Detection of the Water-Binding Sites of the Oxygen-Evolving Complex of Photosystem II Using W‑Band ¹⁷O Electron–Electron Double Resonance-Detected NMR Spectroscopy

Water binding to the Mn₄O₅Ca cluster of the oxygen-evolving complex (OEC) of Photosystem II (PSII) poised in the S₂ state was studied via H₂¹⁷O- and ²H₂O-labeling and high-field electron paramagnetic resonance (EPR) spectroscopy. Hyperfine couplings of coordinating ¹⁷O (<i>I</i> = ⁵/₂) nuclei were detected using W-band (94 GHz) electron–electron double resonance (ELDOR) detected NMR and Davies/Mims electron–nuclear double resonance (ENDOR) techniques. Universal ¹⁵N (<i>I</i> = ¹/₂) labeling was employed to clearly discriminate the ¹⁷O hyperfine couplings that overlap with ¹⁴N (<i>I</i> = 1) signals from the D1-His332 ligand of the OEC (Stich Biochemistry 2011, 50 (34), 7390−7404). Three classes of ¹⁷O nuclei were identified: (i) one μ-oxo bridge; (ii) a terminal Mn–OH/OH₂ ligand; and (iii) Mn/Ca–H₂O ligand(s). These assignments are based on ¹⁷O model complex data, on comparison to the recent 1.9 Å resolution PSII crystal structure (Umena Nature 2011, 473, 55−60), on NH₃ perturbation of the ¹⁷O signal envelope and density functional theory calculations. The relative orientation of the putative ¹⁷O μ-oxo bridge hyperfine tensor to the ¹⁴N­(¹⁵N) hyperfine tensor of the D1-His332 ligand suggests that the exchangeable μ-oxo bridge links the outer Mn to the Mn₃O₃Ca open-cuboidal unit (O4 and O5 in the Umena et al. structure). Comparison to literature data favors the Ca-linked O5 oxygen over the alternative assignment to O4. All ¹⁷O signals were seen even after very short (≤15 s) incubations in H₂¹⁷O suggesting that all exchange sites identified could represent bound substrate in the S₁ state including the μ-oxo bridge. ¹H/²H (<i>I</i> = ¹/₂, 1) ENDOR data performed at Q- (34 GHz) and W-bands complement the above findings. The relatively small ¹H/²H couplings observed require that all the μ-oxo bridges of the Mn₄O₅Ca cluster are deprotonated in the S₂ state. Together, these results further limit the possible substrate water-binding sites and modes within the OEC. This information restricts the number of possible reaction pathways for O–O bond formation, supporting an oxo/oxyl coupling mechanism in S₄

Recording information on protein complexes in an information management system

The Protein Information Management System (PiMS) is a laboratory information management system (LIMS) designed for use with the production of proteins in a research environment. The software is distributed under the CCP4 licence, and so is available free of charge to academic laboratories. Like most LIMS, the underlying PiMS data model originally had no support for protein-protein complexes. To support the SPINE2-Complexes project the developers have extended PiMS to meet these requirements. The modifications to PiMS, described here, include data model changes, additional protocols, some user interface changes and functionality to detect when an experiment may have formed a complex. Example data are shown for the production of a crystal of a protein complex. Integration with SPINE2-Complexes Target Tracker application is also described

The Protein Information Management System (PiMS) : a generic tool for any structural biology research laboratory

The Protein Information Management System (PiMS) is described together with a discussion of how its features make it well suited to laboratories of all sizes

Toxicological and pharmacological assessment of AGEN1884, a novel human IgG1 anti-CTLA-4 antibody

<div><p>CTLA-4 and CD28 exemplify a co-inhibitory and co-stimulatory signaling axis that dynamically sculpts the interaction of antigen-specific T cells with antigen-presenting cells. Anti-CTLA-4 antibodies enhance tumor-specific immunity through a variety of mechanisms including: blockade of CD80 or CD86 binding to CTLA-4, repressing regulatory T cell function and selective elimination of intratumoral regulatory T cells via an Fcγ receptor-dependent mechanism. AGEN1884 is a novel IgG1 antibody targeting CTLA-4. It potently enhanced antigen-specific T cell responsiveness that could be potentiated in combination with other immunomodulatory antibodies. AGEN1884 was well-tolerated in non-human primates and enhanced vaccine-mediated antigen-specific immunity. AGEN1884 combined effectively with PD-1 blockade to elicit a T cell proliferative response in the periphery. Interestingly, an IgG2 variant of AGEN1884 revealed distinct functional differences that may have implications for optimal dosing regimens in patients. Taken together, the pharmacological properties of AGEN1884 support its clinical investigation as a single therapeutic and combination agent.</p></div