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Protein based binding molecules have a broad applicability from therapeutic to technical use. Monoclonal antibodies represent the major class of this type of agents complemented by innovative approaches using scaffold proteins with tailor-made properties. Various concepts for new formats combining antibody chains or antibody fragments and fusions with other entities have been developed recently. This strategy opens up options to design molecules with biophysical, biochemical and pharmacological characteristics in a broad range while simultaneously addressing several targets or epitopes. The demand for such compounds is still growing as reflected by the literature and further new ideas are expected. In this context we developed so called Mabfilin and Fabfilin molecules. The formats synergistically bring together the classical antibody or fragments thereof supplemented with additional binding moieties, the Affilin ® molecules. These are based on the scaffold ubiquitin endowed with novel targeting properties by local randomization and selection from combinatorial libraries. Mab-/Fabfilin variants show advantageous biochemical properties and open a new route for the development of multispecific compounds for flexible applications., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Nuclear factor 90 (NF90) is an RNA-binding protein (RBP) that regulates post-transcriptionally the expression of various mRNAs. NF90 was recently shown to be capable of discriminating between different RNA substrates. This is mediated by an adaptive and co-operative interplay between three RNA-binding motifs (RBMs) in the protein's C-terminus. In many cell types, NF90 exists predominantly in a complex with NF45. Here, we compared the RNA-binding properties of the purified NF90 monomer and the NF90-NF45 heterodimer by biophysical and biochemical means, and demonstrate that the interaction with NF45 considerably affects the characteristics of NF90. Along with a thermodynamic stabilization, complex formation substantially improves the RNA-binding capacity of NF90 by modulating its binding mode and by enhancing its affinity for single- and double-stranded RNA substrates. Our data suggest that features of both the N- and C-termini of NF90 participate in the heterodimerization with NF45 and that the formation of NF90-NF45 changes the conformation of NF90's RBMs to a status in which the co-operative interplay of the RBMs is optimal. NF45 is considered to act as a conformational scaffold for NF90's RBMs, which alters the RNA-binding specificity of NF90. Accordingly, the monomeric NF90 and the NF90-NF45 heterodimer may exert different functions in the cell., (© 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

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.

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 (1)H 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 (1)H NMR, the base pair at the stem's edge was identified as the most stable component of the structure. (1)H 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.

Because wettability is not always examined under standard conditions, we investigated the temperature dependence of saline wettability on unconditioned and conditioned polymethylmethacrylate (PMMA), cellulose acetate butyrate (CAB), and three silicone acrylate lens materials. Sessile drop contact angles were measured in a humidity chamber at 23 degrees C and 34 degrees C using laser-assisted contact angle goniometry. In separate experiments, saline-stored and preconditioned lenses were examined either with or without rinsing. Sessile drop contact angles at 34 degrees C were within 2 degrees to 5 degrees of the room temperature values for both conditioned and unconditioned lenses, demonstrating a negligible temperature dependence. At both temperatures, the conditioned PMMA, CAB, silafocon A, and pasifocon C lenses wet slightly better, by 1 degree to 12 degrees, than unconditioned lenses. However, this increase was only significant with PMMA and silafocon A (P less than 0.05) and reversed when the preconditioned lenses were rinsed repeatedly in saline and reexamined. The results suggest that for these materials: 1) in vitro saline contact angles do not approach those seen on the eye, and this discrepancy can not be explained by temperature or conditioning; and 2) conditioning does not increase material wettability but merely forms a temporary hydrophilic interface that is more wettable than the lens material.