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Contributions of Net Charge on the PlyC Endolysin CHAP Domain

Institute for Bioscience and Biotechnology Research, Rockville, MD 20850, USA
Department of Veterinary Medicine, University of Maryland, College Park, MD 20742, USA
Author to whom correspondence should be addressed.
Antibiotics 2019, 8(2), 70;
Received: 29 April 2019 / Revised: 20 May 2019 / Accepted: 25 May 2019 / Published: 28 May 2019
(This article belongs to the Special Issue Bacteriophages: Alternatives to Antibiotics and Beyond)
Bacteriophage endolysins, enzymes that degrade the bacterial peptidoglycan (PG), have gained an increasing interest as alternative antimicrobial agents, due to their ability to kill antibiotic resistant pathogens efficiently when applied externally as purified proteins. Typical endolysins derived from bacteriophage that infect Gram-positive hosts consist of an N-terminal enzymatically-active domain (EAD) that cleaves covalent bonds in the PG, and a C-terminal cell-binding domain (CBD) that recognizes specific ligands on the surface of the PG. Although CBDs are usually essential for the EADs to access the PG substrate, some EADs possess activity in the absence of CBDs, and a few even display better activity profiles or an extended host spectrum than the full-length endolysin. A current hypothesis suggests a net positive charge on the EAD enables it to reach the negatively charged bacterial surface via ionic interactions in the absence of a CBD. Here, we used the PlyC CHAP domain as a model EAD to further test the hypothesis. We mutated negatively charged surface amino acids of the CHAP domain that are not involved in structured regions to neutral or positively charged amino acids in order to increase the net charge from -3 to a range from +1 to +7. The seven mutant candidates were successfully expressed and purified as soluble proteins. Contrary to the current hypothesis, none of the mutants were more active than wild-type CHAP. Analysis of electrostatic surface potential implies that the surface charge distribution may affect the activity of a positively charged EAD. Thus, we suggest that while charge should continue to be considered for future engineering efforts, it should not be the sole focus of such engineering efforts. View Full-Text
Keywords: endolysin; PlyC CHAP; protein net charge; CBD-independent; FoldX endolysin; PlyC CHAP; protein net charge; CBD-independent; FoldX
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MDPI and ACS Style

Shang, X.; Nelson, D.C. Contributions of Net Charge on the PlyC Endolysin CHAP Domain. Antibiotics 2019, 8, 70.

AMA Style

Shang X, Nelson DC. Contributions of Net Charge on the PlyC Endolysin CHAP Domain. Antibiotics. 2019; 8(2):70.

Chicago/Turabian Style

Shang, Xiaoran, and Daniel C. Nelson 2019. "Contributions of Net Charge on the PlyC Endolysin CHAP Domain" Antibiotics 8, no. 2: 70.

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