Specific arrestin conformations are coupled to distinct downstream effectors, which underlie the functions of many G-protein-coupled receptors (GPCRs). Here, using unnatural amino acid incorporation and fluorine-19 nuclear magnetic resonance (19F-NMR) spectroscopy, we demonstrate that distinct receptor phospho-barcodes are translated to specific β-arrestin-1 conformations and direct selective signalling. With its phosphate-binding concave surface, β-arrestin-1 ‘reads' the message in the receptor phospho-C-tails and distinct phospho-interaction patterns are revealed by 19F-NMR. Whereas all functional phosphopeptides interact with a common phosphate binding site and induce the movements of finger and middle loops, different phospho-interaction patterns induce distinct structural states of β-arrestin-1 that are coupled to distinct arrestin functions. Only clathrin recognizes and stabilizes GRK2-specific β-arrestin-1 conformations. The identified receptor-phospho-selective mechanism for arrestin conformation and the spacing of the multiple phosphate-binding sites in the arrestin enable arrestin to recognize plethora phosphorylation states of numerous GPCRs, contributing to the functional diversity of receptors.

Bioorthogonal chemical reactions together with techniques to expand the genetic code have provided exciting new means for protein labeling and visualization in living systems, [1] as well as for optimizing the efficacy of therapeutic proteins. [2] Toward these goals, amino acids with small bioorthogonal functional groups, such as azide, alkyne, or cyclopropene moieties, [3] as well as larger reactive bioorthogonal groups, [4] such as cyclooctyne, norbornene, trans-cyclooctene, aryltetrazole, or aryltetrazine, have been site-specifically incorporated into proteins, allowing for selective conjugation of biophysical probes through azide-alkyne click chemistry (AAC), tetrazole-alkene photoclick chemistry (TAP), and reverse-electron demand Diels-Alder reactions. [5] The main advantages of the photoclick reaction (Supporting Information, Scheme S1) are: 1) its fast rate (up to 50 m À1 s À1 ); 2) that spatiotemporal control is initiated by a photo-induced reaction; 3) that the photoclick reaction is fluorogenic, allowing for high-contrast fluorescence imaging without tedious washing steps. In previous studies, we reported the site-specific incorporation of p-(2-tetrazole)phenylalanine (p-Tpa) [4a] and N-e-(1-methylcycloprop-2-enecarboxamido)lysine (CpK) [3c] in E. coli and mammalian cells. Subsequent photoirradiation of labeled proteins with UV light facilitates selective conjugation with dimethyl fumarate or diaryltetrazole, respectively.By expanding the genetic code and introducing photoclick chemistry to plants, important problems in plant chemical biology can be addressed, [6] such as photosynthesis and stress response, which can only be studied at the organismal level. Recently, expansion of the genetic code has been used to optimize therapeutic proteins produced in bacteria and mammalian cells. [2] Because plants offer an attractive alternative to microbial fermentation and animal cell cultures for high-yield production of recombinant proteins on an agricultural scale, [8] expanding the genetic code in plants would be useful for producing recombinant therapeutic proteins and enzymes with enhanced properties, better safety, and lower costs. [8] To fully realize the potential of photoclick reaction for tracking fast cellular processes, it is desirable that the unnatural amino acid (UAA) used has a small functional group such that there is minimal perturbation of the target protein, and a very brief exposure to long-wavelength UV light or violet-blue light is used to drive the photoclick reaction to minimize damage to cells and plants. Herein, we addressed these issues by genetically incorporating N-eacryllysine (AcrK, Figure 1 A), in response to an amber stop codon (TAG) in bacterial cells, mammalian cells, and plants. This new strategy was then used to efficiently label proteins both in vitro and in vivo. In comparison to lysine, AcrK has only four extra non-hydrogen atoms, which is significantly less than other UAAs. [3, 4] Replacing one lysine residue with AcrK should cause only minimal perturbation to ...

Advances in RNA research and RNA nanotechnology depend on the ability to manipulate and probe RNA with high precision through chemical approaches, both in vitro and in mammalian cells. However, covalent RNA labeling methods with scope and versatility comparable to those of current protein labeling strategies are underdeveloped. A method is reported for the site- and sequence-specific covalent labeling of RNAs in mammalian cells by using tRNA(Ile2) -agmatidine synthetase (Tias) and click chemistry. The crystal structure of Tias in complex with an azide-bearing agmatine analogue was solved to unravel the structural basis for Tias/substrate recognition. The unique RNA sequence specificity and plastic Tias/substrate recognition enable the site-specific transfer of azide/alkyne groups to an RNA molecule of interest in vitro and in mammalian cells. Subsequent click chemistry reactions facilitate the versatile labeling, functionalization, and visualization of target RNA.

The fucoidan from Ascophyllum nodosum attenuates atherosclerosis by up-regulating reverse cholesterol transport.

Cyclin E/Cdk2 is a key regulator in G 1 -S transition. We have identified a novel cyclin E/Cdk2 substrate called Ankrd17 (ankyrin repeat protein 17) using the TAP tag purification technique. Ankrd17 protein contains two clusters of a total 25 ankyrin repeats at its N terminus, one NES (nuclear exporting signal) and one NLS (nuclear localization signal) in the middle, and one RXL motif at its C terminus. Ankrd17 is expressed in various tissues and associates with cyclin E/Cdk2 in an RXL-dependent manner. It can be phosphorylated by cyclin E/Cdk2 at 3 phosphorylation sites (Ser 1791 , Ser 1794 , and Ser 2150 ). Overexpression of Ankrd17 promotes S phase entry, whereas depletion of Ankrd17 expression by small interfering RNA inhibits DNA replication and blocks cell cycle progression as well as up-regulates the expression of p53 and p21. Ankrd17 is localized to the nucleus and interacts with DNA replication factors including MCM family members, Cdc6 and PCNA. Depletion of Ankrd17 results in decreased loading of Cdc6 and PCNA onto DNA suggesting that Ankrd17 may be directly involved in the DNA replication process. Taken together, these data indicate that Ankrd17 is an important downstream effector of cyclin E/Cdk2 and positively regulates G 1 /S transition.Progression through the cell cycle is driven by the sequential and periodic activation of cyclin/Cdk complexes. Cyclin D/Cdk4/6 4 complexes are active throughout the G 1 phase, cyclin E/Cdk2 at the G 1 /S boundary, cyclin A/Cdk2 during S phase, and cyclin A/Cdk1 and cyclin B/Cdk1 during the G 2 /M transition. Cyclin E/Cdk2 plays a central role in coordinating both the onset of S phase and centrosome duplication in cell cycle (1-4). It presumably exerts most of its biologic activities by phosphorylating its substrates, most frequently through the RXL motif in the substrate that interacts with the cyclin box (5-8). A number of RXL-containing proteins are themselves cell cycle regulatory proteins. In the case of pRb, cyclin E/Cdk2-mediated Rb phosphorylation inactivates Rb by derepressing E2F transcription factors (9, 10), whereas in the case of p27, phosphorylation of p27 by cyclin E/Cdk2 stimulates its degradation by the SCF-Skp2 ubiquitin ligase (11-15).There are a number of proteins identified as cyclin E/Cdk2 substrates that regulate cell division. NPAT is a transcription factor that controls cell cycle-dependent histone gene transcription. The phosphorylation of NPAT by cyclin E/Cdk2 promotes histone transcription (16 -19). Cells without NPAT fail to enter S phase from quiescence (20). CBP/p300 is another protein phosphorylated by cyclin E/Cdk2 at G 1 /S transition to activate its histone acetyltransferase activity (21) and may function as a cofactor for many transcription factors including E2F (22). Interestingly, cyclin E/Cdk2 phosphorylates E2F-5 and increases its transcriptional activity through CBP/p300 recruitment (23). Cyclin E/Cdk2 also phosphorylates centrosomal proteins that regulate centrosome duplication. Phosphorylation of nucleophosmin (NPM) by cyc...

Cysteine dioxygenase (CDO) plays an essential role in sulfur metabolism by regulating homeostatic levels of cysteine. Human CDO contains a post-translationally generated Cys93-Tyr157 cross-linked cofactor. Here, we investigated this Cys-Tyr cross-linking by incorporating unnatural tyrosines in place of Tyr157 via a genetic method. The catalytically active variants were obtained with a thioether bond between Cys93 and the halogen-substituted Tyr157, and we determined the crystal structures of both wild-type and engineered CDO variants in the purely uncross-linked form and with a mature cofactor. Along with mass spectrometry and F NMR, these data indicated that the enzyme could catalyze oxidative C-F or C-Cl bond cleavage, resulting in a substantial conformational change of both Cys93 and Tyr157 during cofactor assembly. These findings provide insights into the mechanism of Cys-Tyr cofactor biogenesis and may aid the development of bioinspired aromatic carbon-halogen bond activation.

Advances in acetylated protein-protein/DNA interactions depend on the development of a novel NMR (nuclear magnetic resonance) probe to study the conformational changes of acetylated proteins. However, the method for detecting the acetylated protein conformation is underdeveloped. Herein, an acetyllysine mimic has been exploited for detecting the conformational changes of acetylated p53-protein/DNA interactions by genetic code expansion and 19F NMR. This 19F NMR probe shows high structural similarity to acetyllysine and could not be deacetylated by sirtuin deacetylase in vitro/vivo. Moreover, acetylation of p53 K164 is reported to be deacetylated by SIRT2 for the first time.

Due to the lack of genetically encoded probes for fluorine-19 nuclear magnetic resonance spectroscopy (19F NMR), its utility for probing eukaryotic membrane protein dynamics is limited. Here we report an efficient method for the genetic incorporation of an unnatural amino acid (UAA), 3′-trifluoro­menthyl-phenyl­alanine (mtfF), into cannabinoid receptor 1 (CB1) in the Baculovirus Expression System. The probe can be inserted at any environmentally sensitive site, while causing minimal structural perturbation to the target protein. Using 19F NMR and X-ray crystallography methods, we discovered that the allosteric modulator Org27569 and agonists synergistically stabilize a previously unrecognized pre-active state. An allosteric modulation model is proposed to explain Org27569’s distinct behavior. We demonstrate that our site-specific 19F NMR labeling method is a powerful tool in decoding the mechanism of GPCR allosteric modulation. This new method should be broadly applicable for uncovering conformational states for many important eukaryotic membrane proteins.