Introduction to magnetic resonance methods in photosynthesis

Electron paramagnetic resonance (EPR) and, more recently, solid-state nuclear magnetic resonance (NMR) have been employed to study photosynthetic processes, primarily related to the light-induced charge separation. Information obtained on the electronic structure, the relative orientation of the cofactors, and the changes in structure during these reactions should help to understand the efficiency of light-induced charge separation. A short introduction to the observables derived from magnetic resonance experiments is given. The relation of these observables to the electronic structure is sketched using the nitroxide group of spin labels as a simple example

Use of the Frank sequence in pulsed EPR

The Frank polyphase sequence has been applied to pulsed EPR of triarylmethyl radicals at 256 MHz (9.1 mT magnetic field), using 256 phase pulses. In EPR, as in NMR, use of a Frank sequence of phase steps permits pulsed FID signal acquisition with very low power microwave/RF pulses (ca. 1.5 mW in the application reported here) relative to standard pulsed EPR. A 0.2 mM aqueous solution of a triarylmethyl radical was studied using a 16 mm diameter cross loop resonator to isolate the EPR signal detection system from the incident pulses

Monitoring Alzheimer Amyloid Peptide Aggregation by EPR

Plaques containing the aggregated β-Amyloid (Aβ) peptide in the brain are the main indicators of Alzheimer’s disease. Fibrils, the building blocks of plaques, can also be produced in vitro and consist of a regular arrangement of the peptide. The initial steps of fibril formation are not well understood and could involve smaller aggregates (oligomers) of Aβ. Such oligomers have even been implicated as the toxic agents. Here, a method to study oligomers on the time scale of aggregation is suggested. We have labeled the 40 residue Aβ peptide variant containing an N-terminal cysteine (cys-Aβ) with the MTSL [1-oxyl-2,2,5,5-tetramethyl-Δ-pyrroline-3-methyl] methanethiosulfonate spin label (SL-Aβ). Fibril formation in solutions of pure SL-Aβ and of SL-Aβ mixed with Aβ was shown by Congo-red binding and electron microscopy. Continuous-wave 9 GHz electron paramagnetic resonance reveals three fractions of different spin-label mobility: one attributed to monomeric Aβ, one to a multimer (8–15 monomers), and the last one to larger aggregates or fibrils. The approach, in principle, allows detection of oligomers on the time scale of aggregation

Phase 1 study of the Pittsburgh compound B derivative 18F-flutemetamol in healthy volunteers and patients with probable Alzheimer disease.

UNLABELLED: (11)C-Pittsburgh compound B (PiB) marks Abeta amyloidosis, a key pathogenetic process in Alzheimer disease (AD). The use of (11)C-PiB is limited to centers with a cyclotron. Development of the (18)F-labeled thioflavin derivative of PiB, (18)F-flutemetamol, could hugely increase the availability of this new technology. The aims of this phase 1 study were to perform brain kinetic modeling of (18)F-flutemetamol, optimize the image acquisition procedure, and compare methods of analysis (step 1) and to compare (18)F-flutemetamol brain retention in AD patients versus healthy controls in a proof-of-concept study (steps 1 and 2). METHODS: In step 1, 3 AD patients (Mini-Mental State Examination, 22-24) and 3 elderly healthy controls were scanned dynamically during windows of 0-90, 150-180, and 220-250 min after injection of approximately 180 MBq of (18)F-flutemetamol, with arterial sampling. We compared different analysis methods (compartmental modeling, Logan graphical analysis, and standardized uptake value ratios) and determined the optimal acquisition window for step 2. In step 2, 5 AD patients (Mini-Mental State Examination, 20-26) and 5 elderly healthy controls were scanned from 80 to 170 min after injection. To determine overall efficacy, steps 1 and 2 were pooled and standardized uptake value ratios were calculated using cerebellar cortex as a reference region. RESULTS: No adverse events were reported. There was a strong correlation between uptake values obtained with the different analysis methods. From 80 min after injection onward, the ratio of neocortical to cerebellar uptake was maximal and only marginally affected by scan start time or duration. AD patients showed significantly increased standardized uptake value ratios in neocortical association zones and striatum, compared with healthy controls, whereas uptake in white matter, cerebellum, and pons did not differ between groups. Two AD patients were (18)F-flutemetamol-negative and 1 healthy control was (18)F-flutemetamol-positive. CONCLUSION: (18)F-flutemetamol uptake can be readily quantified. This phase 1 study warrants further studies to validate this (18)F-labeled derivative of PiB as a biomarker for Abeta amyloidosis

Properties of spin and fluorescent labels at a receptor-ligand interface.

Site-directed labeling was used to obtain local information on the binding interface in a receptor-ligand complex. As a model we have chosen the specific association of the extracellular part of tissue factor (sTF) and factor VIIa (FVIIa), the primary initiator of the blood coagulation cascade. Different spectroscopic labels were covalently attached to an engineered cysteine in position 140 in sTF, a position normally occupied by a Phe residue previously characterized as an important contributor to the sTF:FVIIa interaction. Two spin labels, IPSL [N-(1-oxyl-2,2,5, 5-tetramethyl-3-pyrrolidinyl)iodoacetamide] and MTSSL [(1-oxyl-2,2,5, 5-tetramethylpyrroline-3-methyl)methanethiosulfonate], and two fluorescent labels, IAEDANS [5-((((2-iodoacetyl)amino) ethyl)amino)naphthalene-1-sulfonic acid] and BADAN [6-bromoacetyl-2-dimethylaminonaphthalene], were used. Spectral data from electron paramagnetic resonance (EPR) and fluorescence spectroscopy showed a substantial change in the local environment of all labels when the sTF:FVIIa complex was formed. However, the interaction was probed differently by each label and these differences in spectral appearance could be attributed to differences in label properties such as size, polarity, and/or flexibility. Accordingly, molecular modeling data suggest that the most favorable orientations are unique for each label. Furthermore, line-shape simulations of EPR spectra and calculations based on fluorescence depolarization measurements provided additional details of the local environment of the labels, thereby confirming a tight protein-protein interaction between FVIIa and sTF when the complex is formed. The tightness of this local interaction is similar to that seen in the interior of globular proteins