Crowdsourcing biocuration: The Community Assessment of Community Annotation with Ontologies (CACAO).

Experimental data about gene functions curated from the primary literature have enormous value for research scientists in understanding biology. Using the Gene Ontology (GO), manual curation by experts has provided an important resource for studying gene function, especially within model organisms. Unprecedented expansion of the scientific literature and validation of the predicted proteins have increased both data value and the challenges of keeping pace. Capturing literature-based functional annotations is limited by the ability of biocurators to handle the massive and rapidly growing scientific literature. Within the community-oriented wiki framework for GO annotation called the Gene Ontology Normal Usage Tracking System (GONUTS), we describe an approach to expand biocuration through crowdsourcing with undergraduates. This multiplies the number of high-quality annotations in international databases, enriches our coverage of the literature on normal gene function, and pushes the field in new directions. From an intercollegiate competition judged by experienced biocurators, Community Assessment of Community Annotation with Ontologies (CACAO), we have contributed nearly 5,000 literature-based annotations. Many of those annotations are to organisms not currently well-represented within GO. Over a 10-year history, our community contributors have spurred changes to the ontology not traditionally covered by professional biocurators. The CACAO principle of relying on community members to participate in and shape the future of biocuration in GO is a powerful and scalable model used to promote the scientific enterprise. It also provides undergraduate students with a unique and enriching introduction to critical reading of primary literature and acquisition of marketable skills.

EPR Methods for Biological Cu(II): L-Band CW and NARS.

Copper has many roles in biology that involve the change of coordination sphere and/or oxidation state of the copper ion. Consequently, the study of copper in heterogeneous environments is an important area in biophysics. EPR is a primary technique for the investigation of paramagnetic copper, which is usually the isolated Cu(II) ion, but sometimes as Cu(II) in different oxidation states of multitransition ion clusters. The gross geometry of the coordination environment of Cu(II) can often be determined from a simple inspection of the EPR spectrum, recorded in the traditional X-band frequency range (9-10 GHz). Identification and quantitation of the coordinating ligand atoms, however, is not so straightforward. In particular, analysis of the superhyperfine structure on the EPR spectrum, to determine the number of coordinated nitrogen atoms, is fraught with difficulty at X-band, despite the observation that the overwhelming number of EPR studies of Cu(II) in the literature have been carried out at X-band. Greater reliability has been demonstrated at S-band (3-4 GHz), using the low-field parallel (gz) features. However, analysis relies on clear identification of the outermost superhyperfine line, which has the lowest intensity of all the spectral features. Computer simulations have subsequently indicated that the much more intense perpendicular region of the spectrum can be reliably interpreted at L-band (2 GHz). The present work describes the development of L-band EPR of Cu(II) into a routine method that is applicable to biological samples.

Moving difference (MDIFF) non-adiabatic rapid sweep (NARS) EPR of copper(II).

Non-adiabatic rapid sweep (NARS) EPR spectroscopy has been introduced for application to nitroxide-labeled biological samples (Kittell et al., 2011). Displays are pure absorption, and are built up by acquiring data in spectral segments that are concatenated. In this paper we extend the method to frozen solutions of copper-imidazole, a square planar copper complex with four in-plane nitrogen ligands. Pure absorption spectra are created from concatenation of 170 5-gauss segments spanning 850 G at 1.9 GHz. These spectra, however, are not directly useful since nitrogen superhyperfine couplings are barely visible. Application of the moving difference (MDIFF) algorithm to the digitized NARS pure absorption spectrum is used to produce spectra that are analogous to the first harmonic EPR. The signal intensity is about four times higher than when using conventional 100 kHz field modulation, depending on line shape. MDIFF not only filters the spectrum, but also the noise, resulting in further improvement of the SNR for the same signal acquisition time. The MDIFF amplitude can be optimized retrospectively, different spectral regions can be examined at different amplitudes, and an amplitude can be used that is substantially greater than the upper limit of the field modulation amplitude of a conventional EPR spectrometer, which improves the signal-to-noise ratio of broad lines.

Spin hamiltonian parameters for Cu(II)-prion peptide complexes from L-band electron paramagnetic resonance spectroscopy.

Cu(II) is an essential element for life but is also associated with numerous and serious medical conditions, particularly neurodegeneration. Structural modeling of crystallization-resistant biological Cu(II) species relies on detailed spectroscopic analysis. Electron paramagnetic resonance (EPR) can, in principle, provide spin hamiltonian parameters that contain information on the geometry and ligand atom complement of Cu(II). Unfortunately, EPR spectra of Cu(II) recorded at the traditional X-band frequency are complicated by (i) strains in the region of the spectrum corresponding to the g(∥) orientation and (ii) potentially very many overlapping transitions in the g(⊥) region. The rapid progress of density functional theory computation as a means to correlate EPR and structure, and the increasing need to study Cu(II) associated with biomolecules in more biologically and biomedically relevant environments such as cells and tissue, have spurred the development of a technique for the extraction of a more complete set of spin hamiltonian parameters that is relatively straightforward and widely applicable. EPR at L-band (1-2 GHz) provides much enhanced spectral resolution and straightforward analysis via computer simulation methods. Herein, the anisotropic spin hamiltonian parameters and the nitrogen coordination numbers for two hitherto incompletely characterized Cu(II)-bound species of a prion peptide complex are determined by analysis of their L-band EPR spectra.

Asp-120 locates Zn2 for optimal metallo-beta-lactamase activity.

Metallo-beta-lactamases are zinc-dependent hydrolases that inactivate beta-lactam antibiotics, rendering bacteria resistant to them. Asp-120 is fully conserved in all metallo-beta-lactamases and is central to catalysis. Several roles have been proposed for Asp-120, but so far there is no agreed consensus. We generated four site-specifically substituted variants of the enzyme BcII from Bacillus cereus as follows: D120N, D120E, D120Q, and D120S. Replacement of Asp-120 by other residues with very different metal ligating capabilities severely impairs the lactamase activity without abolishing metal binding to the mutated site. A kinetic study of these mutants indicates that Asp-120 is not the proton donor, nor does it play an essential role in nucleophilic activation. Spectroscopic and crystallographic analysis of D120S BcII, the least active mutant bearing the weakest metal ligand in the series, reveals that this enzyme is able to accommodate a dinuclear center and that perturbations in the active site are limited to the Zn2 site. It is proposed that the role of Asp-120 is to act as a strong Zn2 ligand, locating this ion optimally for substrate binding, stabilization of the development of a partial negative charge in the beta-lactam nitrogen, and protonation of this atom by a zinc-bound water molecule.