Selective Antifolates for Chemically Labeling Proteins in Mammalian Cells

Antifolate labels : Molecules that bind specifically and with high affinity to proteins can be developed into powerful tools for chemical biology. The interaction between substituted 5‐benzyl pyrimidines and dihydrofolate reductase can be exploited for chemically labeling fusion proteins in mammalian cells.

     

Site-Specific Isotopic Labeling (SSIL): Access to High-Resolution Structural and Dynamic Information in Low-Complexity Proteins

Remarkable technical progress in the area of structural biology has paved the way to study previously inaccessible targets. For example, large protein complexes can now be easily investigated by cryo-electron microscopy, and modern high-field NMR magnets have challenged the limits of high-resolution characterization of proteins in solution. However, the structural and dynamic characteristics of certain proteins with important functions still cannot be probed by conventional methods. These proteins in question contain low-complexity regions (LCRs), compositionally biased sequences where only a limited number of amino acids is repeated multiple times, which hamper their characterization. This Concept article describes a site-specific isotopic labeling (SSIL) strategy, which combines nonsense suppression and cell-free protein synthesis to overcome these limitations. An overview on how poly-glutamine tracts were made amenable to high-resolution structural studies is used to illustrate the usefulness of SSIL. Furthermore, we discuss the potential of this methodology to give further insights into the roles of LCRs in human pathologies and liquid–liquid phase separation, as well as the challenges that must be addressed in the future for the popularization of SSIL.     

Strategies for Site-Specific Labeling of Receptor Proteins on the Surfaces of Living Cells by Using Genetically Encoded Peptide Tags

Fluorescence microscopy imaging enables receptor proteins to be investigated within their biological context. A key challenge is to site-specifically incorporate reporter moieties into proteins without interfering with biological functions or cellular networks. Small peptide tags offer the opportunity to combine inducible labeling with small tag sizes that avoid receptor perturbation. Herein, we review the current state of live-cell labeling of peptide-tagged cell-surface proteins. Considering their importance as targets in medicinal chemistry, we focus on membrane receptors such as G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs). We discuss peptide tags that i) are subject to enzyme-mediated modification reactions, ii) guide the complementation of reporter proteins, iii) form coiled-coil complexes, and iv) interact with metal complexes. Given our own contributions in the field, we place emphasis on peptide-templated labeling chemistry.     

Amino Acid‐Selective Segmental Isotope Labeling of Multidomain Proteins for Structural Biology

Current solution NMR techniques enable structural investigations of proteins in molecular particles with sizes up to several hundred kDa. However, the large molecular weight of proteins in such systems results in increased numbers of NMR signals, and the resulting spectral overlap typically imposes limitations. For multidomain proteins, segmental isotope labeling of individual domains facilitates the spectral interpretation by reducing the number of signals, but for large domains with small signal dispersion, signal overlap can persist. To overcome limitations arising from spectral overlap, we present a strategy that combines cell‐free expression and ligation of the expressed proteins to produce multidomain proteins with selective amino acid‐type labeling in individual domains. The bottleneck of intrinsically low cell‐free expression yields of precursor molecules was overcome by introducing new fusion constructs that allowed milligram production of ligation‐competent domains labeled in one or multiple amino acid types. Ligation‐competent unlabeled partner domains were produced in vivo, and subsequent domain ligation was achieved by using an on‐column strategy. This approach is illustrated with two multidomain RNA‐binding proteins, that is, the two C‐terminal RNA‐recognition motifs of the human polypyrimidine tract‐binding protein, and two highly homologous helix–turn–helix domains of the human glutamyl‐prolyl‐tRNA synthetase.     

酪蛋白标签胶研制

介绍了以干酪索为主要成膜物,尿素为分散剂,硫酸锌为螯合剂,淀粉为助剂制备酪蛋白标签胶的制备工艺,讨论了分散剂、螯合剂、淀粉、水溶解性等因索对酪蛋白标签胶的制备与性能的影响,并总结了制备稳定性能最好的酪蛋白标签胶的最佳工艺条件为：每100g标签胶里,分散剂用量为12％,淀粉含量在5％～7％问最合适;螯合剂的用量,以0.4％为最佳,这样实现了标签胶间各项性能的一个均衡,酪紊胶的性能最稳定。     

Reactivity Analysis of New Multivalent Electrophilic Probes for Affinity Labeling of Carbohydrate Binding Proteins

We have designed and synthesized six different multivalent electrophiles as carbohydrate affinity labeling probes. Evaluation of the reactivity of the electrophiles against peanut agglutinin (PNA) and Ricinus communis agglutinin (RCA) showed that p- and m-aryl sulfonyl fluoride are effective protein reactive groups that label carbohydrate binding lectins in a ligand-dependent fashion at a nanomolar probe concentration. Analysis of the selectivity of affinity labeling in the presence of excess BSA as a nonspecific protein indicated that m-arylsulfonyl fluoride is a more selective protein-reactive group, albeit with attenuated reactivity. Further analysis showed that the labeling efficiency of the multivalent electrophilic probes can be improved by employing reaction conditions involving 25 °C instead of typically employed 4 °C. Both isomers of arylsulfonyl fluoride groups together represent promising affinity labels for target identification studies that could serve as more efficient alternatives to photoreactive groups.     

Site-specific protein modification on living cells catalyzed by Sortase

The use of enzymes is a promising approach for site-specific protein modification on living cells owing to their substrate specificity. Herein we describe a general strategy for the site-specific modification of cell surface proteins with synthetic molecules by using Sortase, a transpeptidase from Staphylococcus aureus. The short peptide tag LPETGG is genetically introduced to the C terminus of the target protein, expressed on the cell surface. Subsequent addition of Sortase and an N-terminal triglycine-containing probe results in the site-specific labeling of the tagged protein. We were successful in the C-terminal-specific labeling of osteoclast differentiation factor (ODF) with a biotin- or fluorophore-containing short peptide on the living cell surface. The labeling reaction occurred efficiently in serum-containing medium, as well as serum-free medium or PBS. The labeled products were detected after incubation for 5 min. In addition, site-specific protein-protein conjugation was successfully demonstrated on a living cell surface by the Sortase-catalyzed reaction. This strategy provides a powerful tool for cell biology and cell surface engineering.     

Labeling of Phosphatidylinositol Lipid Products in Cells through Metabolic Engineering by Using a Clickable myo-Inositol Probe

Phosphatidylinositol (PI) lipids control critical biological processes, so aberrant biosynthesis often leads to disease. As a result, the capability to track the production and localization of these compounds in cells is vital for elucidating their complex roles. Herein, we report the design, synthesis, and application of clickable myo-inositol probe 1 a for bioorthogonal labeling of PI products. To validate this platform, we initially conducted PI synthase assays to show that 1 a inhibits PI production in vitro. Fluorescence microscopy experiments next showed probe-dependent imaging in T-24 human bladder cancer and Candida albicans cells. Growth studies in the latter showed that replacement of myo-inositol with probe 1 a led to an enhancement in cell growth. Finally, fluorescence-based TLC analysis and mass spectrometry experiments support the labeling of PI lipids. This approach provides a promising means for tracking the complex biosynthesis and trafficking of these lipids in cells.     

水基干酪素标签胶贮存稳定性的研究

以干酪素、淀粉为主要成膜物，以尿素作为分散剂合成了一种用于粘贴标签的水基干酪素胶。探讨了胶粘剂的组成对粘度及其稳定性的影响。     

Design of a Multiuse Photoreactor To Enable Visible-Light Photocatalytic Chemical Transformations and Labeling in Live Cells

Despite the growing use of visible-light photochemistry in both chemistry and biology, no general low-heat photoreactor for use across these different disciplines exists. Herein, we describe the design and use of a standardized photoreactor for visible-light-driven activation and photocatalytic chemical transformations. Using this single benchtop photoreactor, we performed photoredox reactions across multiple visible light wavelengths, a high-throughput photocatalytic cross-coupling reaction, and in vitro labeling of proteins and live cells. Given the success of this reactor in all tested applications, we envision that this multi-use photoreactor will be widely used in biology, chemical biology, and medicinal chemistry settings.     

Versatile Interacting Peptide (VIP) Tags for Labeling Proteins with Bright Chemical Reporters

Fluorescence microscopy is an essential tool for the biosciences, enabling the direct observation of proteins in their cellular environment. New methods that facilitate attachment of photostable synthetic fluorophores with genetic specificity are needed to advance the frontiers of biological imaging. Here, we describe a new set of small, selective, genetically encoded tags for proteins based on a heterodimeric coiled‐coil interaction between two peptides: CoilY and CoilZ. Proteins expressed as a fusion to CoilZ were selectively labeled with the complementary CoilY fluorescent probe peptide. Fluorophore‐labeled target proteins were readily detected in cell lysates with high specificity and sensitivity. We found that these versatile interacting peptide (VIP) tags allowed rapid and specific delivery of bright organic dyes or quantum dots to proteins displayed on living cells. Additionally, we validated that either CoilY or CoilZ could serve as the VIP tag, which enabled us to observe two distinct cell‐surface protein targets with this one heterodimeric pair.     

生物标记用发光材料研究现状

综述了生物标记用发光材料研究进展及其应用现状,对各种材料的特点进行了分析,指出以绿色荧光蛋白等为代表的荧光材料将是未来生物标记的主流.     

Accurate Determination of Human CPR Conformational Equilibrium by smFRET Using Dual Orthogonal Noncanonical Amino Acid Labeling

Conjugation of fluorescent dyes to proteins—a prerequisite for the study of conformational dynamics by single-molecule (sm) FRET—can lead to substantial changes in a dye's photophysical properties, ultimately biasing the determination of inter-dye distances. In particular, cyanine dyes and their derivatives, the most commonly used dyes in smFRET experiments, exhibit such behavior. To overcome this, we developed a general strategy to equip proteins site-specifically with FRET pairs through chemoselective reactions with two distinct noncanonical amino acids simultaneously incorporated through genetic code expansion in Escherichia coli. Application of this technique to human NADPH-cytochrome P450 reductase (CPR) demonstrated the importance of homogenously labeled samples for accurate determination of FRET efficiencies and unveiled the effect of NADP⁺ on the ionic-strength-dependent modulation of the conformational equilibrium of CPR. Thanks to its generality and accuracy, the presented methodology establishes a new benchmark for deciphering of complex molecular dynamics in single molecules.     

Bivalent SMAC Mimetics for Treating Cancer by Antagonizing Inhibitor of Apoptosis Proteins

Inhibitors of apoptosis proteins (IAPs) inhibit caspase activity, allowing various cancers to reduce programmed cell death (apoptosis) and resist drug treatment. The second mitochondrial-derived activator of caspases (SMAC) protein is an endogenous IAP antagonist, which can be considered as a potential anticancer therapy. Small-molecule SMAC mimetics based on the Ala-Val-Pro-Ile motif have been validated as potent IAP antagonists. In particular, most bivalent SMAC mimetics, which target both the baculovirus IAP repeat 2 (BIR2) and BIR3 domains in X-linked IAP (XIAP), antagonize IAPs better than the corresponding monovalent mimetics. Here we focus on strategies for designing bivalent small-molecule SMAC mimetics and progress in using them to antagonize IAPs. We also consider their clinical potential. Our discussion will hopefully help guide further study of these interesting mimetics.     

[18F]SiFA‐isothiocyanate: A New Highly Effective Radioactive Labeling Agent for Lysine‐Containing Proteins

A highly efficient ¹⁸F‐labeling synthon for universal protein labeling is reported. Diverse ¹⁸F‐labeled proteins of 66–144 kDa were prepared with [¹⁸F]SiFA‐isothiocyanate synthesized by an isotopic ¹⁹F for ¹⁸F exchange at the silicon atom. Overall preparative radiochemical yields were 20–40 % after 40–50 min. No bone uptake of ¹⁸F radioactivity was detected until 90 min post‐injection of ¹⁸F‐SiFA‐RSA; this demonstrates the metabolic stability of the [¹⁸F]SiFA moiety.