Investigation of protein-ligand interactions using new approaches on actin model

Actin, the most abundant protein in eukaryotic cells, can be found in two forms; globular (G-monomer) or filamentous (F-chain). Monomer actin with molecular weight 43 kDa consists of 375 aminoacids and four subunits (subdomain). Post-synthesis, G-acin undergoes modifications like ADP-ribosilation, acetilation and phosphorilation. Actin interacts with; drug molecules (phalloidin, cucurbitacin, colchicines), many proteins of membrane, cytoskeleton and plasma and protein synthesis factors (elongation factors 1 and 2, ribosomes, tRNA and apoptose related DNAse I). Globular and filamentous actin takes part in many cellular functions (cytoskeleton, mobility, division, transportation, signalization, apoptosis and protein synthesis) after such interactions. Cellular thermal shift assay (CETSA and TSA) are methods used for researching protein-ligand interactions. They can be utilized to determine intracellular interactions, time of effect and disposal periods of drugs, to design drugs and reveal data on protein-ligand binding affinities. Protein thermal stability increases with ligand/drug binding and a righ shift in denaturation curves (melting point; Tm). Cucurbitacins are triterpenoid formed molecules extracted from family cucurbitaceae. Actin is shown to interact with cucurbitacins however the binding sites are mostly unknown. Natural or synthetic cucurbitacins can be used in cancer treatment. This study examines protein-ligand interactions with TSA, a novel approach, in actin model, specificially G-actin with cucurbitacin. immunofluorescence techniques revealed that cytoskeleton degrades and aggregates with cucurbitacin I. TSA results show increase in Tm and decrease in fluorescence intensity showing interactions between actin and cucurbitacin I. In vivo and in vitro interactions are used for drug design and to research drug activity mechanisms so the results can ve used for potential pre-clinic research. Keywords: Actin, binding kinetics, protein-ligand interactions, thermal shift assay, cucurbitacin I.