Sedimentation of large, soluble proteins up to 140 kDa for 1H-detected MAS NMR and 13C DNP NMR - practical aspects.

Solution NMR is typically applied to biological systems with molecular weights < 40 kDa whereas magic-angle-spinning (MAS) solid-state NMR traditionally targets very large, oligomeric proteins and complexes exceeding 500 kDa in mass, including fibrils and crystalline protein preparations. Here, we propose that the gap between these size regimes can be filled by the approach presented that enables investigation of large, soluble and fully protonated proteins in the range of 40-140 kDa. As a key step, ultracentrifugation produces a highly concentrated, gel-like state, resembling a dense phase in spontaneous liquid-liquid phase separation (LLPS). By means of three examples, a Sulfolobus acidocaldarius bifurcating electron transfer flavoprotein (SaETF), tryptophan synthases from Salmonella typhimurium (StTS) and their dimeric ß-subunits from Pyrococcus furiosus (PfTrpB), we show that such samples yield well-resolved proton-detected 2D and 3D NMR spectra at 100 kHz MAS without heterogeneous broadening, similar to diluted liquids. Herein, we provide practical guidance on centrifugation conditions and tools, sample behavior, and line widths expected. We demonstrate that the observed chemical shifts correspond to those obtained from µM/low mM solutions or crystalline samples, indicating structural integrity. Nitrogen line widths as low as 20-30 Hz are observed. The presented approach is advantageous for proteins or nucleic acids that cannot be deuterated due to the expression system used, or where relevant protons cannot be re-incorporated after expression in deuterated medium, and it circumvents crystallization. Importantly, it allows the use of low-glycerol buffers in dynamic nuclear polarization (DNP) NMR of proteins as demonstrated with the cyanobacterial phytochrome Cph1.

Sedimentation of large, soluble proteins up to 140 kDa for 1H-detected MAS NMR and 13C DNP NMR - practical aspects.

Solution NMR is typically applied to biological systems with molecular weights < 40 kDa whereas magic-angle-spinning (MAS) solid-state NMR traditionally targets very large, oligomeric proteins and complexes exceeding 500 kDa in mass, including fibrils and crystalline protein preparations. Here, we propose that the gap between these size regimes can be filled by the approach presented that enables investigation of large, soluble and fully protonated proteins in the range of 40-140 kDa. As a key step, ultracentrifugation produces a highly concentrated, gel-like state, resembling a dense phase in spontaneous liquid-liquid phase separation (LLPS). By means of three examples, a Sulfolobus acidocaldarius bifurcating electron transfer flavoprotein (SulfETF), tryptophan synthases from Salmonella typhimurium (StTS) and the dimeric ß-subunits from Pyrococcus furiosus (PfTrpB), we show that such samples yield well-resolved proton-detected 2D and 3D NMR spectra at 100 kHz MAS without heterogeneous broadening, similar to diluted liquids. Herein, we provide practical guidance on centrifugation conditions and tools, sample behavior, and line widths expected. We demonstrate that the observed chemical shifts correspond to those obtained from µM/low mM solutions or crystalline samples, indicating structural integrity. Nitrogen line widths as low as 20-30 Hz are observed. The presented approach is advantageous for proteins or nucleic acids that cannot be deuterated due to the expression system used, or where relevant protons cannot be re-incorporated after expression in deuterated medium, and it circumvents crystallization. Importantly, it allows the use of low-glycerol buffers in dynamic nuclear polarization (DNP) NMR of proteins as demonstrated with the cyanobacterial phytochrome Cph1.

TapA acts as specific chaperone in TasA filament formation by strand complementation.

Studying mechanisms of bacterial biofilm generation is of vital importance to understanding bacterial cell-cell communication, multicellular cohabitation principles, and the higher resilience of microorganisms in a biofilm against antibiotics. Biofilms of the nonpathogenic, gram-positive soil bacterium Bacillus subtilis serve as a model system with biotechnological potential toward plant protection. Its major extracellular matrix protein components are TasA and TapA. The nature of TasA filaments has been of debate, and several forms, amyloidic and non-Thioflavin T-stainable have been observed. Here, we present the three-dimensional structure of TapA and uncover the mechanism of TapA-supported growth of nonamyloidic TasA filaments. By analytical ultracentrifugation and NMR, we demonstrate TapA-dependent acceleration of filament formation from solutions of folded TasA. Solid-state NMR revealed intercalation of the N-terminal TasA peptide segment into subsequent protomers to form a filament composed of ß-sandwich subunits. The secondary structure around the intercalated N-terminal strand ß0 is conserved between filamentous TasA and the Fim and Pap proteins, which form bacterial type I pili, demonstrating such construction principles in a gram-positive organism. Analogous to the chaperones of the chaperone-usher pathway, the role of TapA is in donating its N terminus to serve for TasA folding into an Ig domain-similar filament structure by donor-strand complementation. According to NMR and since the V-set Ig fold of TapA is already complete, its participation within a filament beyond initiation is unlikely. Intriguingly, the most conserved residues in TasA-like proteins (camelysines) of Bacillaceae are located within the protomer interface.

Mechanism of threonine ADP-ribosylation of F-actin by a Tc toxin.

Tc toxins deliver toxic enzymes into host cells by a unique injection mechanism. One of these enzymes is the actin ADP-ribosyltransferase TccC3, whose activity leads to the clustering of the cellular cytoskeleton and ultimately cell death. Here, we show in atomic detail how TccC3 modifies actin. We find that the ADP-ribosyltransferase does not bind to G-actin but interacts with two consecutive actin subunits of F-actin. The binding of TccC3 to F-actin occurs via an induced-fit mechanism that facilitates access of NAD+ to the nucleotide binding pocket. The following nucleophilic substitution reaction results in the transfer of ADP-ribose to threonine-148 of F-actin. We demonstrate that this site-specific modification of F-actin prevents its interaction with depolymerization factors, such as cofilin, which impairs actin network turnover and leads to steady actin polymerization. Our findings reveal in atomic detail a mechanism of action of a bacterial toxin through specific targeting and modification of F-actin.

Multimodal Imaging of Patients With Gliomas Confirms 11C-MET PET as a Complementary Marker to MRI for Noninvasive Tumor Grading and Intraindividual Follow-Up After Therapy.

The value of combined L-( methyl-[11C]) methionine positron-emitting tomography (MET-PET) and magnetic resonance imaging (MRI) with regard to tumor extent, entity prediction, and therapy effects in clinical routine in patients with suspicion of a brain tumor was investigated. In n = 65 patients with histologically verified brain lesions n = 70 MET-PET and MRI (T1-weighted gadolinium-enhanced [T1w-Gd] and fluid-attenuated inversion recovery or T2-weighted [FLAIR/T2w]) examinations were performed. The computer software "visualization and analysis framework volume rendering engine (Voreen)" was used for analysis of extent and intersection of tumor compartments. Binary logistic regression models were developed to differentiate between World Health Organization (WHO) tumor types/grades. Tumor sizes as defined by thresholding based on tumor-to-background ratios were significantly different as determined by MET-PET (21.6 ± 36.8 cm3), T1w-Gd-MRI (3.9 ± 7.8 cm3), and FLAIR/T2-MRI (64.8 ± 60.4 cm3; P < .001). The MET-PET visualized tumor activity where MRI parameters were negative: PET positive tumor volume without Gd enhancement was 19.8 ± 35.0 cm3 and without changes in FLAIR/T2 10.3 ± 25.7 cm3. FLAIR/T2-MRI visualized greatest tumor extent with differences to MET-PET being greater in posttherapy (64.6 ± 62.7 cm3) than in newly diagnosed patients (20.5 ± 52.6 cm3). The binary logistic regression model differentiated between WHO tumor types (fibrillary astrocytoma II n = 10 from other gliomas n = 16) with an accuracy of 80.8% in patients at primary diagnosis. Combined PET and MRI improve the evaluation of tumor activity, extent, type/grade prediction, and therapy-induced changes in patients with glioma and serve information highly relevant for diagnosis and management.

Velocity mapping of the aortic flow at 9.4 T in healthy mice and mice with induced heart failure using time-resolved three-dimensional phase-contrast MRI (4D PC MRI).

OBJECTIVES: In this study, we established and validated a time-resolved three-dimensional phase-contrast magnetic resonance imaging method (4D PC MRI) on a 9.4 T small-animal MRI system. Herein we present the feasibility of 4D PC MRI in terms of qualitative and quantitative flow pattern analysis in mice with transverse aortic constriction (TAC). MATERIALS AND METHODS: 4D PC FLASH images of a flow phantom and mouse heart were acquired at 9.4 T using a four-point phase-encoding scheme. The method was compared with slice-selective PC FLASH and ultrasound using Bland-Altman analysis. Advanced 3D streamlines were visualized utilizing Voreen volume-rendering software. RESULTS: In vitro, 4D PC MRI flow profiles showed the transition between laminar and turbulent flow with increasing velocities. In vivo, 4D PC MRI data of the ascending aorta and the pulmonary artery were confirmed by ultrasound, resulting in linear regressions of R (2) > 0.93. Magnitude- and direction-encoded streamlines differed substantially pre- and post-TAC surgery. CONCLUSIONS: 4D PC MRI is a feasible tool for in vivo velocity measurements on high-field small-animal scanners. Similar to clinical measurement, this method provides a complete spatially and temporally resolved dataset of the murine cardiovascular blood flow and allows for three-dimensional flow pattern analysis.

About the influence of illumination models on image comprehension in direct volume rendering.

In this paper, we present a user study in which we have investigated the influence of seven state-of-the-art volumetric illumination models on the spatial perception of volume rendered images. Within the study, we have compared gradient-based shading with half angle slicing, directional occlusion shading, multidirectional occlusion shading, shadow volume propagation, spherical harmonic lighting as well as dynamic ambient occlusion. To evaluate these models, users had to solve three tasks relying on correct depth as well as size perception. Our motivation for these three tasks was to find relations between the used illumination model, user accuracy and the elapsed time. In an additional task, users had to subjectively judge the output of the tested models. After first reviewing the models and their features, we will introduce the individual tasks and discuss their results. We discovered statistically significant differences in the testing performance of the techniques. Based on these findings, we have analyzed the models and extracted those features which are possibly relevant for the improved spatial comprehension in a relational task. We believe that a combination of these distinctive features could pave the way for a novel illumination model, which would be optimized based on our findings.