Single-cell FRET imaging of transferrin receptor trafficking dynamics by Sfp-catalyzed, site-specific protein labeling

Fluorescence imaging of living cells depends on an efficient and specific method for labeling the target cellular protein with fluorophores. Here we show that Sfp phosphopantetheinyl transferase-catalyzed protein labeling is suitable for fluorescence imaging of membrane proteins that spend at least part of their membrane trafficking cycle at the cell surface. In this study, transferrin receptor 1 (TfR1) was fused to peptide carrier protein (PCP), and the TfR1-PCP fusion protein was specifically labeled with fluorophore Alexa 488 by Sfp. The trafficking of transferrin-TfR1-PCP complex during the process of transferrin-mediated iron uptake was imaged by fluorescence resonance energy transfer between the fluorescently labeled transferrin ligand and TfR1 receptor. We thus demonstrated that Sfp-catalyzed small molecule labeling of the PCP tag represents a practical and efficient tool for molecular imaging studies in living cells.     

Phagemid encoded small molecules for high throughput screening of chemical libraries

A new strategy for monovalently displaying small molecules on phage surfaces was developed and applied to high throughput screening for molecules with high binding affinity to the target protein. Peptidyl carrier protein (PCP) excised from nonribosomal peptide synthetase was monovalently displayed on the surface of M13 phage as pIII fusion proteins. Small molecules of diverse structures were conjugated to coenzyme A (CoA) and then covalently attached to the phage displayed PCP by Sfp phosphopantetheinyl transferase. Because Sfp is broadly promiscuous for the transfer of small molecule linked phosphopantetheinyl moieties to apo PCP domains, this approach will enable displaying libraries of small molecules on phage surfaces. Unique 20-base-pair (bp) DNA sequences were also incorporated into the phagemid DNA so that each compound displayed on the phage surface was encoded by a DNA bar code encapsulated inside the phage coat protein. Single round selection of phage displayed small molecules achieved more than 2000-fold enrichment of small molecules with nM binding affinity to the target protein. The selection process is further accelerated by the use of DNA decoding arrays for identifying the selected small molecules.     

Direct site-selective covalent protein immobilization catalyzed by a phosphopantetheinyl transferase

Immobilization of proteins onto solid supports is important in the preparation of functional protein microarrays and in the development of bead-based bioassays, biosensors, and industrial biocatalysts. In order to generate the stable, functional, and homogeneous materials required for these applications, attention has focused on methods that enable the efficient and site-specific covalent immobilization of recombinant proteins onto a wide range of platforms. To this end, the phosphopantetheinyl transferase Sfp was employed to catalyze the direct immobilization of recombinant proteins bearing the small, genetically encoded ybbR tag onto surfaces functionalized with CoA. Using mass spectrometry, it was shown that the Sfp catalyzes immobilization of a model acyl carrier protein (ACP) onto CoA-derivatized PEGA resin beads through specific covalent bond formation. Luciferase (Luc) and glutathione-S-transferase (GST) ybbR-fusion proteins were similarly immobilized onto PEGA resin retaining high levels of enzyme activity. This strategy was also successfully applied for the immobilization of the ACP, as well as ybbR-Luc, -GST, and -thioredoxin fusion proteins, on hydrogel microarray slides. Overall, the Sfp-catalyzed surface ligation is mild, quantitative, and rapid, occurring in a single step without prior chemical modification of the target protein. Immobilization of the target proteins directly from a cell lysate mixture was also demonstrated.     

New aldehyde tag sequences identified by screening formylglycine generating enzymes in vitro and in vivo

Formylglycine generating enzyme (FGE) performs a critical posttranslational modification of type I sulfatases, converting cysteine within the motif CxPxR to the aldehyde-bearing residue formylglycine (FGly). This concise motif can be installed within heterologous proteins as a genetically encoded "aldehyde tag" for site-specific labeling with aminooxy- or hydrazide-functionalized probes. In this report, we screened FGEs from M. tuberculosis and S. coelicolor against synthetic peptide libraries and identified new substrate sequences that diverge from the canonical motif. We found that E. coli's FGE-like activity is similarly promiscuous, enabling the use of novel aldehyde tag sequences for in vivo modification of recombinant proteins.     

Non-enzymatic covalent protein labeling using a reactive tag

We describe herein a new method for covalent labeling of proteins using a complementary recognition pair of peptide tag and synthetic molecular probe. The rapid and specific covalent labeling of a tag-fused protein was achieved by the reaction on the tag site with the probe through their selective molecular recognition. The advantages of this method involve the facile functional modification and the high labeling specificity of the tag-fused protein, which are demonstrated in the labeling experiments in various conditions even inside cells.     

Oligo-Asp tag/Zn(II) complex probe as a new pair for labeling and fluorescence imaging of proteins

To accomplish the selective labeling of a specific protein in complicated biological systems, a peptide tag incorporated into the protein and a complementary small molecular probe are required. Although a variety of peptide tag/probe pairs have been developed as molecular tools for protein analyses, the availability of pairs suitable for real-time imaging of proteins is still limited. We now report a new peptide tag/artificial probe pair composed of a genetically encodable oligo-aspartate sequence (D4 tag, (D4)n, n = 1-3) and the corresponding multinuclear Zn(II) complexes (Zn(II)-DpaTyrs). The strong binding affinity of the Zn(II)-DpaTyr probes with the D4 tag was a result of the multiple coordination bonds and the multivalent effect. It was measured quantitatively by isothermal titration calorimetry. The high affinity between the tag and the probe, indispensable for the selective protein labeling, enabled the pair to be used for the labeling and fluorescence imaging of a membrane-bound receptor protein tethering a triply repeated D4 tag ((D4)3) in an intact cell configuration without significantly affecting the receptor signal transduction.     

Site‐Selective Cysteine–Cyclooctyne Conjugation

We report a site‐selective cysteine–cyclooctyne conjugation reaction between a seven‐residue peptide tag (DBCO‐tag, Leu‐Cys‐Tyr‐Pro‐Trp‐Val‐Tyr) at the N or C terminus of a peptide or protein and various aza‐dibenzocyclooctyne (DBCO) reagents. Compared to a cysteine peptide control, the DBCO‐tag increases the rate of the thiol–yne reaction 220‐fold, thereby enabling selective conjugation of DBCO‐tag to DBCO‐linked fluorescent probes, affinity tags, and cytotoxic drug molecules. Fusion of DBCO‐tag with the protein of interest enables regioselective cysteine modification on proteins that contain multiple endogenous cysteines; these examples include green fluorescent protein and the antibody trastuzumab. This study demonstrates that short peptide tags can aid in accelerating bond‐forming reactions that are often slow to non‐existent in water.     

A dipicolinic acid tag for rigid lanthanide tagging of proteins and paramagnetic NMR spectroscopy

A new lanthanide tag was designed for site-specific labeling of proteins with paramagnetic lanthanide ions. The tag, 4-mercaptomethyl-dipicolinic acid, binds lanthanide ions with nanomolar affinity, is readily attached to proteins via a disulfide bond, and avoids the problems of diastereomer formation associated with most of the conventional lanthanide tags. The high lanthanide affinity of the tag opens the possibility to measure residual dipolar couplings in a single sample containing a mixture of paramagnetic and diamagnetic lanthanides. Using the DNA-binding domain of the E. coli arginine repressor as an example, it is demonstrated that the tag allows immobilization of the lanthanide ion in close proximity of the protein by additional coordination of the lanthanide by a carboxyl group of the protein. The close proximity of the lanthanide ion promotes accurate determinations of magnetic susceptibility anisotropy tensors. In addition, the small size of the tag makes it highly suitable for studies of intermolecular interactions.     

In vivo reporter labeling of proteins via metabolic delivery of coenzyme A analogues

We demonstrate site-specific reporter labeling of proteins within live cells. By accessing the coenzyme A (CoA) metabolic pathway with a cell-permeable pantetheine analogue, we deliver CoA-bound reporter molecules for post-translational protein modification in vivo. These methods may be applied to natural product pathway manipulation as well as applications in conventional molecular and cellular biology.     

化学标记与蛋白质/多肽的识别检测

化学标记技术可以实现选择性地标记蛋白质/多肽分子,从而极大地提高了对蛋白质/多肽的识别效率和检测灵敏度,是突破蛋白质/多肽化学组成局限和仪器分析检测能力瓶颈的有效途径.本文对目前这一领域的研究现状扼要地进行了综述,主要包括针对蛋白质/多肽分子中内源氨基酸残基的标记策略、蛋白质/多肽分子中翻译后修饰基团的标记策略、基因编码表达肽段的标记策略以及配体/抗体亲和标记策略.透过这些研究所取得的成果,可以断定化学标记技术将会不断发展并将在蛋白质及蛋白质组学研究中发挥重要作用.     

Cloning and characterization of a phosphopantetheinyl transferase from Streptomyces verticillus ATCC15003, the producer of the hybrid peptide-polyketide antitumor drug bleomycin.

BACKGROUND: Phosphopantetheinyl transferases (PPTases) catalyze the posttranslational modification of carrier proteins by the covalent attachment of the 4'-phosphopantetheine (P-pant) moiety of coenzyme A to a conserved serine residue, a reaction absolutely required for the biosynthesis of natural products including fatty acids, polyketides, and nonribosomal peptides. PPTases have been classified according to their carrier protein specificity. In organisms containing multiple P-pant-requiring pathways, each pathway has been suggested to have its own PPTase activity. However, sequence analysis of the bleomycin biosynthetic gene cluster in Streptomyces verticillus ATCC15003 failed to reveal an associated PPTase gene. RESULTS: A general approach for cloning PPTase genes by PCR was developed and applied to the cloning of the svp gene from S. verticillus. The svp gene is mapped to an independent locus not clustered with any of the known NRPS or PKS clusters. The Svp protein was overproduced in Escherichia coli, purified to homogeneity, and shown to be a monomer in solution. Svp is a PPTase capable of modifying both type I and type II acyl carrier proteins (ACPs) and peptidyl carrier proteins (PCPs) from either S. verticillus or other Streptomyces species. As compared to Sfp, the only 'promiscuous' PPTase known previously, Svp displays a similar catalytic efficiency (k(cat)/K(m)) for the BlmI PCP but a 346-fold increase in catalytic efficiency for the TcmM ACP. CONCLUSIONS: PPTases have recently been re-classified on a structural basis into two subfamilies: ACPS-type and Sfp-type. The development of a PCR method for cloning Sfp-type PPTases from actinomycetes, the recognition of the Sfp-type PPTases to be associated with secondary metabolism with a relaxed carrier protein specificity, and the availability of Svp, in addition to Sfp, should facilitate future endeavors in engineered biosynthesis of peptide, polyketide, and, in particular, hybrid peptide-polyketide natural products.     

Protein Organic Chemistry and Applications for Labeling and Engineering in Live‐Cell Systems

The modification of proteins with synthetic probes is a powerful means of elucidating and engineering the functions of proteins both in vitro and in live cells or in vivo. Herein we review recent progress in chemistry‐based protein modification methods and their application in protein engineering, with particular emphasis on the following four strategies: 1) the bioconjugation reactions of amino acids on the surfaces of natural proteins, mainly applied in test‐tube settings; 2) the bioorthogonal reactions of proteins with non‐natural functional groups; 3) the coupling of recognition and reactive sites using an enzyme or short peptide tag–probe pair for labeling natural amino acids; and 4) ligand‐directed labeling chemistries for the selective labeling of endogenous proteins in living systems. Overall, these techniques represent a useful set of tools for application in chemical biology, with the methods 2–4 in particular being applicable to crude (living) habitats. Although still in its infancy, the use of organic chemistry for the manipulation of endogenous proteins, with subsequent applications in living systems, represents a worthy challenge for many chemists.     

标签谷胱甘肽S-转移酶的二价亲和标记试剂的制备与应用

设计合成融合表达标签谷胱甘肽S-转移酶(GST)的二价亲和标记试剂,用于功能化磁珠后位点选择性固定化标签GST,为磁分离筛选配体混合物库提供固定化融合靶蛋白的候选方案。 为减少疏水配体在标签GST活性位点的结合,需同时占据标签GST双活性中心内疏水结合位点并发生共价修饰的二价亲和标记试剂。以双苯环为疏水定位基、溴乙酰基为巯基修饰基团、羧基为连接官能团得单价标记试剂,以二乙基三胺为连接臂将单价标记试剂与连接臂两端伯胺连接得标签GST的对称二价亲和标记试剂,再以线性三胺连接臂中间的氨基与羧基磁珠偶联得功能化磁珠。 表征目标化合物对标签GST的标记动力学、结合比;功能化磁珠对标签GST的不可逆固定化动力学和固载容量,及将磁珠表面二价亲和标记试剂转变成还原型谷胱甘肽(GSH)加合物后对标签GST可逆固定化的效果;以碱性磷酸酶及疏水荧光配体为模型考察磁珠固定化标签GST后的非特异结合。 目标化合物对标签GST半抑制浓度为(22±0.2) μmol/L,其与GSH的饱和加合物半抑制浓度为(0.41±0.06) μmol/L,二者与标签GST二聚体结合比接近1：1。 功能化磁珠对标签GST不可逆及可逆固定化的容量均接近25 mg/g磁珠。 偶联GST的磁珠对蛋白非特异吸附很弱,再进一步用单价亲和标记试剂和GSH加合物封闭固定化标签GST剩余的活性位点后对疏水小分子也无显著结合。 结果表明,所设计二价亲和标记试剂功能化磁珠适合用于标签GST及其融合表达蛋白的位点选择性固定化。     

Study of molecular interactions between lipids and proteins using dynamic surface tension measurements: a review

Molecular interactions between small molecules and proteins, such as binding of lipids to proteins, are of fundamental importance in various biological processes. A recently-developed method based on dynamic surface tension measurement is efficient and versatile in detecting such molecular interactions: Axisymmetric Drop Shape Analysis (ADSA) provides a tool for measuring the surface tension (γ) response to surface area changes. Through the analysis of the γ response pattern, surface competitive adsorption between small organic molecules and protein molecules can be detected. Surface squeeze-out of small molecules by proteins can also be observed. Molecular binding of lipids to proteins manifests itself in a modification of the γ response which is not compatible with a simple superposition of the two individual patterns. The specific binding can be studied in terms of dose effects and specificity.     

Sensitive detection of proteins in polyacrylamide gel via isatoic anhydride derivatization: Introduction of a low‐cost fluorescent prelabeling procedure

Here, we introduce isatoic anhydride as a sensitive and commodious fluorescent prelabel for detection of proteins in one‐dimensional polyacrylamide gels. High reactivity of isatoic anhydride with nucleophiles in mild alkaline environments makes it an appropriate tag for labeling of biomolecules. In this study, we show that preelectrophoresis labeling of proteins with isatoic anhydride for few minutes at room temperature allows detection of 2–4 ng of standard proteins, BSA and lysozyme, per band. Proteins were successfully labeled in the presence of a wide range of common biological reagents and in crude cell extract. The labeled proteins have the same electrophoretic migration in comparison to unlabeled proteins; however the application of saturation labeling method results in slight band broadening. Compatibility of the method with downstream processes was assessed by tryptic digestion of labeled proteins and study of peptide mixture using gel electrophoresis which revealed partial digestion of labeled proteins due to lysine modification. The present procedure is sensitive, rapid, and inexpensive and is a promising alternative for current protein staining procedures, where downstream processes are not desired.     

Synthesis and characterization of a 'fluorous' (fluorinated alkyl) affinity reagent that labels primary amine groups in proteins/peptides

Strong non-covalent interactions such as biotin-avidin affinity play critical roles in protein/peptide purification. A new type of 'fluorous' (fluorinated alkyl) affinity approach has gained popularity due especially to its low level of non-specific binding to proteins/peptides. We have developed a novel water-soluble fluorous labeling reagent that is reactive (via an active sulfo-N-hydroxylsuccinimidyl ester group) to primary amine groups in proteins/peptides. After fluorous affinity purification, the bulky fluorous tag moiety and the long oligoethylene glycol (OEG) spacer of this labeling reagent can be trimmed via the cleavage of an acid labile linker. Upon collision-induced dissociation, the labeled peptide ion yields a characteristic fragment that can be retrieved from the residual portion of the fluorous affinity tag, and this fragment ion can serve as a marker to indicate that the relevant peptide has been successfully labeled. As a proof of principle, the newly synthesized fluorous labeling reagent was evaluated for peptide/protein labeling ability in phosphate-buffered saline (PBS). Results show that both the aqueous environment protein/peptide labeling and the affinity enrichment/separation process were highly efficient.     

Genetic Encoding of bicyclononynes and trans-cyclooctenes for site-specific protein labeling in vitro and in live mammalian cells via rapid fluorogenic Diels-Alder reactions

Rapid, site-specific labeling of proteins with diverse probes remains an outstanding challenge for chemical biologists. Enzyme-mediated labeling approaches may be rapid but use protein or peptide fusions that introduce perturbations into the protein under study and may limit the sites that can be labeled, while many "bioorthogonal" reactions for which a component can be genetically encoded are too slow to effect quantitative site-specific labeling of proteins on a time scale that is useful for studying many biological processes. We report a fluorogenic reaction between bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) and tetrazines that is 3-7 orders of magnitude faster than many bioorthogonal reactions. Unlike the reactions of strained alkenes, including trans-cyclooctenes and norbornenes, with tetrazines, the BCN-tetrazine reaction gives a single product of defined stereochemistry. We have discovered aminoacyl-tRNA synthetase/tRNA pairs for the efficient site-specific incorporation of a BCN-containing amino acid, 1, and a trans-cyclooctene-containing amino acid 2 (which also reacts extremely rapidly with tetrazines) into proteins expressed in Escherichia coli and mammalian cells. We demonstrate the rapid fluorogenic labeling of proteins containing 1 and 2 in vitro, in E. coli , and in live mammalian cells. These approaches may be extended to site-specific protein labeling in animals, and we anticipate that they will have a broad impact on labeling and imaging studies.     

Specific labeling of cell surface proteins with chemically diverse compounds

The specific and covalent labeling of fusion proteins with synthetic molecules opens up new ways to study protein function in the living cell. Here we present a novel method that allows for the specific and exclusive extracellular labeling of proteins on the surfaces of live cells with a large variety of synthetic molecules including fluorophores, protein ligands, or quantum dots. The approach is based on the specific labeling of fusion proteins of acyl carrier protein with synthetic molecules through post-translational modification catalyzed by phosphopantetheine transferase. The specificity and versatility of the labeling should allow it to become an important tool for studying and manipulating cell surface proteins and for complementing existing approaches in cell surface engineering.     

Versatile and Efficient Site‐Specific Protein Functionalization by Tubulin Tyrosine Ligase

A novel chemoenzymatic approach for simple and fast site‐specific protein labeling is reported. Recombinant tubulin tyrosine ligase (TTL) was repurposed to attach various unnatural tyrosine derivatives as small bioorthogonal handles to proteins containing a short tubulin‐derived recognition sequence (Tub‐tag). This novel strategy enables a broad range of high‐yielding and fast chemoselective C‐terminal protein modifications on isolated proteins or in cell lysates for applications in biochemistry, cell biology, and beyond, as demonstrated by the site‐specific labeling of nanobodies, GFP, and ubiquitin.     

Targetable fluorescent sensors for advanced cell function analysis

Chemistry-based bioimaging techniques have contributed to the elucidation of intracellular physiological events. During the last few decades, many fluorescent sensors have been developed and used in live cell experiments. Owing to immense efforts by numerous research groups, several strategies have been developed to design fluorescent sensors based on various components such as small molecules and fluorescent proteins. Recently, site-specific targeting of fluorescent sensors has attracted increasing attention. Strategies for fluorescent sensor targeting were surveyed in this review with the aims to expand current knowledge on chemistry-based bioimaging and aid in the emergence of related innovative technologies. The first discussed strategy is based on the intrinsic properties of small molecules for localization at specific organelles, such as mitochondria, nuclei, and lysosomes. As a further elaboration of the topic, our recent study about in vivo targeting of pH sensors was briefly introduced. The second strategy exploits genetically encoded tags and their specific ligands. Here, fluorescent sensors with commercially available tags and corresponding ligands were mainly reviewed. As the final topic, our original protein labeling technique, which enables fluorogenic labeling as an advanced technology, was introduced.