Biology subjects

Viegas, A.

Publications and source records attributed to Viegas, A..

3 recordsLinked to original sources

bioRxiv2026

NMR reveals specific remodelling of protein folding landscapes in ionic liquids

Ionic liquids (ILs) are designer solvents with tunable properties that have emerged as powerful cosolutes in protein science and biotechnology. While ILs are known to stabilize or destabilize proteins, their molecular mechanisms remain poorly defined, particularly regarding effects on the unfolded ensemble. Here, we use the metastable SH3 domain of the Drosophila adaptor protein Drk, which coexists in a slow two-state equilibrium between folded and unfolded states, as a model to dissect how ILs modulate protein conformational equilibria. Using nuclear magnetic resonance (NMR) spectroscopy, we show that cholinium glutamate ([Ch][Glu]) stabilizes the folded state by preferential exclusion from the protein surface, raising the barrier to unfolding in a manner reminiscent of osmolytes and crowding agents. In contrast, 1-butyl-3-methylimidazolium dicyanamide ([Bmim][dca]) shifts the equilibrium toward the unfolded state, not by destabilizing the native fold, but by stabilizing a compact, non-native -helical conformation within the unfolded ensemble. Notably, this concentration-dependent mechanism differs from traditional denaturants such as urea or guanidinium chloride, which promote random-coil unfolded states. Kinetic measurements further reveal that [Ch][Glu] slows unfolding, whereas [Bmim][dca] slows folding, indicating that the two ILs reshape the folding landscape in opposite directions. These findings challenge the prevailing view that cosolutes act primarily on the folded state and establish unfolded-state stabilization as a critical determinant of protein stability. More broadly, this work provides a molecular framework for understanding how ILs reshape protein folding landscapes and offers design principles for tailoring ILs as stabilizers or destabilizers in biotechnology and therapeutic applications. Significance StatementProteins exist in a delicate balance between folded and unfolded states, and cosolutes such as salts are generally viewed as acting only on the folded state. Here, we show that ionic liquids (ILs) are able to reshape this balance and uniquely modulate both folded and unfolded protein states through distinct molecular mechanisms. These results challenge the conventional view of protein stabilization/destabilization and demonstrate that unfolded-state modulation is a critical determinant of protein behavior. By establishing principles for how ILs alter protein folding landscapes, this work provides a framework for designing ILs with tailored effects, with implications for biocatalysis, protein engineering, and therapeutic formulation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=178 SRC="FIGDIR/small/700775v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@372d3borg.highwire.dtl.DTLVardef@7a7fadorg.highwire.dtl.DTLVardef@543f4eorg.highwire.dtl.DTLVardef@1084836_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2024

Substrate-dependent oxidative inactivation of a W-dependent formate dehydrogenase involving selenocysteine displacement.

Metal-dependent formate dehydrogenases are very promising targets for enzyme optimization and design of bio-inspired catalysts for CO2 reduction, towards novel strategies for climate change mitigation. For effective application of these enzymes, the catalytic mechanism must be fully understood, and the molecular determinants clarified. Despite numerous studies, several doubts persist, namely regarding the role played by the possible dissociation of the SeCys ligand from the Mo/W active site. Additionally, the O2 sensitivity of these enzymes must also be understood as it poses an important obstacle for biotechnological applications. Here we present a combined biochemical, spectroscopic, and structural characterization of Desulfovibrio vulgaris FdhAB (DvFdhAB) when exposed to oxygen in the presence of a substrate (formate or CO2). This study reveals that O2 inactivation is promoted by the presence of either substrate and involves forming a new species in the active site, captured in the crystal structures, where the SeCys ligand is displaced from tungsten coordination and replaced by a dioxygen or peroxide molecule. This new form was reproducibly obtained and supports the conclusion that, although W-DvFdhAB can catalyze the oxidation of formate in the presence of oxygen for some minutes, it gets irreversibly inactivated after prolonged O2 exposure in the presence of either substrate. These results reveal that oxidative inactivation does not require reduction of the metal, as widely assumed, as it can also occur in the oxidized state in the presence of CO2.

biochemistry↗

bioRxiv2021

Biophysical and pharmacokinetic characterization of a small-molecule inhibitor of RUNX1/ETO tetramerization with anti-leukemic effects

Acute myeloid leukemia (AML) is a malignant disease of immature myeloid cells and the most prevalent acute leukemia among adults. The oncogenic homo-tetrameric fusion protein RUNX1/ETO results from the chromosomal translocation t(8;21) and is found in AML patients. The nervy homology region 2 (NHR2) domain of ETO mediates tetramerization; this oligomerization is essential for oncogenic activity. Previously, we identified the first-in-class small-molecule inhibitor of NHR2 tetramer formation, 7.44, which was shown to specifically interfere with NHR2, restore gene expression down-regulated by RUNX1/ETO, inhibit the proliferation of RUNX1/ETO-depending SKNO-1 cells, and reduce the RUNX1/ETO-related tumor growth in a mouse model. However, no biophysical and structural characterization of 7.44 binding to the NHR2 domain has been reported. Likewise, the compound has not been characterized as to physicochemical, pharmacokinetic, and toxicological properties. Here, we characterize the interaction between the NHR2 domain of RUNX1/ETO and 7.44 by biophysical assays and show that 7.44 interferes with NHR2 tetramer stability and leads to an increase in the dimer population of NHR2. The affinity of 7.44 with respect to binding to NHR2 is Klig = 3.95 {+/-} 1.28 M. By NMR spectroscopy combined with molecular dynamics simulations, we show that 7.44 binds with both heteroaromatic moieties to NHR2 and interacts with or leads to conformational changes in the N-termini of the NHR2 tetramer. Finally, we demonstrate that 7.44 has favorable physicochemical, pharmacokinetic, and toxicological properties. Together with biochemical, cellular, and in vivo assessments, the results reveal 7.44 as a lead for further optimization towards targeted therapy of t(8;21) AML.

biophysics↗