We investigated imidazolium-based ionic liquid (IL) interactions with human serum albumin (HSA) to discern the level of cation interactions towards protein stability. STD-NMR spectroscopy was used to observe the imidazolium IL protons involved in direct binding and to identify the interactions responsible for changes in Tm as accessed by differential scanning calorimetry (DSC). Cations influence protein stability less than anions but still significantly. It was found that longer alkyl side chains of imidazolium-based ILs (more hydrophobic) are associated with a higher destabilisation effect on HSA than short-alkyl groups (less hydrophobic). The reason for such destabilisation lies on the increased surface contact area of the cation with the protein, particularly on the hydrophobic contacts promoted by the terminus of the alkyl chain. The relevance of the hydrophobic contacts is clearly demonstrated by the introduction of a polar moiety in the alkyl chain: a methoxy or alcohol group. Such structural modification reduces the degree of hydrophobic contacts with HSA explaining the lesser extent of protein destabilisation when compared to longer alkyl side chain groups: above [C2mim](+). Competition STD-NMR experiments using [C2mim](+), [C4mim](+) and [C2OHmim](+) also validate the importance of the hydrophobic interactions. The combined effect of cation and anion interactions was explored using (35)Cl NMR. Such experiments show that the nature of the cation has no influence on the anion-protein contacts, still the nature of the anion modulates the cation-protein interaction. Herein we propose that more destabilising anions are likely to be a result of a partial contribution from the cation as a direct consequence of the different levels of interaction (cation-anion pair and cation-protein).

Gram-positive bacteria homeostasis and antibiotic resistance mechanisms are dependent on the intricate architecture of the cell wall, where amidated peptidoglycan plays an important role. The amidation reaction is carried out by the bi-enzymatic complex MurT-GatD, for which biochemical and structural information is very scarce. In this work, we report the first crystal structure of the glutamine amidotransferase member of this complex, GatD from Staphylococcus aureus, at 1.85 Å resolution. A glutamine molecule is found close to the active site funnel, hydrogen-bonded to the conserved R128. In vitro functional studies using 1H-NMR spectroscopy showed that S. aureus MurT-GatD complex has glutaminase activity even in the absence of lipid II, the MurT substrate. In addition, we produced R128A, C94A and H189A mutants, which were totally inactive for glutamine deamidation, revealing their essential role in substrate sequestration and catalytic reaction. GatD from S. aureus and other pathogenic bacteria share high identity to enzymes involved in cobalamin biosynthesis, which can be grouped in a new sub-family of glutamine amidotransferases. Given the ubiquitous presence of GatD, these results provide significant insights into the molecular basis of the so far undisclosed amidation mechanism, contributing to the development of alternative therapeutics to fight infections.

Understanding protein structure and dynamics, which govern key cellular processes, is crucial for basic and applied research. Intrinsically disordered protein (IDP) regions display multifunctionality via alternative transient conformations, being key players in disease mechanisms. IDP regions are abundant, namely in small viruses, allowing a large number of functions out of a small proteome. The relation between protein function and structure is thus now seen from a different perspective: as IDP regions enable transient structural arrangements, each conformer can play different roles within the cell. However, as IDP regions are hard and time-consuming to study via classical techniques (optimized for globular proteins with unique conformations), new methods are required. Here, employing the dengue virus (DENV) capsid (C) protein and the immunoglobulin-binding domain of streptococcal protein G, we describe a straightforward NMR method to differentiate the solvent accessibility of single amino acid N-H groups in structured and IDP regions. We also gain insights into DENV C flexible fold region biological activity. The method, based on minimal pH changes, uses the well-established 1H-15N HSQC pulse sequence and is easily implementable in current protein NMR routines. The data generated are simple to interpret, with this rapid approach being an useful first-choice IDPs characterization method.

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The structural determinants of the interaction of the G-quadruplex (G4) motif found in precursor miRNA 149 (rG4) with the acridine orange derivative C 8 , a G4 ligand stabilizer possessing anticancer activity, and the protein nucleolin (overexpressed in cancer cells) were investigated by Nuclear Magnetic Resonance (NMR) spectroscopy. For the rG4/C 8 complex, the results revealed a strong stabilizing interaction between the aromatic core and the iodinated ring of the C 8 ligand with the rG4 structure. The NMR study revealed also different interaction patterns between nucleolin and rG4 and nucleolin and rG4/C 8 complex. In the absence of the ligand, rG4 establishes interactions with polar residues of the protein while for the rG4/C 8 complex, these contacts are mainly established with amino acids that have hydrophobic side chains. However, nucleolin chemical shift perturbation studies in the presence of rG4 or rG4/C 8 reveal the same location between domains 1 and 2 of the protein, which suggests that the rG4 and rG4/C 8 complex bind in this region. This puzzling structural study opens a new framework to study rG4/ligand/ nucleolin complexes that might impact the biogenesis of miRNA 149.

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A mística que envolve o culto a Dioniso influenciou de forma contundente o pensamento ocidental. Muitos filósofos – desde a Antiguidade até a Filosofia Contemporânea – encontraram nas diferentes vertentes do dionisismo elementos para fundamentar sua interpretação da realidade. Dentre os elementos da mística dionisíaca que mais chamaram a atenção destaca-se o estrangeirismo. Dioniso sempre se manifesta como um forasteiro. Suas dádivas, tais como o vinho, provém de terras distantes, do oriente. A loucura, o excesso, a música extática, a dança frenética e a mântica orgiástica de seus ritos são bárbaros. Trata-se de uma religião originalmente bárbara que para ser amplamente aceita foi necessário se helenizar. Sabendo disso, este trabalho discorrerá a respeito de alguns aspectos da proveniência estrangeira da mística dionisíaca. Para tal, vamos considerar os argumentos dos filólogos Erwin Rohde e Martin Person Nilsson.