Structural Stability Analysis of T7 Endolysin by Mutating the Each Core Histidine into Lysine

Abstract

An amidase protein called T7 endolysin (T7L) encoded by T7 bacteriophage cleaves the amide bond between N-acetylmuramoyl residue and L-alanine of the bacterial peptidoglycan layer and helps in the release of the mature phages. The pH dependent conformational studies have shown that the CD curves of the protein at pH 6 mostly overlaps with the curves at pH 7. When the pH reaches 5, secondary structure of the protein starts to melt. The protein remains as a partially denatured form at pH 3 [1]. Studies based on NMR and other biophysical technique have shown that the protein contains a network of 4 histidine molecules (H18, H48, H69, H123) in its core structure which is responsible for this pH dependent structural transition [1]. To know which histidine molecule of the core region renders the highest amount of structural stability to the protein, 4 different recombinants of T7L were isolated where one but different histidine of the core region is replaced with lysine in each recombinant. The pH dependent structural transition studies of the four recombinants including the wild type through various biophysical techniques can reveal which recombinant remains stable as we decrease the pH. These observations can help us to evaluate which histidine is the major contributor for the stability of the core structure in various pH range.