Extended ɛ34 Phage TSP Renatures After Urea-acid Unfolding

In antimicrobial-peptide/protein engineering, understanding the peptide/protein’s adaptability to harsh environmental conditions such as urea, proteases, fluctuating temperatures, high salts provide enormous insight into the pharmacokinetics and pharmacodynamics of the engineered peptide/protein and its ability to survive the harsh internal environment of the human body such as the gut or the harsh external environment to which they are applied. A previous work in our laboratory demonstrated that our cloned Eɛ34 TSP showed potent antimicrobial activity against Salmonella newington, and more so, could prevent biofilm formation on decellularized tissue. In this work, the effects of urea-acid on the Eɛ34 stability is studied, and the results demonstrates that at lower pHs of 3 and 4 with urea the protein was denatured into monomeric species. However, the protein withstood urea denaturation above pH of 5 and thus remained as trimeric protein. The mechanism of denaturation of Eɛ34 TSP seems to show that urea denatures proteins by depleting hydrophobic core of the protein by directly binding to the amide units via hydrogen bonds. The results of our in-silico investigation determined that urea binds with Eɛ34 TSP with relative free energies range of -3.4 to -2.9 kcal/mol at the putative globular head binding domain of the protein. The urea molecules interacts with with the protein’s predicted hydrophobic core, thus, disrupting and exposing the shielded hydrophobic moieties of Eɛ34 TSP to the solvent. We further showed that after the unfolding of Eɛ34 TSP via urea-acid, renaturation of the protein to its native conformation was possible within few hours. This unique characteristic of refolding of Eɛ34 TSP which is similar to that of the P22 phage tailspike protein is of special interest to protein scientists and can also be exploited in antimicrobial-protein engineering.