the widely used fluorescent proteins. Here we describe pharmacokinetically optimized, fluorescent trimethoprim (TMP) analogues that can be used to specifically label recombinant proteins fused to E. coli dihydrofolate reductase (eDHFR) in living, wild-type mammalian cells. These improved fluorescent tags exhibited high specificity and fast labeling kinetics, and they could be detected at a high signal-to-noise ratio by using fluorescence microscopy and fluorescence-activated cell sorting (FACS). We also show that fluorescent TMP-eDHFR complexes are complements to green fluorescent protein (GFP) for two-color protein labeling experiments in cells.     

Development of SNAP-tag fluorogenic probes for wash-free fluorescence imaging

The ability to specifically attach chemical probes to individual proteins represents a powerful approach to the study and manipulation of protein function in living cells. It provides a simple, robust and versatile approach to the imaging of fusion proteins in a wide range of experimental settings. However, a potential drawback of detection using chemical probes is the fluorescence background from unreacted or nonspecifically bound probes. In this report we present the design and application of novel fluorogenic probes for labeling SNAP-tag fusion proteins in living cells. SNAP-tag is an engineered variant of the human repair protein O(6)-alkylguanine-DNA alkyltransferase (hAGT) that covalently reacts with benzylguanine derivatives. Reporter groups attached to the benzyl moiety become covalently attached to the SNAP tag while the guanine acts as a leaving group. Incorporation of a quencher on the guanine group ensures that the benzylguanine probe becomes highly fluorescent only upon labeling of the SNAP-tag protein. We describe the use of intramolecularly quenched probes for wash-free labeling of cell surface-localized epidermal growth factor receptor (EGFR) fused to SNAP-tag and for direct quantification of SNAP-tagged β-tubulin in cell lysates. In addition, we have characterized a fast-labeling variant of SNAP-tag, termed SNAP(f), which displays up to a tenfold increase in its reactivity towards benzylguanine substrates. The presented data demonstrate that the combination of SNAP(f) and the fluorogenic substrates greatly reduces the background fluorescence for labeling and imaging applications. This approach enables highly sensitive spatiotemporal investigation of protein dynamics in living cells.     

Cover Picture: A Powerful Combinatorial Screen to Identify High‐Affinity Terbium(III)‐Binding Peptides (ChemBioChem 4/2003)

The cover picture shows the chemistry and biology used to develop a new protein labeling strategy based on lanthanide‐binding peptides. Focused peptide libraries are screened to identify oligopeptide motifs of dual function: the peptides bind Tb^III with remarkably high affinity and promote intense Tb^III luminescence. The peptide sequences are then genetically encoded to create recombinant fusion proteins that possess a site‐specific and minimally invasive fluorophore. Further information can be found in the two articles by Imperiali and co‐workers on pp. 265–271 and pp. 272–276.     

A powerful combinatorial screen to identify high-affinity terbium(III)-binding peptides

Lanthanide-binding tags (LBTs) are protein fusion partners consisting of encoded amino acids that bind lanthanide ions with high affinity. Herein, we present a new screening methodology for the identification of new LBT sequences with high affinity for Tb(3+) ions and intense luminescence properties. This methodology utilizes solid-phase split-and-pool combinatorial peptide synthesis. Orthogonally cleavable linkers allow an efficient two-step screening procedure. The initial screen avoids the interference caused by on-bead screening by photochemically releasing a portion of the peptides into an agarose matrix for evaluation. The secondary screen further characterizes each winning sequence in a defined aqueous solution. Employment of this methodology on a series of focused combinatorial libraries yielded a linear peptide sequence of 17 encoded amino acids that demonstrated a 140-fold increase in affinity (57 nM dissociation constant, K(D)) over previously reported lanthanide-binding peptides. This linear sequence was macrocyclized by introducing a disulfide bond between flanking cysteine residues to produce a peptide with a 2-nM apparent dissociation constant for Tb(3+) ions.Supporting information for this article is available on the WWW under http://www.chemphyschem.org or from the author.     

Glycine fluoromethylketones as SENP-specific activity based probes

We report here the synthesis and biochemical properties of a new peptidyl activity-based probe 1 for SUMO proteases, SENPs. The activity-based probe has at its C terminus a glycine-derived fluoromethylketone moiety as a reactive group designed to target the active-site cysteine of SENPs. Based on a study of the interactions between SENPs and SUMOs, we introduced further design elements that allow the activity-based probe to selectively target SENPs at low micromolar to high nanomolar concentrations. Moreover, 1 out-competes SUMO1 from the reversible SUMO1-SENP1 complex, thus suggesting that 1 and SUMO1 share a common binding site on SENP1.     

Specificity of Lipoprotein Chaperones for the Characteristic Lipidated Structural Motifs of their Cognate Lipoproteins

Lipoprotein‐binding chaperones mediate intracellular transport of lipidated proteins and determine their proper localisation and functioning. Understanding of the exact structural parameters that determine recognition and transport by different chaperones is of major interest. We have synthesised several lipid‐modified peptides, representative of different lipoprotein classes, and have investigated their binding to the relevant chaperones PDEδ, UNC119a, UNC119b, and galectins‐1 and ‐3. Our results demonstrate that PDEδ recognises S‐isoprenylated C‐terminal peptidic structures but not N‐myristoylated peptides. In contrast, UNC119 proteins bind only mono‐N‐myristoylated, but do not recognise doubly lipidated and S‐isoprenylated peptides at the C terminus. For galectins‐1 and ‐3, neither binding to N‐acylated, nor to C‐terminally prenylated peptides could be determined. These results shed light on the specificity of the chaperone‐mediated cellular lipoprotein transport systems.     

Multiplexed Delivery of Synthetic (Un)Conjugatable Ubiquitin and SUMO2 Enables Simultaneous Monitoring of Their Localization and Function in Live Cells

Ubiquitin (Ub) and its related small Ub like modifier (SUMO) are among the most influential protein post-translational modifications in eukaryotes. Unfortunately, visualizing these modifications in live cells is a challenging task. Chemical protein synthesis offers great opportunities in studying and further understanding Ub and SUMO biology. Nevertheless, the low cell permeability of proteins limits these studies mainly for in vitro applications. Here, we introduce a multiplexed protein cell delivery approach, termed MBL (multiplexed bead loading), for simultaneous loading of up to four differentially labeled proteins with organic fluorophores. We applied MBL to visualize ubiquitination and SUMOylation events in live and untransfected cells without fluorescent protein tags or perturbation to their endogenous levels. Our study reveals unprecedented involvements of Ub and SUMO2 in lysosomes depending on conjugation states. We envision that this approach will improve our understanding of dynamic cellular processes such as formation and disassembly of membraneless organelles.     

Seeing is believing: peptide-based fluorescent sensors of protein tyrosine kinase activity

Protein tyrosine kinases are key biochemical effectors of the signaling pathways that drive both normal and aberrant cell behavior. The ability to visualize the activity of tyrosine kinases in both a continuous and sensitive fashion will have a dramatic impact on the identification and characterization of inhibitors, the elucidation of the biochemical role of protein tyrosine kinases in various biological processes, and the imaging of kinase action in cells, tissues, and whole organisms. Several chemical strategies have recently been described that translate the formation of a phosphorylated tyrosine residue into a fluorescent readout. The challenges associated with the design of protein tyrosine kinase sensors, as well as the scope and limitations of the currently available sensors, are described.     

Site-specific chemical modification of proteins with a prelabelled cysteine tag using the artificially split Mxe GyrA intein

The selective modification of proteins with a synthetic probe is of central interest for many aspects of protein chemistry. We have recently reported a new approach in which a short cysteine-containing tag (CysTag) fused to one part of a split intein is first modified with a sulfhydryl-reactive probe. In a second step, protein trans-splicing is used to link the labelled CysTag to a target protein that has been expressed in fusion with the complementary split intein fragment. Here, we present the generation and biochemical characterisation of the artificially split Mycobacterium xenopi GyrA intein. We show that this split intein is active without a renaturation step and that it provides a significant improvement for the CysTag protein-labelling approach in terms of product yields and target protein tolerance. Two proteins with multiple cysteine residues, human growth hormone and a multidomain nonribosomal peptide synthetase, were site-specifically modified with high yields. Our approach combines the benefits of the plethora of commercially available cysteine-reactive probes with a straightforward route for their site-specific incorporation even into complex and cysteine-rich proteins.