Unraveling the differential structural stability and dynamics features of T7 endolysin partially folded conformations

Abstract

Background: Characterization of partially collapsed protein conformations at atomic level is a daunting task due to their inherent flexibility and conformational heterogeneity. T7 bacteriophage endolysin (T7L) is a single-domain amidase that facilitates the lysis of Gram-negative bacteria. T7L exhibits a pH-dependent structural transition from native state to partially folded (PF) conformation. In the pH range 5–3, T7L PF states display differential ANS binding characteristics. Methods: CD, fluorescence, NMR spectroscopy and lysis assays were used to investigate the structure-stability- dynamics relationships of T7L PF conformations. Results: Structural studies indicated a partial loss of secondary/tertiary structures compared to its native state. The loss in the tertiary structure and the hydrophobic core opening increases upon decrease of pH from 5 to 3. Thermal denaturation experiments delineated that the pH 5 conformation is thermally irreversible in contrast to pH 3, depicting that hydrophobic core opening is essential for thermal reversibility. Further, urea dependent unfolding features of PF state at pH 5 and 4 evidenced for a collapsed conformation at intermediate urea concentrations. Residue level studies revealed that ?1-helix and ?3-?4 segment of T7L are the major contributors for such a structural collapse and inherent dynamics. Conclusions: The results suggested that the low pH PF states of T7L are heterogeneous and exhibits differential structural, unfolding, thermal reversibility, and dynamic features. General significance: Unraveling the structure-stability characteristics of different endolysin conformations is essential for designing novel chimeric and engineered phage endolysins as broadband antimicrobial agents over a varied pH range. © 2018 Elsevier B.V.