In-depth analysis of Gαs protein activity by probing different fluorescently labeled guanine nucleotides.

G proteins are interacting partners of G protein-coupled receptors (GPCRs) in eukaryotic cells. Upon G protein activation, the ability of the Gα subunit to exchange GDP for GTP determines the intracellular signal transduction. Although various studies have successfully shown that both Gαs and Gαi have an opposite effect on the intracellular cAMP production, with the latter being commonly described as "more active", the functional analysis of Gαs is a comparably more complicated matter.

Design, synthesis, and analysis of macrobicyclic peptides for targeting the Gαi protein.

Bicyclic peptides are important chemical tools that can function, for example, as bioactive ligands switching on/off signaling pathways mediated by guanine nucleotide-binding proteins as bicycles are more broadly applicable. Despite their relevance in medicinal chemistry, the synthesis of such peptides is challenging, and the final yield is highly dependent on the chemical composition and physicochemical properties of the scaffold. We recently discovered novel, state-specific peptide modulators targeting the Gαi protein, namely, GPM-2/GPM-3, by screening a one-bead-two-compound combinatorial library.

Quality Assessment of Selected Protein Structures Derived from Homology Modeling and AlphaFold.

With technology advancing, many prediction algorithms have been developed to facilitate the modeling of inherently dynamic and flexible macromolecules such as proteins. Improvements in the prediction of protein structures have attracted a great deal of attention due to the advantages they offer, e.g.

Bicyclic Peptide Library Screening for the Identification of Gαi Protein Modulators.

Noncanonical G protein activation and inactivation, particularly for the Gαi/s protein subfamilies, have long been a focus of chemical research. Combinatorial libraries were already effectively applied to identify modulators of the guanine-nucleotide exchange, as can be exemplified with peptides such as KB-752 and GPM-1c/d, the so-called guanine-nucleotide exchange modulators. In this study, we identified novel bicyclic peptides from a combinatorial library screening that show prominent properties as molecular switch-on/off modulators of Gαi signaling.

Fluorescence Anisotropy Assay with Guanine Nucleotides Provides Access to Functional Analysis of Gαi1 Proteins.

Gα proteins as part of heterotrimeric G proteins are molecular switches essential for G protein-coupled receptor- mediated intracellular signaling. The role of the Gα subunits has been examined for decades with various guanine nucleotides to elucidate the activation mechanism and Gα protein-dependent signal transduction. Several approaches describe fluorescent ligands mimicking the GTP function, yet lack the efficient estimation of the proteins' GTP binding activity and the fraction of active protein.