Enhanced dynamics of conformationally heterogeneous T7 bacteriophage lysozyme native state attenuates its stability and activity.

Proteins are dynamic in nature and exists in a set of equilibrium conformations on various timescale motions. The flexibility of proteins governs various biological functions, and therefore elucidation of such functional dynamics is essential. In this context, we have studied the structure-dynamics-stability-activity relationship of bacteriophage T7 lysozyme/endolysin (T7L) native state ensemble in the pH range 6-8. Our studies established that T7L native state is conformationally heterogeneous, as several residues of its C-terminal half are present in two conformations (major and minor) in the slow exchange time scale of NMR. Structural and dynamic studies suggested that the residues belonging to minor conformations do exhibit native-like structural and dynamic features. Further, the NMR relaxation experiments unravelled that the native state is highly dynamic and the dynamic behaviour is regulated by the pH, as the pH 6 conformation exhibited enhanced dynamics compared to pH 7/8. The stability measurments and cell based activity studies on T7L indicated that the native protein at pH 6 is ~ 2 kcal less stable, and is ~ 50 % less active to those of pH 7/8. A comprehensive analysis of the T7L active site, unfolding initation sites and the residues with altered dynamics outlined that, the attenuation of stability and activity is a resultant of its enhanced dynamic properties, which in turn can be attributed to the protonation/deprotonation of its partially buried His residues. Our study on T7L structure-dynamics-activity paradigm could assist in engineering novel amidase based endolysins with enhanced activity and stability over a broad pH range.