Probing the Secondary Structure of Membrane-Bound gp28 Using Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy.

Abstract

Membrane proteins play a vital role in various cellular functions and are important targets for drug interactions. However, determining their local secondary structure is challenging due to their hydrophobic nature and limited experimental techniques. This study focuses on the use of electron spin echo envelope modulation (ESEEM) spectroscopy, in combination with site-directed spin labeling (SDSL) and ²H-labeled amino acid side chain (d₁₀-Leu), to study the local secondary structure of a recently discovered phage-encoded lytic protein, gp28. gp28 is a membrane protein with three predicted helices that plays a crucial role in the lysis process of bacteriophages that lack spanins. gp28 is an antimicrobial protein specifically involved in disrupting the outer membrane of the host cell. Through the synthesis of nine constructs of gp28 peptides, we systematically probed the three predicted helices. The local secondary structure of the gp28 protein in POPC/POPG vesicles was investigated using ESEEM spectroscopy. Additionally, the global secondary structure was verified using CD spectroscopy. Subsequently, the ESEEM technique allowed us to determine the local secondary structure within the three predicted alpha helices of gp28 in a membrane. This study revealed the presence of alpha helical structural components in all three predicted helices of gp28. These results not only enhance our comprehension of the local secondary structure of gp28 but also showcase the effectiveness of the ESEEM spectroscopic technique in studying membrane protein systems encoded by bacteriophages.