The phosphorylation of Ser15 in the transactivation domain (TAD) of the tumor suppressor protein 53 (p53) by ataxia-telangiectasia mutated (ATM) kinase is a crucial step in the tumor suppressor function of p53. An understanding of the factors that affect the rate of Ser15 phosphorylation may provide new strategies for the manipulation of the ATM-p53 pathway in cancer therapy. In this study, the effect of electrostatic interactions between ATM and p53 was investigated by measuring the phosphorylation of Ser15 at varying pH ranges from 5 to 9. To achieve this, two different kinase assay methods were utilized: the ELISA technique, which directly quantifies the phosphorylated Ser15, and the Universal Kinase Assay, which assesses the formation of ADP. The results revealed that Ser15 phosphorylation was pH-dependent, with higher phosphorylation rates observed in the alkaline range. To ascertain whether the lower phosphorylation rates observed at acidic pH were due to protein denaturation, a pH-dependent solubility profile was generated using the CamSol server. The obtained results demonstrated comparable solubility rates within the pH range of the kinase assays performed. Furthermore, the significance of negatively charged residues in TAD1-39 was evaluated by substituting Asp and Glu residues with hydrophobic and uncharged hydrophilic residues in TAD1-39 using ChimeraX and subsequently comparing their interactions with the ATM using the protein-protein docking server HADDOCK2.4. The results of the docking simulations indicated that the alteration of negatively charged residues with uncharged ones resulted in a reduction in the efficiency of the interaction between the ATM and TAD1-39. In conclusion, it can be stated that electrostatic interactions between the ATM and TAD are important for optimal Ser15 phosphorylation.

Staphylococci are pathogens that cause serious infections in humans and animals. Nosocomial infections caused by staphylococci, particularly methicillin-resistant Staphylococcus aureus (MRSA) strains, are mostly transmitted through healthcare workers, patients, or contaminated materials and food. In recent years, studies have been carried out to develop alternative antimicrobial strategies due to the inadequacy of existing antibiotics in the prevention of systemic, skin and implant-related biofilm infections caused by these multi-antibiotic resistant strains. One of these new approaches is the development of products containing the bacteriophage endolysin, which is particularly effective against multi-antibiotic-resistant bacteria. In this study, endolysin genes of bacteriophages (prophages) integrated into the chromosomes of Staphylococcus strains were amplified by polymerase chain reaction (PCR) and cloned into pET SUMO and pET-30b(+) vectors and produced recombinantly in E. coli. Anti-staphylococcal and antibiofilm activity of recombinant endolysins against S. aureus, S. epidermidis, and S. haemolyticus strains isolated from clinical specimens, were demonstrated using turbidity reduction, biofilm removal in microwell plates by crystal violet method, and capacity of endolysins to kill biofilm-forming bacteria by confocal microscopy imaging by live-dead staining. The combination of endolysin was shown to reduce bacterial culture turbidity by at least 50% at 60 minutes and biofilms by approximately 70% at 12 hours. These results show that endolysins have the potential to be used in the prevention of staphylococcal infections.