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.

The Impact of -Acryloyllysine Piperazides on the Conformational Dynamics of Transglutaminase 2.

In addition to the classic functions of proteins, such as acting as a biocatalyst or binding partner, the conformational states of proteins and their remodeling upon stimulation need to be considered. A prominent example of a protein that undergoes comprehensive conformational remodeling is transglutaminase 2 (TGase 2), the distinct conformational states of which are closely related to particular functions. Its involvement in various pathophysiological processes, including fibrosis and cancer, motivates the development of theranostic agents, particularly based on inhibitors that are directed toward the transamidase activity.

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.

Application of a Fluorescence Anisotropy-Based Assay to Quantify Transglutaminase 2 Activity in Cell Lysates.

Transglutaminase 2 (TGase 2) is a multifunctional protein which is involved in various physiological and pathophysiological processes. The latter also include its participation in the development and progression of malignant neoplasms, which are often accompanied by increased protein synthesis. In addition to the elucidation of the molecular functions of TGase 2 in tumor cells, knowledge of its concentration that is available for targeting by theranostic agents is a valuable information.

Distinct 3-disulfide-bonded isomers of tridegin differentially inhibit coagulation factor XIIIa: The influence of structural stability on bioactivity.

Tridegin is a 66mer cysteine-rich coagulation factor XIIIa (FXI-IIa) inhibitor from the giant amazon leech Haementeria ghilianii of yet unknown disulfide connectivity. This study covers the structural and functional characterization of five different 3-disulfide-bonded tridegin isomers. In addition to three previously identified isomers, one isomer containing the inhibitory cystine knot (ICK, knottin) motif, and one isomer with the leech antihemostatic protein (LAP) motif were synthesized in a regioselective manner.