Class I glutaredoxins are enzymatically active, glutathione-dependent oxidoreductases, whilst class II glutaredoxins are typically enzymatically inactive, Fe-S cluster-binding proteins. Enzymatically active glutaredoxins harbor both a glutathione-scaffold site for reacting with glutathionylated disulfide substrates and a glutathione-activator site for reacting with reduced glutathione. Here, using yeast ScGrx7 as a model protein, we comprehensively identified and characterized key residues from four distinct protein regions, as well as the covalently bound glutathione moiety, and quantified their contribution to both interaction sites. Additionally, we developed a redox-sensitive GFP2-based assay, which allowed the realtime assessment of glutaredoxin structure-function relationships inside living cells. Finally, we employed this assay to rapidly screen multiple glutaredoxin mutants, ultimately enabling us to convert enzymatically active and inactive glutaredoxins into each other. In summary, we have gained a comprehensive understanding of the mechanistic underpinnings of glutaredoxin catalysis and have elucidated the determinant structural differences between the two main classes of glutaredoxins.

We describe the first extensive energetic evaluation of GPCR dimerization on the atomistic level by means of potential of mean force (PMF) computations and implicit solvent/implicit membrane end‐point free energy calculations (MM‐PBSA approach). Free energies of association computed from the PMFs show that the formation of both the 1/8 and 4/5 interface is energetically favorable for TGR5, the first GPCR known to be activated by hydrophobic bile acids and neurosteroids. Furthermore, formation of the 1/8 interface is favored over that of the 4/5 interface. Both results are in line with our previous FRET experiments in live cells. Differences in lipid‐protein interactions are identified to contribute to the observed differences in free energies of association. A per‐residue decomposition of the MM‐PBSA effective binding energy reveals hot spot residues specific for both interfaces that form clusters. This knowledge may be used to guide the design of dimerization inhibitors or perform mutational studies to explore physiological consequences of distorted TGR5 association. Finally, we characterized the role of Y111, located in the conserved (D/E)RY motif, as a facilitator of TGR5 interactions. The types of computations performed here should be transferable to other transmembrane proteins that form dimers or higher oligomers as long as good structural models of the dimeric or oligomeric states are available. Such computations may help to overcome current restrictions due to an imperfect energetic representation of protein association at the coarse‐grained level. © 2019 Wiley Periodicals, Inc.

The mechanical properties of red blood cells (RBCs) are fundamental for their physiological role as gas transporters. RBC flexibility and elasticity allow them to survive the hemodynamic changes in the different regions of the vascular tree, to dynamically contribute to the flow thereby decreasing vascular resistance, and to deform during the passage through narrower vessels. RBC mechanoproperties are conferred mainly by the structural characteristics of their cytoskeleton, which consists predominantly of a spectrin scaffold connected to the membrane via nodes of actin, ankyrin and adducin. Changes in redox state and treatment with thioltargeting molecules decrease the deformability of RBCs and affect the structure and stability of the spectrin cytoskeleton, indicating that the spectrin cytoskeleton may contain redox switches. In this perspective review, we revise current knowledge about the structural and functional characterization of spectrin cysteine redox switches and discuss the current lines of research aiming to understand the role of redox regulation on RBC mechanical properties. These studies may provide novel functional targets to modulate RBC function, blood viscosity and flow, and tissue perfusion in disease conditions.

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