Backbone resonance assignments of the C-terminal thioesterase domain of tyrocidine synthetase C.

Natural macrocyclic peptides produced by microorganisms serve as valuable resources for therapeutic compounds, including antibiotics, anticancer agents, and immune suppressive agents. Nonribosomal peptide synthetases (NRPSs) are responsible for the biosynthesis of macrocyclic peptides. NRPSs are large multimodular enzymes, and each module recognizes and incorporates one specific amino acid into the polypeptide product.

Direct evidence for a deprotonated lysine serving as a H-bond "acceptor" in a photoreceptor protein.

Deprotonation or suppression of the pa of the amino group of a lysine sidechain is a widely recognized phenomenon whereby the sidechain amino group transiently can act as a nucleophile at the active site of enzymatic reactions. However, a deprotonated lysine and its molecular interactions have not been directly experimentally detected. Here, we demonstrate a deprotonated lysine stably serving as an "acceptor" in a H-bond between the photosensor protein RcaE and its chromophore.

Insight into the C-terminal SH3 domain mediated binding of Drosophila Drk to Sos and Dos.

Drk, a Drosophila homologue of human GRB2, interacts with Sevenless (Sev) receptor via its SH2 domain, while the N- and C-terminal SH3 domains (Drk-NSH3 and Drk-CSH3, respectively) are responsible for the interaction with proline-rich motifs (PRMs) of Son of sevenless (Sos) or Daughter of Sevenless (Dos). Drk-NSH3 on its own has a conformational equilibrium between folded and unfolded states, and the folded state is stabilised by the association with a Sos-derived proline-rich peptide with PxxPxR motif. In contrast, Drk-CSH3 is supposed to bind PxxxRxxKP motifs in Dos.

Novel Oleanane-Type Triterpene Glycosides from the L. Seeds and Apoptosis-Inducing Activity via Mitochondria.

L., commonly known as "Soapwort", is a rich source of triterpene glycosides; however, the chemical constituents of seeds have not been fully identified. In this study, we conducted a systematic phytochemical investigation of the seeds of and obtained 17 oleanane-type triterpene glycosides (-), including seven new glycosides (-).

Raman Spectroscopy of an Atypical C15-, Bilin Chromophore in Cyanobacteriochrome RcaE.

Cyanobacteriochromes (CBCRs) belong to the phytochrome superfamily of photoreceptors, the members of which utilize a linear tetrapyrrole (bilin) as a chromophore. RcaE is a representative member of a green/red-type CBCR subfamily that photoconverts between a green-absorbing dark state and red-absorbing photoproduct (Pr). Our recent crystallographic study showed that the phycocyanobilin (PCB) chromophore of RcaE adopts a unique C15-, configuration in the Pr state, unlike the typical C15-, configuration for the phytochromes and other CBCRs.

Syntheses and structure of dinuclear metal complexes containing naphthyl-Ir bichromophore.

A series of novel metal complexes were synthesized containing an Ir-cyclometalated bichromophore as a visible-light sensitizer. A new bichromophoric unit containing a naphthyl substituent and methyl substituents on the 2-phenylpyridine chelating ligand was synthesized and characterized for the first time. According to the increased crystallinity of the bichromophoric unit, novel Ir-M metal complexes (M = Pd, Mn, and Ir) were synthesized and fully characterized.

Structural basis of the protochromic green/red photocycle of the chromatic acclimation sensor RcaE.

Cyanobacteriochromes (CBCRs) are bilin-binding photosensors of the phytochrome superfamily that show remarkable spectral diversity. The green/red CBCR subfamily is important for regulating chromatic acclimation of photosynthetic antenna in cyanobacteria and is applied for optogenetic control of gene expression in synthetic biology. It is suggested that the absorption change of this subfamily is caused by the bilin C15/C15- photoisomerization and a subsequent change in the bilin protonation state.

Mechanism of self/nonself-discrimination in Brassica self-incompatibility.

Self-incompatibility (SI) is a breeding system that promotes cross-fertilization. In Brassica, pollen rejection is induced by a haplotype-specific interaction between pistil determinant SRK (S receptor kinase) and pollen determinant SP11 (S-locus Protein 11, also named SCR) from the S-locus. Although the structure of the B.

Domain selective labeling for NMR studies of multidomain proteins by domain ligation using highly active sortase A.

Structural study of multidomain proteins using NMR is an emerging issue for understanding biological functions. To this end, domain-specific labeling is expected to be a key technology for facilitating the NMR-assignment process and for collecting distance information via spin labeling. To obtain domain-specific labeled samples, use of sortase A as a protein ligation tool is a viable approach.

High-Resolution Protein 3D Structure Determination in Living Eukaryotic Cells.

Proteins in living cells interact specifically or nonspecifically with an enormous number of biomolecules. To understand the behavior of proteins under intracellular crowding conditions, it is indispensable to observe their three-dimensional (3D) structures at the atomic level in a physiologically natural environment. We demonstrate the first de novo protein structure determinations in eukaryotes with the sf9 cell/baculovirus system using NMR data from living cells exclusively.

Structural insights into ubiquitin phosphorylation by PINK1.

Mutations of PTEN-induced putative kinase 1 (PINK1) and the E3 ubiquitin (Ub) ligase parkin can cause familial parkinsonism. These two proteins are essential for ubiquitylation of damaged mitochondria and subsequent degradation. PINK1 phosphorylates Ser65 of Ub and the Ub-like (UBL) domain of parkin to allosterically relieve the autoinhibition of parkin.

Parkinson's disease-related DJ-1 functions in thiol quality control against aldehyde attack in vitro.

DJ-1 (also known as PARK7) has been identified as a causal gene for hereditary recessive Parkinson's disease (PD). Consequently, the full elucidation of DJ-1 function will help decipher the molecular mechanisms underlying PD pathogenesis. However, because various, and sometimes inconsistent, roles for DJ-1 have been reported, the molecular function of DJ-1 remains controversial.

Chemical shift assignments of the first and second RRMs of Nrd1, a fission yeast MAPK-target RNA binding protein.

Negative regulator differentiation 1 (Nrd1), a fission yeast RNA binding protein, modulates cytokinesis and sexual development and contributes to stress granule formation in response to environmental stresses. Nrd1 comprises four RRM domains and binds and stabilizes Cdc4 mRNA that encodes the myosin II light chain. Nrd1 binds the Cpc2 fission-yeast RACK1 homolog, and the interaction promotes Nrd1 localization to stress granules.

Improved in-cell structure determination of proteins at near-physiological concentration.

Investigating three-dimensional (3D) structures of proteins in living cells by in-cell nuclear magnetic resonance (NMR) spectroscopy opens an avenue towards understanding the structural basis of their functions and physical properties under physiological conditions inside cells. In-cell NMR provides data at atomic resolution non-invasively, and has been used to detect protein-protein interactions, thermodynamics of protein stability, the behavior of intrinsically disordered proteins, etc. in cells.

A new carbamidemethyl-linked lanthanoid chelating tag for PCS NMR spectroscopy of proteins in living HeLa cells.

Structural analyses of proteins under macromolecular crowding inside human cultured cells by in-cell NMR spectroscopy are crucial not only for explicit understanding of their cellular functions but also for applications in medical and pharmaceutical sciences. In-cell NMR experiments using human cultured cells however suffer from low sensitivity, thus pseudocontact shifts from protein-tagged paramagnetic lanthanoid ions, analysed using sensitive heteronuclear two-dimensional correlation NMR spectra, offer huge potential advantage in obtaining structural information over conventional NOE-based approaches. We synthesised a new lanthanoid-chelating tag (M8-CAM-I), in which the eight-fold, stereospecifically methylated DOTA (M8) scaffold was retained, while a stable carbamidemethyl (CAM) group was introduced as the functional group connecting to proteins.

Evaluation of the reliability of the maximum entropy method for reconstructing 3D and 4D NOESY-type NMR spectra of proteins.

Despite their advantages in analysis, 4D NMR experiments are still infrequently used as a routine tool in protein NMR projects due to the long duration of the measurement and limited digital resolution. Recently, new acquisition techniques for speeding up multidimensional NMR experiments, such as nonlinear sampling, in combination with non-Fourier transform data processing methods have been proposed to be beneficial for 4D NMR experiments. Maximum entropy (MaxEnt) methods have been utilised for reconstructing nonlinearly sampled multi-dimensional NMR data.

Efficient and cost effective production of active-form human PKB using silkworm larvae.

Protein kinase B (PKB) also known as Akt is involved in many signal transduction pathways. As alterations of the PKB pathway are found in a number of human malignancies, PKB is considered an important drug target for cancer therapy. However, production of sufficient amounts of active PKB for biochemical and structural studies is very costly because of the necessity of using a higher organism expression system to obtain phosphorylated PKB.

Structural insights into the recruitment of SMRT by the corepressor SHARP under phosphorylative regulation.

The transcriptional corepressors SMRT/NCoR, components of histone deacetylase complexes, interact with nuclear receptors and many other transcription factors. SMRT is a target for the ubiquitously expressed protein kinase CK2, which is known to phosphorylate a wide variety of substrates. Increasing evidence suggests that CK2 plays a regulatory role in many cellular events, particularly, in transcription.

An in-cell NMR study of monitoring stress-induced increase of cytosolic Ca2+ concentration in HeLa cells.

Recent developments in in-cell NMR techniques have allowed us to study proteins in detail inside living eukaryotic cells. The lifetime of in-cell NMR samples is however much shorter than that in culture media, presumably because of various stresses as well as the nutrient depletion in the anaerobic environment within the NMR tube. It is well known that Ca(2+)-bursts occur in HeLa cells under various stresses, hence the cytosolic Ca(2+) concentration can be regarded as a good indicator of the healthiness of cells in NMR tubes.

Structure of the second RRM domain of Nrd1, a fission yeast MAPK target RNA binding protein, and implication for its RNA recognition and regulation.

Negative regulator of differentiation 1 (Nrd1) is known as a negative regulator of sexual differentiation in fission yeast. Recently, it has been revealed that Nrd1 also regulates cytokinesis, in which physical separation of the cell is achieved by a contractile ring comprising many proteins including actin and myosin. Cdc4, a myosin II light chain, is known to be required for cytokinesis.

Divergent DNA-binding specificities of a group of ETHYLENE RESPONSE FACTOR transcription factors involved in plant defense.

Transcription factors (TFs) recognize target DNA sequences with distinct DNA-binding domains (DBDs). The DBD of Arabidopsis (Arabidopsis thaliana) ETHYLENE RESPONSE FACTOR1 (AtERF1) uses three consecutive β-strands to recognize a GCC-containing sequence, but tobacco (Nicotiana tabacum) ERF189 and periwinkle (Catharanthus roseus) Octadecanoid-derivative Responsive Catharanthus AP2-domain protein3 (ORCA3) of the same TF subgroup appear to target similar but divergent DNA sequences. Here, we examined how DNA-binding specificities of these TFs have diverged in each plant lineage to regulate distinct defense metabolisms.

High-resolution heteronuclear multidimensional NMR of proteins in living insect cells using a baculovirus protein expression system.

Recent developments in in-cell NMR techniques have allowed us to study proteins in detail inside living eukaryotic cells. In order to complement the existing protocols, and to extend the range of possible applications, we introduce a novel approach for observing in-cell NMR spectra using the sf9 cell/baculovirus system. High-resolution 2D (1)H-(15)N correlation spectra were observed for four model proteins expressed in sf9 cells.