Isothermal titration calorimetry of TNF-alpha and the ubiquitin variant 10F.

<p>10F at 135 µM was injected into the measurement cell containing TNF-alpha at 35.6 µM. The titration was carried out at 25°C. Experiments were conducted in duplicate. The upper panel shows the raw data and the lower panel shows the integrated enthalpy data corrected for dilution heat of a single experiment. Fitting the data to a single-site binding model (solid line), we determined K_D = 180±50 nM, stoichiometry factor N = 0.35±0.04, ΔH = −57.3±2.5 kJ mol⁻¹ and TΔS = −18.8±3.2 kJ mol⁻¹.</p

2D-NMR characterization of the scaffold ubiquitin F45W and the variant 10F.

<p>¹H-¹⁵N fHSQC spectra of the scaffold ubiquitin F45W (black, 900 µM) and its variant 10F (red, 400 µM) were acquired on an 800 MHz NMR spectrometer at 25°C. Both proteins were dissolved in PBS, 1 mM EDTA at pH 7.4. Superimposition of the spectra shows significant deviation of chemical shifts for almost all amino acids including the conserved ones. Note, that most residues between 130 ppm and 137 ppm are aliased along the ¹⁵N-dimension.</p

Coordinated Action of Two Double-Stranded RNA Binding Motifs and an RGG Motif Enables Nuclear Factor 90 To Flexibly Target Different RNA Substrates

The mechanisms of how RNA binding proteins (RBP) bind to and distinguish different RNA molecules are yet uncertain. Here, we performed a comprehensive analysis of the RNA binding properties of multidomain RBP nuclear factor 90 (NF90) by investigating specifically the functional activities of two double-stranded RNA binding motifs (dsRBM) and an RGG motif in the protein’s unstructured C-terminus. By comparison of the RNA binding affinities of several NF90 variants and their modes of binding to a set of defined RNA molecules, the activities of the motifs turned out to be very different. While dsRBM1 contributes little to RNA binding, dsRBM2 is essential for effective binding of double-stranded RNA. The protein’s immediate C-terminus, including the RGG motif, is indispensable for interactions of the protein with single-stranded RNA, and the RGG motif decisively contributes to NF90’s overall RNA binding properties. Conformational studies, which compared wild-type NF90 with a variant that contains a pseudophosphorylated residue in the RGG motif, suggest that the NF90 C-terminus is involved in conformational changes in the protein after RNA binding, with the RGG motif acting as a central regulatory element. In summary, our data propose a concerted action of all RNA binding motifs within the frame of the full-length protein, which may be controlled by regulation of the activity of the RGG motif, e.g., by phosphorylation. This multidomain interplay enables the RBP NF90 to discriminate RNA features by dynamic and adaptable interactions

Amino acid positions chosen for randomization in library construction.

<p>Based on the scaffold ubiquitin (shown here in cartoon representation), in particular with an F45W substitution, a library for the selection of artificial binding proteins was generated. For this purpose the six surface-exposed amino acid residues K6, L8, R42, I44, H68 and V70 (highlighted in red), located in the beta-sheet region of the scaffold, were chosen to be randomized for library construction. After <i>in vitro</i> selection against TNF-alpha, in the ubiquitin variant named 10F the residues D58 and Y59 (highlighted in blue) were found deleted. This figure was generated using pdb entry 1UBI and the software PyMOL version 0.99rc6 (DeLano Scientific LLC, South San Francisco, CA).</p

Size exclusion chromatography (SEC) of the complex of TNF-alpha and the ubiquitin variant 10F.

<p>(A) SEC in presence of detergent Tween-20. Samples of fluorescein-labeled 10F (10 µM), TNF-alpha (60 µM), and a mixture of both, were pre-incubated for 10 min at 4°C and loaded on a Superose 12 size-exclusion column. Experiments were performed with sample and running buffer, both containing 0.05% (v/v) Tween-20, and detection wavelengths of 280 nm (blue line) and 495 nm (red dashed line). Note that there is no significant shift of the TNF-alpha elution volume when comparing the elution profiles of TNF-alpha alone and the mixture. Fluorescein itself did not co-elute with TNF-alpha (data not shown). (B) SEC in absence of detergent Tween-20. For analysis of Tween-20 independent complex formation, a detergent-free mixture of unlabeled 10F (10 µM) and TNF-alpha (60 µM) was incubated at 4°C and aliquots were analyzed as described before using running buffer without Tween-20. Note that the area of the 10F peak (16.5 ml) decreases during prolonged incubation. Dashed lines mark the borders of the fraction used for SDS-PAGE analysis. (C) SDS-PAGE analysis of the detergent-free SEC complex fractions. Aliquots of appropriate SEC fractions were analyzed by SDS-PAGE (4–12% gradient gel) followed by Coomassie staining. M: Fermentas PageRuler Unstained, 1: control TNF-alpha (2 µg), 2: control 10F (2 µg), 3: 0.17 h preincubation, 4: 1 h, 5: 2 h*, 6: 6 h, 7: 12 h, 8: 24 h*, 9: 44 h. *To reduce complexity chromatograms were not included in (B).</p

ELISA analysis of the TNF-alpha binding ubiquitin variant 10F.

<p>(A) Specificity of ubiquitin variant 10F. The interaction of the purified, His₆-tagged binder with different immobilized target proteins (500 ng per well, human serum was used without dilution) is shown. (NGF: nerve growth factor, nPAC-1Rs: short splice form of N-terminal domain of human PACAP-receptor 1) (B) Characterization of the specificity by competition of the interaction of TNF-alpha and 10F. Biotinylated TNF-alpha (1 µM), preincubated for 1 h with competitor (etanercept: TNF-receptor 2 dimerized by fusion to IgG F_c), was incubated with immobilized 10F (100 ng per well). Detection by avidin-peroxidase demonstrates competition by etanercept. (C) Concentration-dependent competition ELISA. 10F (100 nM, His₆-tagged), preincubated with different concentrations of TNF-alpha, was incubated with immobilized TNF-alpha (150 ng per well). An IC₅₀ value of 289±53 nM was determined.</p

NMR analysis of the titration of 10F with TNF-alpha.

<p>¹H-¹⁵N fHSQC spectra of ¹⁵N-labeled ubiquitin variant 10F in presence of increasing concentration of unlabeled TNF-alpha were recorded in PBS, 1 mM EDTA pH 7.4 with 0.05% Tween-20. The relative intensity decrease of two amid resonances was plotted against molar ratio of both proteins. As determined by the intersection point of the linear fits of the initial part and the plateau part of the curve 2.88±0.18 TNF-alpha monomers are bound per 10F molecule.</p

Initiation of RNA Synthesis by the Hepatitis C Virus RNA-Dependent RNA Polymerase Is Affected by the Structure of the RNA Template

The hepatitis C virus (HCV) RNA-dependent RNA polymerase NS5B is a central enzyme of the intracellular replication of the viral (+)­RNA genome. Here, we studied the individual steps of NS5B-catalyzed RNA synthesis by a combination of biophysical methods, including real-time 1D ¹H NMR spectroscopy. NS5B was found to bind to a nonstructured and a structured RNA template in different modes. Following NTP binding and conversion to the catalysis-competent ternary complex, the polymerase revealed an improved affinity for the template. By monitoring the folding/unfolding of 3′(−)­SL by ¹H NMR, the base pair at the stem’s edge was identified as the most stable component of the structure. ¹H NMR real-time analysis of NS5B-catalyzed RNA synthesis on 3′(−)­SL showed that a pronounced lag phase preceded the processive polymerization reaction. The presence of the double-stranded stem with the edge base pair acting as the main energy barrier impaired RNA synthesis catalyzed by NS5B. Our observations suggest a crucial role of RNA-modulating factors in the HCV replication process

Examples for the determination of radial magnification errors.

<p>(A) Radial intensity profile measured in scans of the precision mask. Blue lines are experimental scans, and shaded areas indicate the regions expected to be illuminated on the basis of the known mask geometry. In this example, the increasing difference between the edges corresponds to a calculated radial magnification error of -3.1%. (B—D) Examples for differences between the experimentally measured positions of the light/dark transitions (blue circles, arbitrarily aligned for absolute mask position) and the known edge distances of the mask. The solid lines indicate the linear or polynomial fit. (B) Approximately linear magnification error with a slope corresponding to an error of -0.04%. Also indicated as thin lines are the confidence intervals of the linear regression. (C) A bimodal shift pattern of left and right edges, likely resulting from out-of-focus location of the mask, with radial magnification error of -1.7%. (D) A non-linear distortion leading to a radial magnification error of -0.53% in the <i>s</i>-values from the analysis of back-transformed data. The thin grey lines in C and D indicate the best linear fit through all data points.</p

Absence of a long-term trend in <i>s</i>_{<i>20T</i>,<i>t</i>,<i>r</i>,<i>v</i>}-values of the BSA monomer with time of experiment for the three kits (blue, green, and magenta).

<p>Highlighted as bold solid line is the overall average, and the grey area indicates one standard deviation.</p