Large-scale protein conformational motions on nanosecond-microsecond time scales are important for many biological processes, but remain largely unexplored because of methodological limitations. NMR relaxation methods can access these time scales if protein tumbling is prevented, but the isotropy required for high-resolution solution NMR is then lost. However, if the immobilized protein molecules are randomly oriented, the water 2H and 17O spins relax as in a solution of freely tumbling protein molecules, with the crucial difference that they now sample motions on all time scales up to approximately 100 micros. In particular, the exchange rates of internal water molecules can be determined directly from the 2H or 17O magnetic relaxation dispersion (MRD) profile. This possibility opens up a new window for characterizing the motions of individual internal water molecules as well as the large-scale protein conformational fluctuations that govern the exchange rates of structural water molecules. We introduce and validate this new NMR method by presenting and analyzing an extensive set of 2H and 17O MRD data from cross-linked gels of two model proteins: bovine pancreatic trypsin inhibitor and ubiquitin. We determine residence times and order parameters of four internal water molecules in these proteins and show that they are quantitatively consistent with the information available from crystallography and solution MRD. We also show how slow motions of side-chains bearing labile hydrogens can be monitored by the same approach. Proteins of any size can be studied at physiological hydration levels with this method.

Water-biomolecule interactions have been extensively studied in dilute solutions, crystals, and rehydrated powders, but none of these model systems may capture the behavior of water in the highly organized intracellular milieu. Because of the experimental difficulty of selectively probing the structure and dynamics of water in intact cells, radically different views about the properties of cell water have proliferated. To resolve this long-standing controversy, we have measured the 2 H spin relaxation rate in living bacteria cultured in D2O. The relaxation data, acquired in a wide magnetic field range (0.2 mT-12 T) and analyzed in a modelindependent way, reveal water dynamics on a wide range of time scales. Contradicting the view that a substantial fraction of cell water is strongly perturbed, we find that Ϸ85% of cell water in Escherichia coli and in the extreme halophile Haloarcula marismortui has bulk-like dynamics. The remaining Ϸ15% of cell water interacts directly with biomolecular surfaces and is motionally retarded by a factor 15 ؎ 3 on average, corresponding to a rotational correlation time of 27 ps. This dynamic perturbation is three times larger than for small monomeric proteins in solution, a difference we attribute to secluded surface hydration sites in supramolecular assemblies. The relaxation data also show that a small fraction (Ϸ0.1%) of cell water exchanges from buried hydration sites on the microsecond time scale, consistent with the current understanding of protein hydration in solutions and crystals. biomolecular hydration ͉ buried water molecules ͉ Escherichia coli ͉ Haloarcula marismortui ͉ in vivo NMR W ater, the ubiquitous biosolvent, mediates or modulates the intermolecular forces that govern the self-assembly of biological cells, it controls the rates of substrate diffusion and conformational transitions, and it participates in molecular recognition and enzyme catalysis (1-4). It is therefore imperative to characterize and understand any differences between cell water and bulk water. Biopolymers and other solutes make up one-third of the mass of a typical cell, so this difference could be substantial. The few experimental techniques that can monitor the molecular properties of water in vivo have suffered from interpretational ambiguities, allowing widely discordant views about cell water structure and dynamics to coexist for a long time (5-7). NMR spectroscopy can provide information about cell water via the spin relaxation times of the dominant water-1 H signal (8, 9). In fact, tissue-specific variations in water relaxation times provided the impetus for developing magnetic resonance imaging (10). Unfortunately, the interpretation of water-1 H relaxation data from biological samples is confounded by crossrelaxation, intermolecular paramagnetic couplings, and protonexchange modulation of the nuclear shielding (11,12). Here, we circumvent these complications by measuring the relaxation rate of the longitudinal water-2 H magnetization from cells cultured in D 2 O. We have chosen to study...

Water 2H and 17O spin relaxation is used to study water dynamics in the hydration layers of two small peptides, two globular proteins and in living cells of two microorganisms. The dynamical heterogeneity of hydration water is characterized by performing relaxation measurements over a wide temperature range, extending deeply into the supercooled regime, or by covering a wide frequency range. Protein hydration layers can be described by a power-law distribution of rotational correlation times with an exponent close to 2. This distribution comprises a small fraction of protein-specific hydration sites, where water rotation is strongly retarded, and a dominant fraction of generic hydration sites, where water rotation is as fast as in the hydration shells of small peptides. The generic dynamic perturbation factor is less than 2 at room temperature and exhibits a maximum near 260 K. The dynamic perturbation is induced by H-bond constraints that interfere with the cooperative mechanism that facilitates rotation in bulk water. Because these constraints are temperature-independent, hydration water does not follow the super-Arrhenius temperature dependence of bulk water. Water in living cells behaves as expected from studies of simpler model systems, the only difference being a larger fraction of secluded (strongly perturbed) hydration sites associated with the supramolecular organization in the cell. Intracellular water that is not in direct contact with biopolymers has essentially the same dynamics as bulk water. There is no significant difference in cell water dynamics between mesophilic and halophilic organisms, despite the high K+ and Na+ concentrations in the latter.

Purpose: The superior soft tissue contrast of magnetic resonance imaging (MRI) compared to computed tomography (CT) has urged the integration of MRI and elimination of CT in radiotherapy treatment (RT) for prostate. An intraprostatic gold fiducial marker (GFM) appears hyperintense on CT. On T2-weighted (T2w) MRI target delineation images, the GFM appear as a small signal void similar to calcifications and post biopsy fibrosis. It can therefore be difficult to identify the markers without CT. Detectability of GFMs can be improved using additional MR images, which are manually registered to target delineation images. This task requires manual labor, and is associated with interoperator differences and image registration errors. The aim of this work was to develop and evaluate an automatic method for identification of GFMs directly in the target delineation images without the need for image registration. Methods: T2w images, intended for target delineation, and multiecho gradient echo (MEGRE) images intended for GFM identification, were acquired for prostate cancer patients. Signal voids in the target delineation images were identified as GFM candidates. The GFM appeared as round, symmetric, signal void with increasing area for increasing echo time in the MEGRE images. These image features were exploited for automatic identification of GFMs in a MATLAB model using a patient training dataset (n = 20). The model was validated on an independent patient dataset (n = 40). The distances between the identified GFM in the target delineation images and the GFM in CT images were measured. A human observatory study was conducted to validate the use of MEGRE images. Results: The sensitivity, specificity, and accuracy of the automatic method and the observatory study was 84%, 74%, 81% and 98%, 94%, 97%, respectively. The mean absolute difference in the GFM distances for the automatic method and observatory study was 1.28 AE 1.25 mm and 1.14 AE 1.06 mm, respectively. Conclusions: Multiecho gradient echo images were shown to be a feasible and reliable way to perform GFM identification. For clinical practice, visual inspection of the results from the automatic method is needed at the current stage.

Results of the study show that the sCT conversion method can be used clinically, with minimal differences between sCT and CT dose distributions for target and relevant organs at risk. The small differences seen are consistent between centers, indicating that an MR imaging-only workflow using MriPlanner is robust for a variety of field strengths, vendors, and treatment techniques.

Protein-based microarrays are among the novel class of rapidly emerging proteomic technologies that will allow us to efficiently perform global proteome analysis. However, the process of designing adequate protein microarrays is a major inherent problem. In this study, we have evaluated a protein microarray platform based on nonpurified affinity-tagged single-chain (sc) Fv antibody fragments to generate proof-of-principle and to demonstrate the specificity and sensitivity of the array design. To this end, we used our human recombinant scFv antibody library genetically constructed around one framework, the n-CoDeR library containing 2 x 10(10) clones, as a source for our probes. The probes were immobilized via engineered C-terminal affinity tags, his- or myc-tags, to either Ni(2+)-coated slides or anti-tag antibody coated substrates. The results showed that highly functional microarrays were generated and that nonpurified scFvs readily could be applied as probes. Specific and sensitive microarrays were obtained, providing a limit of detection in the pM to fM range, using fluorescence as the mode of detection. Further, the results showed that spotting the analyte on top of the arrayed probes, instead of incubating the array with large sample volumes (333 pL vs. 40 microL), could reduce the amount of analyte required 4000 times, from 1200 attomole to 300 zeptomole. Finally, we showed that a highly complex proteome, such as human sera containing several thousand different proteins, could be directly fluorescently labeled and successfully analyzed without compromising the specificity and sensitivity of the antibody microarrays. This is a prerequisite for the design of high-density antibody arrays applied in high-throughput proteomics.

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Dosimetric errors in a magnetic resonance imaging (MRI) only radiotherapy workflow may be caused by system specific geometric distortion from MRI. The aim of this study was to evaluate the impact on planned dose distribution and delineated structures for prostate patients, originating from this distortion. A method was developed, in which computer tomography (CT) images were distorted using the MRI distortion field. The displacement map for an optimized MRI treatment planning sequence was measured using a dedicated phantom in a 3 T MRI system. To simulate the distortion aspects of a synthetic CT (electron density derived from MR images), the displacement map was applied to CT images, referred to as distorted CT images. A volumetric modulated arc prostate treatment plan was applied to the original CT and the distorted CT, creating a reference and a distorted CT dose distribution. By applying the inverse of the displacement map to the distorted CT dose distribution, a dose distribution in the same geometry as the original CT images was created. For 10 prostate cancer patients, the dose difference between the reference dose distribution and inverse distorted CT dose distribution was analyzed in isodose level bins. The mean magnitude of the geometric distortion was 1.97 mm for the radial distance of 200-250 mm from isocenter. The mean percentage dose differences for all isodose level bins, were  ⩽0.02% and the radiotherapy structure mean volume deviations were <0.2%. The method developed can quantify the dosimetric effects of MRI system specific distortion in a prostate MRI only radiotherapy workflow, separated from dosimetric effects originating from synthetic CT generation. No clinically relevant dose difference or structure deformation was found when 3D distortion correction and high acquisition bandwidth was used. The method could be used for any MRI sequence together with any anatomy of interest.