A method was developed to determine the interspin distances of two or more nitroxide spin labels attached to specific sites in proteins. This method was applied to different conformations of spin-labeled insulins. The electron paramagnetic resonance (EPR) line broadening due to dipolar interaction is determined by fitting simulated EPR powder spectra to experimental data, measured at temperatures below 200 K to freeze the protein motion. The experimental spectra are composed of species with different relative nitroxide orientations and interspin distances because of the flexibility of the spin label side chain and the variety of conformational substates of proteins in frozen solution. Values for the average interspin distance and for the distance distribution width can be determined from the characteristics of the dipolar broadened line shape. The resulting interspin distances determined for crystallized insulins in the R6 and T6 structure agree nicely with structural data obtained by x-ray crystallography and by modeling of the spin-labeled samples. The EPR experiments reveal slight differences between crystal and frozen solution structures of the B-chain amino termini in the R6 and T6 states of hexameric insulins. The study of interspin distances between attached spin labels can be applied to obtain structural information on proteins under conditions where other methods like two-dimensional nuclear magnetic resonance spectroscopy or x-ray crystallography are not applicable.

The increasing number of mouse models of human disease used in biomedical research applications has led to an enhanced interest in non-invasive imaging of mice, e.g. using MRI for phenotyping. However, MRI of small rodents puts high demands on the sensitivity of data acquisition. This requirement can be addressed by using cryogenic radio-frequency (RF) detection devices. The aim of this work was to investigate the in vivo performance of a 400 MHz cryogenic transmit/receive RF probe (CryoProbe) designed for MRI of the mouse brain. To characterize this novel probe, MR data sets were acquired with both the CryoProbe and a matched conventional receive-only surface coil operating at room temperature (RT) using conventional acquisition protocols (gradient and spin echo) with identical parameter settings. Quantitative comparisons in phantom and in vivo experiments revealed gains in the signal-to-noise ratio (SNR) of 2.4 and 2.5, respectively. The increased sensitivity of the CryoProbe was invested to enhance the image quality of high resolution structural images acquired in scan times compatible with routine operation (< 45 min). In high resolution (30 x 30 x 300 microm(3)) structural images of the mouse cerebellum, anatomical details such as Purkinje cell and molecular layers could be identified. Similarly, isotropic (60 x 60 x 60 microm(3)) imaging of mouse cortical and subcortical areas revealed anatomical structures smaller than 100 microm. Finally, 3D MR angiography (52 x 80 x 80 microm(3)) of the brain vasculature enabled the detailed reconstruction of intracranial vessels (anterior and middle cerebral artery). In conclusion, this low temperature detection device represents an attractive option to increase the performance of small animal MR systems operating at 9.4 Tesla.

Photo-excited structural changes of the light-driven proton pump bacteriorhodopsin were monitored using double-site-directed spin labeling combined with electron paramagnetic resonance (EPR) spectroscopy. The inter-spin distances between nitroxides attached at residue positions 100 and 226, 101 and 160, and 101 and 168 were determined for the BR initial state and the trapped M photo-intermediate. Distance changes that occur during the photocycle were followed with millisecond time resolution under physiological conditions at 293 K. The kinetic analysis of the EPR data and comparison with the absorbance changes in the visible spectrum reveal an outward movement of helix F during the late M intermediate and a subsequent approach of helix G toward the proton channel. The displacements of the cytoplasmic moieties of these helices amount to 0.1-0.2 nm. We propose that the resulting opening of the proton channel decreases the pK of the proton donor D96 and facilitates proton transfer to the Schiff base during the M-to-N transition.

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In prostate cancers (PCa), the formation of malignant stroma may substantially influence tumor phenotype and aggressiveness. Thus, the impact of the orthotopic and subcutaneous implantations of hormone-sensitive (H), hormone-insensitive (HI), and anaplastic (AT1) Dunning PCa in rats on growth, microcirculation, and metabolism was investigated. For this purpose, dynamic contrast-enhanced magnetic resonance imaging and (1)H magnetic resonance spectroscopy ([(1)H]MRS) were applied in combination with histology. Consistent observations revealed that orthotopic H tumors grew significantly slower compared to subcutaneous ones, whereas the growth of HI and AT1 tumors was comparable at both locations. Histologic analysis indicated that glandular differentiation and a close interaction of tumor cells and smooth muscle cells (SMC) were associated with slow tumor growth. Furthermore, there was a significantly lower SMC density in subcutaneous H tumors than in orthotopic H tumors. Perfusion was observed to be significantly lower in orthotopic H tumors than in subcutaneous H tumors. Regional blood volume and permeability-surface area product showed no significant differences between tumor models and their implantation sites. Differences in growth between subcutaneous and orthotopic H tumors can be attributed to tumor-stroma interaction and perfusion. Here, SMC, may stabilize glandular structures and contribute to the maintenance of differentiated phenotype.