Dipolar EPR Spectroscopy Research Articles

Orientation Resolving Dipolar High-Field EPR Spectroscopy on Disordered Solids: II. Structure of Spin-Correlated Radical Pairs in Photosystem I

The distance and relative orientation of functional groups within protein domains and their changes during chemical reactions determine the efficiency of biological processes. In this work on electron transfer proteins, we report the results of orientation resolving dipolar high-field EPR spectroscopy on the charge-separated state P700•+ A1•– (P700, primary electron donor; A1, phylloquinone electron acceptor) in Photosystem I (PS I). Pulsed high-field EPR spectroscopy at W-band (95 GHz, 3.4 T) with extensions to PELDOR (pulsed electron–electron double resonance) and RIDME (relaxation-induced dipolar modulation enhancement) was utilized to obtain the parameters describing the three-dimensional structure of the laser-flash-induced transient radical pair P700•+ A1•– in a frozen solution of deuterated PS I from the cyanobacterium Synechocystis sp. PCC 6803, which is performing oxygenic photosynthesis. The measured distances and relative orientations of the weakly coupled radical ions in the radical pair P700•+ A1•– are compared with previously reported geometries and with those of the precursor cofactors P700 and A1 known from X-ray crystallography. Cyclic electron transfer was found to proceed exclusively via the A-branch of the cofactor chain of PS I at cryogenic temperature. The position and orientation of the reduced phylloquinone coincide with those of the precursor, revealing that no substantial orientational changes of the phylloquinone molecule upon charge separation occur. Several distinct orientations of the P700•+ g-tensor axes with respect to the molecular frame of the primary donor were found experimentally, which we explain by several conformational substates of the P700•+ radical structure having slightly different electron spin density distributions.

Entrance to a lipoxygenase substrate cavity is defined

A persistent puzzle about lipoxygenase enzymes is how specificity for position and stereochemistry of oxygen addition to polyunsaturated fatty acids is achieved. In lipoxygenase structures, a curved substrate channel approaches the centrally located active site, with ends of the curve near different surface locations. A long‐standing suggestion is that varied specificity results from reversed placement of the polar and methyl ends of the substrate within highly similar protein structures. We approach this problem by placing a spin label on the polar end of a lipoxygenase substrate analog and use paramagnetic distance geometry to locate the substrate spin relative to selected protein positions. Soybean seed lipoxygenase‐1 has been substituted with spin labels at five sites. A five‐point representation of the structure is obtained by measuring ten inter‐site distances by pulsed dipolar EPR spectroscopy (PDS) in doubly spin labeled enzyme constructs. Refinement of spin label coordinates results from modeling and fits to the native protein structure. Finally, the substrate analog polar end is found with five more distance measurements between substrate and protein spins. The spin of the substrate analog is just outside the conjunction of helices‐2 and ‐11. Detailed spin labeling of helix‐2 reveals flexibility permitting entrance to the internal cavity. 1NIH GM65268 and 2NIH RR016292

Orientation-Resolving Pulsed Electron Dipolar High-Field EPR Spectroscopy on Disordered Solids: I. Structure of Spin-Correlated Radical Pairs in Bacterial Photosynthetic Reaction Centers

Distance and relative orientation of functional groups within protein domains and their changes during chemical reactions determine the efficiency of biological processes. In this work on disordered solid-state electron-transfer proteins, it is demonstrated that the combination of pulsed high-field EPR spectroscopy at the W band (95 GHz, 3.4 T) with its extensions to PELDOR (pulsed electron-electron double resonance) and RIDME (relaxation-induced dipolar modulation enhancement) offers a powerful tool for obtaining not only information on the electronic structure of the redox partners but also on the three-dimensional structure of radical-pair systems with large interspin distances (up to about 5 nm). Strategies are discussed both in terms of data collection and data analysis to extract unique solutions for the full radical-pair structure with only a minimum of additional independent structural information. By this novel approach, the three-dimensional structure of laser-flash-induced transient radical pairs P(865)(*+)Q(A)(*-) in frozen-solution reaction centers (RCs) from the photosynthetic bacterium Rhodobacter (Rb.) sphaeroides is solved. The measured positions and relative orientations of the weakly coupled ion radicals P(865)(*+) and Q(A)(*-) are compared with those of the precursor cofactors P865 and QA known from X-ray crystallography. A small but significant reorientation of the reduced ubiquinone QA is revealed and interpreted as being due to the photosynthetic electron transfer. In contrast to the large conformational change of Q(B)(*-) upon light illumination of the RCs, the small light-induced reorientation of Q(A)(*-) had escaped previous attempts to detect structural changes of photosynthetic cofactors upon charge separation. Although small, they still may be of functional importance for optimizing the electronic coupling of the redox partners in bacterial photosynthesis both for the charge-separation and charge-recombination processes.

Unappreciation of a priori identifiability in software packages causes ambiguities in numerical estimates

ARTICLESUnappreciation of a priori identifiability in software packages causes ambiguities in numerical estimatesC. Cobelli, and M. P. SaccomaniC. Cobelli, and M. P. SaccomaniPublished Online:01 Jun 1990https://doi.org/10.1152/ajpendo.1990.258.6.E1058MoreSectionsPDF (323 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat "Unappreciation of a priori identifiability in software packages causes ambiguities in numerical estimates." American Journal of Physiology-Endocrinology and Metabolism, 258(6), pp. E1058–E1059 Previous Back to Top Download PDF FiguresReferencesRelatedInformation Cited ByCompactness regularization in the analysis of dipolar EPR spectroscopy dataJournal of Magnetic Resonance, Vol. 339Practical identifiability in the frame of nonlinear mixed effects models: the example of the in vitro erythropoiesis4 October 2021 | BMC Bioinformatics, Vol. 22, No. 1Calibration, Selection and Identifiability Analysis of a Mathematical Model of the in vitro Erythropoiesis in Normal and Perturbed ContextsIn Silico Biology, Vol. 13, No. 1-2The Union between Structural and Practical Identifiability Makes Strength in Reducing Oncological Model Complexity: A Case StudyComplexity, Vol. 2018Identifiability of Nonlinear ODE Models in Systems Biology: Results from Both Structural and Data-Based MethodsStructural identifiability and indistinguishability of compartmental models16 April 2009 | Expert Opinion on Drug Metabolism & Toxicology, Vol. 5, No. 3Development of a compartmental model of human zinc metabolism: identifiability and multiple studies analysesLeland V. Miller, Nancy F. Krebs, and K. Michael Hambidge1 November 2000 | American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, Vol. 279, No. 5 More from this issue > Volume 258Issue 6June 1990Pages E1058-E1059 Copyright & PermissionsCopyright © 1990 the American Physiological Societyhttps://doi.org/10.1152/ajpendo.1990.258.6.E1058PubMed2360625History Published online 1 June 1990 Published in print 1 June 1990 Metrics