Synthesis, screening, and sequencing of cysteine-rich one-bead one-compound peptide libraries.

Cysteine-rich peptides are valued as tags for biarsenical fluorophores and as environmentally important reagents for binding toxic heavy metals. Due to the inherent difficulties created by cysteine, the power of one-bead one-compound (OBOC) libraries has never been applied to the discovery of short cysteine-rich peptides. We have developed the first method for the synthesis, screening, and sequencing of cysteine-rich OBOC peptide libraries. First, we synthesized a heavily biased cysteine-rich OBOC library, incorporating 50% cysteine at each position (Ac-X8-KM-TentaGel). Then, we developed conditions for cysteine alkylation, cyanogen bromide cleavage, and direct MS/MS sequencing of that library at the single bead level. The sequencing efficiency of this library was comparable to a traditional cysteine-free library. To validate screening of cysteine-rich OBOC libraries, we reacted a library with the biarsenical FlAsH and identified beads bearing the known biarsenical-binding motif (CCXXCC). These results enable OBOC libraries to be used in high-throughput discovery of cysteine-rich peptides for protein tagging, environmental remediation of metal contaminants, or cysteine-rich pharmaceuticals

Documenting marine species traits in the World Register of Marine Species (WoRMS): current status, future plans and encountered challenges

The importance of describing species patterns and the underlying processes explaining these patterns is essential to assess the status and future evolution of marine ecosystems. This requires biological information on functional and structural species traits such as feeding ecology, body size, reproduction, life history, etc.To accommodate this need, the World Register of Marine Species (WoRMS) (WoRMS Editorial Board 2017) is expanding its content with trait information (Costello et al. 2015), subdivided into 3 main categories: (1) taxonomy related traits, e.g. paraphyletic groups, (2) biological and ecological traits-specific characteristics of a taxon, e.g. body size or feeding type and (3) human defined traits, e.g. the legal protection status of species, whether a species is introduced, harmful, or used as an ecological indicator.Initially, priority was given to the inclusion of traits that could be applied to the majority of marine taxa and where the information was easily available. The main driver for this approach was that the inclusion of these traits should result in new research, which in turn would drive improvements in the quality and quantity of trait information. Pilot projects were carried out for different species groups, allowing a thorough documentation of a selection of traits. In parallel, a standard vocabulary was put together (http://www.marinespecies.org/traits/wiki/), based on already existing resources to cover all marine life. All documented traits needed to be compliant with this vocabulary, in order to make the data as widely useable as possible, across groups. Defining a trait across all marine life is not trivial, as scientists can use terms in a different way between groups. This stresses the importance for users to realize these differences in terminology, before they analyse a trait across all taxa.Some traits were thought to be quite straightforward to document, although practice proved otherwise. Such a trait is body size, where the aim was to document the numerical value of the ‘maximum body size in length’. In reality, a lot of variation is possible (e.g. for fish: fork length versus standard length) and maximum size is not always considered relevant from an ecological point of view. On the other hand, documenting numerical body size for each marine species is quite time consuming. Therefore, a complementary size trait will be documented, indicating whether taxa are considered as micro, meio, macro or mega.Whereas the initial approach was to complete the register for each tackled trait relevant for all marine species, we now complement this by (1) documenting several traits within a specific group, regardless whether this trait is also present in other taxon groups, and (2) documenting one specific trait, covering a variety – but not all – taxonomic groups, e.g. the composition of the skeleton for calcareous animals.Where possible, we aim to document a trait on a higher taxonomic level to allow the work to progress more rapidly. As the database allows top-down inheritance of traits, exceptions can easily be documented. In addition, collaborations are sought with already running initiatives such as Encyclopedia of Life.Very soon, all the documented traits will be searchable through the Marine Species Traits Portal. The human-defined traits are already accessible through the EMODnet Biology Portal (http://www.emodnet-biology.eu/toolbox), in combination with distribution information from the European Ocean Biogeographic Information System (EurOBIS; www.eurobis.org; Vandepitte et al. 2011; Vandepitte et al. 2015) and taxonomy from WoRMS (www.marinespecies.org). Through the LifeWatch Taxonomic Backbone (LW-TaxBB) (http://www.lifewatch.be/data-services/), services are offered to access these traits, combined with data and information from other resources such as WoRMS and (Eur)OBIS.We would like to acknowledge the EMODnet Biology and the LifeWatch project, in which the Flanders Marine Institute (VLIZ) – host institute of WoRMS – is responsible for the development of the LW-TaxBB. Both projects provide funding for the documentation of trait data and development of services allowing researchers to easily access the available data, in combination with data from other sources

NRG-CING: integrated validation reports of remediated experimental biomolecular NMR data and coordinates in wwPDB

For many macromolecular NMR ensembles from the Protein Data Bank (PDB) the experiment-based restraint lists are available, while other experimental data, mainly chemical shift values, are often available from the BioMagResBank. The accuracy and precision of the coordinates in these macromolecular NMR ensembles can be improved by recalculation using the available experimental data and present-day software. Such efforts, however, generally fail on half of all NMR ensembles due to the syntactic and semantic heterogeneity of the underlying data and the wide variety of formats used for their deposition. We have combined the remediated restraint information from our NMR Restraints Grid (NRG) database with available chemical shifts from the BioMagResBank and the Common Interface for NMR structure Generation (CING) structure validation reports into the weekly updated NRG-CING database (http://nmr.cmbi.ru.nl/NRG-CING). Eleven programs have been included in the NRG-CING production pipeline to arrive at validation reports that list for each entry the potential inconsistencies between the coordinates and the available experimental NMR data. The longitudinal validation of these data in a publicly available relational database yields a set of indicators that can be used to judge the quality of every macromolecular structure solved with NMR. The remediated NMR experimental data sets and validation reports are freely available online

Human resource requirements for quality-assured electronic data capture of the tuberculosis case register

<p>Abstract</p> <p>Background</p> <p>The tuberculosis case register is the data source for the reports submitted by basic management units to the national tuberculosis program. Our objective was to measure the data entry time required to complete and double-enter one record, and to estimate the time for the correction of errors in the captured information from tuberculosis case registers in Cambodia and Viet Nam. This should assist in quantifying the additional requirements in human resources for national programs moving towards electronic recording and reporting.</p> <p>Methods</p> <p>Data from a representative sample of tuberculosis case registers from Cambodia and Viet Nam were double-entered and discordances resolved by rechecking the original case register. Computer-generated data entry time recorded the time elapsed between opening of a new record and saving it to disk.</p> <p>Results</p> <p>The dataset comprised 22,732 double-entered records of 11,366 patients (37.1% from Cambodia and 62.9% from Viet Nam). The mean data entry times per record were 97.5 (95% CI: 96.2-98.8) and 66.2 (95% CI: 59.5-73.0) seconds with medians of 90 and 31 s respectively in Cambodia and in Viet Nam. The percentage of records with an error was 6.0% and 39.0% respectively in Cambodia and Viet Nam. Data entry time was inversely associated with error frequency. We estimate that approximately 118-person-hours were required to produce 1,000 validated records.</p> <p>Conclusions</p> <p>This study quantifies differences between two countries for data entry time for the tuberculosis case register and frequencies of data entry errors and suggests that higher data entry speed is partially offset by requiring revisiting more records for corrections.</p

Use of Docking Peptides to Design Modular Substrates with High Efficiency for Mitogen-Activated Protein Kinase Extracellular Signal-Regulated Kinase

The mitogen-activated protein (MAP) kinase ERK plays a key role in the regulation of cellular proliferation. Mutations in the ERK cascade occur in 30% of malignant tumors. Thus understanding how the kinase identifies its cognate substrates as well as monitoring the activity of ERK is central to cancer research and therapeutic development. ERK binds to its protein targets, both downstream substrates and upstream activators, via a binding site distinct from the catalytic site of ERK. The substrate sequences that bind, or dock, to these sites on ERK influence the efficiency of phosphorylation. For this reason, simple peptide substrates containing only phosphorylation sequences typically possess low efficiencies for ERK. Appending short docking peptides derived from full-length protein substrates and activators of ERK to a phosphorylation sequence increased the affinity of ERK for the phosphorylation sequence by as much as 200-fold, while only slightly diminishing the maximal velocity of the reaction. The efficiency of the phosphorylation reaction was increased by up to 150 fold, while the specificity of the substrate for ERK was preserved. Simple, modular peptide substrates which can be easily tailored to possess high phosphorylation efficiencies will enhance our understanding of the regulation of ERK and provide a tool for the development of new kinase assays

FAD binding, cobinamide binding and active site communication in the corrin reductase (CobR)

Adenosylcobalamin, the coenzyme form of vitamin B12, is one Nature's most complex coenzyme whose de novo biogenesis proceeds along either an anaerobic or aerobic metabolic pathway. The aerobic synthesis involves reduction of the centrally chelated cobalt metal ion of the corrin ring from Co(II) to Co(I) before adenosylation can take place. A corrin reductase (CobR) enzyme has been identified as the likely agent to catalyse this reduction of the metal ion. Herein, we reveal how Brucella melitensis CobR binds its coenzyme FAD (flavin dinucleotide) and we also show that the enzyme can bind a corrin substrate consistent with its role in reduction of the cobalt of the corrin ring. Stopped-flow kinetics and EPR reveal a mechanistic asymmetry in CobR dimer that provides a potential link between the two electron reduction by NADH to the single electron reduction of Co(II) to Co(I)

Structure of human Fe–S assembly subcomplex reveals unexpected cysteine desulfurase architecture and acyl-ACP–ISD11 interactions

In eukaryotes, sulfur is mobilized for incorporation into multiple biosynthetic pathways by a cysteine desulfurase complex that consists of a catalytic subunit (NFS1), LYR protein (ISD11), and acyl carrier protein (ACP). This NFS1-ISD11-ACP (SDA) complex forms the core of the iron-sulfur (Fe-S) assembly complex and associates with assembly proteins ISCU2, frataxin (FXN), and ferredoxin to synthesize Fe-S clusters. Here we present crystallographic and electron microscopic structures of the SDA complex coupled to enzyme kinetic and cell-based studies to provide structure-function properties of a mitochondrial cysteine desulfurase. Unlike prokaryotic cysteine desulfurases, the SDA structure adopts an unexpected architecture in which a pair of ISD11 subunits form the dimeric core of the SDA complex, which clarifies the critical role of ISD11 in eukaryotic assemblies. The different quaternary structure results in an incompletely formed substrate channel and solvent-exposed pyridoxal 5'-phosphate cofactor and provides a rationale for the allosteric activator function of FXN in eukaryotic systems. The structure also reveals the 4'-phosphopantetheine-conjugated acyl-group of ACP occupies the hydrophobic core of ISD11, explaining the basis of ACP stabilization. The unexpected architecture for the SDA complex provides a framework for understanding interactions with acceptor proteins for sulfur-containing biosynthetic pathways, elucidating mechanistic details of eukaryotic F e-S cluster biosynthesis, and clarifying how defects in Fe-S cluster assembly lead to diseases such as Friedreich's ataxia. Moreover, our results support a lock-and-key model in which LYR proteins associate with acyl-ACP as a mechanism for fatty acid biosynthesis to coordinate the expression, Fe-S cofactor maturation, and activity of the respiratory complexes. Keywords: LYR; ACP; iron-sulfur cluster; PLP; frataxi