Thermal Characterization and Interaction of the Subunits from the Multimeric Bacteriophage Endolysin PlyC

Author:

Hoopes J. Todd12,Heselpoth Ryan D.1ORCID,Schwarz Frederick P.12,Nelson Daniel C.13ORCID

Affiliation:

1. Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, MD 20850, USA

2. National Institute of Standards and Technology, Gaithersburg, MD 20899, USA

3. Department of Veterinary Medicine, University of Maryland, College Park, MD 20740, USA

Abstract

Bacteriophage endolysins degrade the bacterial peptidoglycan and are considered enzymatic alternatives to small-molecule antibiotics. In particular, the multimeric streptococcal endolysin PlyC has appealing antibacterial properties. However, a comprehensive thermal analysis of PlyC is lacking, which is necessary for evaluating its long-term stability and downstream therapeutic potential. Biochemical and kinetic-based methods were used in combination with differential scanning calorimetry to investigate the structural, kinetic, and thermodynamic stability of PlyC and its various subunits and domains. The PlyC holoenzyme structure is irreversibly compromised due to partial unfolding and aggregation at 46 °C. Unfolding of the catalytic subunit, PlyCA, instigates this event, resulting in the kinetic inactivation of the endolysin. In contrast to PlyCA, the PlyCB octamer (the cell wall-binding domain) is thermostable, denaturing at ~75 °C. The isolation of PlyCA or PlyCB alone altered their thermal properties. Contrary to the holoenzyme, PlyCA alone unfolds uncooperatively and is thermodynamically destabilized, whereas the PlyCB octamer reversibly dissociates into monomers and forms an intermediate state at 74 °C in phosphate-buffered saline with each subunit subsequently denaturing at 92 °C. Adding folded PlyCA to an intermediate state PlyCB, followed by cooling, allowed for in vitro reconstitution of the active holoenzyme.

Funder

National Institutes of Health

Publisher

MDPI AG

Subject

General Agricultural and Biological Sciences,General Immunology and Microbiology,General Biochemistry, Genetics and Molecular Biology

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