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Allosteric regulation of the Tet repressor (TetR) homodimer relies on tetracycline binding that abolishes the affinity for the DNA operator. Previously, interpretation of circular dichroism data called for unfolding of the α-helical DNA-binding domains in absence of binding to DNA or tetracycline. Our small angle X-ray scattering of TetR(D) in solution contradicts this unfolding as a physiological process. Instead, in the core domain crystal structures analyses show increased immobilisation of helix α9 and two C-terminal turns of helix α8 upon tetracycline binding. Tetracycline complexes of TetR(D) and four single-site alanine variants were characterised by isothermal titration calorimetry, fluorescence titration, X-ray crystal structures, and melting curves. Five crystal structures confirm that Thr103 is a key residue for the allosteric events of induction, with the T103A variant lacking induction by any tetracycline. The T103A variant shows anti-cooperative inducer binding, and a melting curve of the tetracycline complex different to TetR(D) and other variants. For the N82A variant inducer binding is clearly anti-cooperative but triggers the induced conformation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Antifreeze proteins (AFPs) provide protection for organisms subjected to the presence of ice crystals. The psychrophilic diatom Fragilariopsis cylindrus which is frequently found in polar sea ice carries a multitude of AFP isoforms. In this study we report the heterologous expression of two antifreeze protein isoforms from F. cylindrus in Escherichia coli. Refolding from inclusion bodies produced proteins functionally active with respect to crystal deformation, recrystallization inhibition and thermal hysteresis. We observed a reduction of activity in the presence of the pelB leader peptide in comparison with the GS-linked SUMO-tag. Activity was positively correlated to protein concentration and buffer salinity. Thermal hysteresis and crystal deformation habit suggest the affiliation of the proteins to the hyperactive group of AFPs. One isoform, carrying a signal peptide for secretion, produced a thermal hysteresis up to 1.53°C±0.53°C and ice crystals of hexagonal bipyramidal shape. The second isoform, which has a long preceding N-terminal sequence of unknown function, produced thermal hysteresis of up to 2.34°C±0.25°C. Ice crystals grew in form of a hexagonal column in presence of this protein. The different sequences preceding the ice binding domain point to distinct localizations of the proteins inside or outside the cell. We thus propose that AFPs have different functions in vivo, also reflected in their specific TH capability., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Secretory phospholipase A(2) is involved in inflammatory processes and was previously shown to be inhibited by lipophilic tetracyclines such as minocycline (minoTc) and doxycycline. Lipophilic tetracyclines might be a new lead compound for the design of specific inhibitors of secretory phospholipase A(2), which play a crucial role in inflammatory processes. Our X-ray crystal structure analysis at 1.65 A resolution of the minoTc complex of phospholipase A(2) (PLA(2)) of the Indian cobra (Naja naja naja) is the first example of nonantibiotic tetracycline interactions with a protein. MinoTc interferes with the conformation of the active-site Ca(2+)-binding loop, preventing Ca(2)(+) binding, and shields the active site from substrate entrance, resulting in inhibition of the enzyme. MinoTc binding to PLA(2) is dominated by hydrophobic interactions quite different from antibiotic recognition of tetracyclines by proteins or the ribosome. The affinity of minoTc for PLA(2) was determined by surface plasmon resonance, resulting in a dissociation constant K(d)=1.8 x 10(-)(4) M., ((c) 2010 Elsevier Ltd. All rights reserved.)