Mechanism of Ferrous Iron Binding and Oxidation by Ferritin from a Pennate Diatom

A novel ferritin was recently found in Pseudo-nitzschia multiseries (PmFTN), a marine pennate diatom that plays a major role in global primary production and carbon sequestration into the deep ocean. Crystals of recombinant PmFTN were soaked in iron and zinc solutions, and the structures were solved to 1.65–2.2-Å resolution. Three distinct iron binding sites were identified as determined from anomalous dispersion data from aerobically grown ferrous soaked crystals. Sites A and B comprise the conserved ferroxidase active site, and site C forms a pathway leading toward the central cavity where iron storage occurs. In contrast, crystal structures derived from anaerobically grown and ferrous soaked crystals revealed only one ferrous iron in the active site occupying site A. In the presence of dioxygen, zinc is observed bound to all three sites. Iron oxidation experiments using stopped-flow absorbance spectroscopy revealed an extremely rapid phase corresponding to Fe(II) oxidation at the ferroxidase site, which is saturated after adding 48 ferrous iron to apo-PmFTN (two ferrous iron per subunit), and a much slower phase due to iron core formation. These results suggest an ordered stepwise binding of ferrous iron and dioxygen to the ferroxidase site in preparation for catalysis and a partial mobilization of iron from the site following oxidation.     

Efficient isotopic tryptophan labeling of membrane proteins by an indole controlled process conduct

A protocol for the efficient isotopic labeling of large G protein‐coupled receptors with tryptophan in Escherichia coli as expression host was developed that sufficiently suppressed the naturally occurring L‐tryptophan indole lyase, which cleaves tryptophan into indole, pyruvate, and ammonia resulting in scrambling of the isotopic label in the protein. Indole produced by the tryptophanase is naturally used as messenger for cell–cell communication. Detailed analysis of different process conducts led to the optimal expression strategy, which mimicked cell–cell communication by the addition of indole during expression. Discrete concentrations of indole and ¹⁵N₂‐L‐tryptophan at dedicated time points in the fermentation drastically increased the isotopic labeling efficiency. Isotope scrambling was only observed in glutamine, asparagine, and arginine side chains but not in the backbone. This strategy allows producing specifically tryptophan labeled membrane proteins at high concentrations avoiding the disadvantages of the often low yields of auxotrophic E. coli strains. In the fermentation process carried out according to this protocol, we produced ～15 mg of tryptophan labeled neuropeptide Y receptor type 2 per liter medium. Biotechnol. Bioeng. 2013; 110: 1681–1690. © 2013 Wiley Periodicals, Inc.     

Modified method for determination of sulfur metabolites in plant tissues by stable isotope dilution-based liquid chromatography–electrospray ionization–tandem mass spectrometry

A wide variety of sulfur metabolites play important roles in plant functions. We have developed a precise and sensitive method for the simultaneous measurement of several sulfur metabolites based on liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) and ³⁴S metabolic labeling of sulfur-containing metabolites in Arabidopsis thaliana seedlings. However, some sulfur metabolites were unstable during the extraction procedure. Our proposed method does not allow for the detection of the important sulfur metabolite homocysteine because of its instability during sample extraction. Stable isotope-labeled sulfur metabolites of A. thaliana shoot were extracted and utilized as internal standards for quantification of sulfur metabolites with LC–MS/MS using S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), methionine (Met), glutathione (GSH), and glutathione disulfide (GSSG) as example metabolites. These metabolites were detected using electrospray ionization in positive mode. Standard curves were linear (r² > 0.99) over a range of concentrations (SAM 0.01–2.0 μM, SAH 0.002–0.10 μM, Met 0.05–4.0 μM, GSH 0.17–20.0 μM, GSSG 0.07–20.0 μM), with limits of detection for SAM, SAH, Met, GSH, and GSSG of 0.83, 0.67, 10, 0.56, and 1.1 nM, respectively; and the within-run and between-run coefficients of variation based on quality control samples were less than 8%.     

Affinity-based fluorogenic labeling of ATP-binding proteins with sequential photoactivatable cross-linkers

A specific illumination approach has been developed for identification of adenosine triphosphate (ATP)-binding proteins. This strategy utilizes a tandem photoactivatable unit that consists of a diazirine group as a carbene precursor and an o-hydroxycinnamate moiety as a coumarin precursor. The photolysis of diazirine induces a specific cross-link on target proteins and is followed by photoactivation of coumarin generation with a concomitant release of the pre-installed affinity ligand. The ATP, installed with this cross-linker at the γ-position, successfully transferred a coumarin onto ATP-binding proteins using only UV-irradiation.     

Structure–activity studies on the upstream splice junction of a semisynthetic intein

Protein trans-splicing by split inteins holds great potential for the chemical modification and semisynthesis of proteins. However, the structural requirements of the extein sequences immediately flanking the intein are only poorly understood. This knowledge is of particular importance for protein labeling, when synthetic moieties are to be attached to the protein of interest as seamlessly as possible. Using the semisynthetic Ssp DnaB intein both in form of its wild-type sequence and its evolved M86 mutant, we systematically varied the sequence upstream of the short synthetic Int^N fragment using both proteinogenic amino acids and unnatural building blocks. We could show for the wild-type variant that the native N-extein sequence could be reduced to the glycine residue at the (?1) position directly flanking the intein without significant loss of activity. The glycine at this position is strongly preferred over building blocks containing a phenyl group or extended alkyl chain adjacent to the scissile amide bond of the N-terminal splice junction. Despite their negative effects on the splicing yields, these unnatural substrates were well processed in the N–S acyl shift to form the respective thioesters and did not result in an increased decoupling of the asparagine cyclization step at the C-terminal splicing junction. Therefore, the transesterification step appeared to be the bottleneck of the protein splicing pathway. The fluorophore 7-hydroxycoumarinyl-4-acetic acid as a minimal N-extein was efficiently ligated to the model protein, in particular with the M86 mutant, probably because of its higher resemblance to glycine with an aliphatic c-α carbon atom at the (?1) position. This finding indicates a way for the virtually traceless labeling of proteins without inserting extra flanking residues. Due to its overall higher activity, the M86 mutant appears most promising for many protein labeling and chemical modification schemes using the split intein approach.     

Molecular recognition of a model globular protein apomyoglobin by synthetic receptor cyclodextrin: effect of fluorescence modification of the protein and cavity size of the receptor in the interaction

Labelling of proteins with some extrinsic probe is unavoidable in molecular biology research. Particularly, spectroscopic studies in the optical region require fluorescence modification of native proteins by attaching polycyclic aromatic fluoroprobe with the proteins under investigation. Our present study aims to address the consequence of the attachment of a fluoroprobe at the protein surface in the molecular recognition of the protein by selectively small model receptor. A spectroscopic study involving apomyoglobin (Apo‐Mb) and cyclodextrin (CyD) of various cavity sizes as model globular protein and synthetic receptors, respectively, using steady‐state and picosecond‐resolved techniques, is detailed here. A study involving Förster resonance energy transfer, between intrinsic amino acid tryptophan (donor) and N, N‐dimethyl naphthalene moiety of the extrinsic dansyl probes at the surface of Apo‐Mb, precisely monitor changes in donor acceptor distance as a consequence of interaction of the protein with CyD having different cavity sizes (β and γ variety). Molecular modelling studies on the interaction of tryptophan and dansyl probe with β‐CyD is reported here and found to be consistent with the experimental observations. In order to investigate structural aspects of the interacting protein, we have used circular dichroism spectroscopy. Temperature‐dependent circular dichroism studies explore the change in the secondary structure of Apo‐Mb in association with CyD, before and after fluorescence modification of the protein. Overall, the study well exemplifies approaches to protein recognition by CyD as a synthetic receptor and offers a cautionary note on the use of hydrophobic fluorescent labels for proteins in biochemical studies involving recognition of molecules. Copyright © 2013 John Wiley & Sons, Ltd.     

General Properties of Endo-N-acetylmuramidase of Bacillus subtilis YT-25

The enzymatic behaviour, amino acid composition and some physical properties of a new endo-N-acetylmuramidase (B-enzyme) of Bacillus subtilis YT–25 were determined and compared with hen’s egg white lysozyme. The molecular weight was estimated to be about 13000 by the sedimentation equilibrium method. The isoelectric point was pH 9.8. The amino acid composition indicates that the enzyme is rich in basic amino acids, especially lysin. Maximal activity on the lysis of cell walls of M. lysodeikticus occurred at pH 6.2. The enzyme was stable at pH 3.5 ~ 6.0. The specific activity for the lysis of cell walls of M. lysodeikticus was less than fourth part of that of hen’s egg white lysozyme. Digest of cell walls of M. lysodeikticus with B-enzyme consisted greater numbers of high molecular products than digest with egg white lysozyme. Substrate specificity of B-enzyme seemed to be different from that of egg white lysozyme.     

低分子肝素的绿色荧光蛋白荧光标记及其活性检测

增强型绿色荧光蛋白（EGFP）是生物领域常用的标记物. 本实验首先利用高碘酸法活化低分子肝素（LMWH）,与EGFP连接得到LMWH EGFP.然后用Sephacryl S-200 HR对其进行初步分离,再用Sephadex G-10 HR进行脱盐纯化. 采用Sephacryl S 200 HR检测LMWH EGFP的纯度,为单一对称峰. 经检测,LMWH-EGFP具有良好的热稳定性和耐碱性. 通过荧光分光检测器检测LMWH-EGFP的λEx为488 nm,λEm为509 nm. 通过抗凝实验发现LMWH EGFP仍具有抗凝活性. 本实验建立了LWMH荧光标记的方法,为多糖的荧光标记提供了新的选择.     

Protein C‐terminal enzymatic labeling identifies novel caspase cleavages during the apoptosis of multiple myeloma cells induced by kinase inhibition

Caspase activation and proteolytic cleavages are the major events in the early stage of apoptosis. Identification of protein substrates cleaved by caspases will reveal the occurrence of the early events in the apoptotic process and may provide potential drug targets for cancer therapy. Although several N‐terminal MS‐based proteomic approaches have been developed to identify proteolytic cleavages, these methods have their inherent drawbacks. Here we apply a previously developed proteomic approach, protein C‐terminal enzymatic labeling (ProC‐TEL), to identify caspase cleavage events occurring in the early stage of the apoptosis of a myeloma cell line induced by kinase inhibition. Both previously identified and novel caspase cleavage sites are detected and the reduction of the expression level of several proteins is confirmed biochemically upon kinase inhibition although the current ProC‐TEL procedure is not fully optimized to provide peptide identifications comparable to N‐terminal labeling approaches. The identified cleaved proteins form a complex interaction network with central hubs determining morphological changes during the apoptosis. Sequence analyses show that some ProC‐TEL identified caspase cleavage events are unidentifiable when traditional N‐terminomic approaches are utilized. This work demonstrates that ProC‐TEL is a complementary approach to the N‐terminomics for the identification of proteolytic cleavage events such as caspase cleavages in signaling pathways.