Probing the Structural Topology and Dynamic Properties of gp28 Using Continuous Wave Electron Paramagnetic Resonance Spectroscopy.

Lysis of Gram-negative bacteria by dsDNA phages is accomplished through either the canonical holin-endolysin pathway or the pinholin-SAR endolysin pathway. During lysis, the outer membrane (OM) is disrupted, typically by two-component spanins or unimolecular spanins. However, in the absence of spanins, phages use alternative proteins called Disruptin to disrupt the OM. The Disruptin family includes the cationic antimicrobial peptide gp28, which is found in the virulent podophage φKT. In this study, EPR spectroscopy was used to analyze the dynamics and topology of gp28 incorporated into a lipid bilayer, revealing differences in mobility, depth parameter, and membrane interaction among different segments and residues of the protein. Our results indicate that multiple points of helix 2 and helix 3 interact with the phospholipid membrane, while others are solvent-exposed, suggesting that gp28 is a surface-bound peptide. The CW-EPR power saturation data and helical wheel analysis confirmed the amphipathic-helical structure of gp28. Additionally, course-grain molecular dynamics simulations were further used to develop the structural model of the gp28 peptide associated with the lipid bilayers. Based on the data obtained in this study, we propose a structural topology model for gp28 with respect to the membrane. This work provides important insights into the structural and dynamic properties of gp28 incorporated into a lipid bilayer environment.

Determining the helical tilt angle and dynamic properties of the transmembrane domains of pinholin S2168 using mechanical alignment EPR spectroscopy.

The lytic cycle of bacteriophage φ21 for the infected E. coli is initiated by pinholin S21, which determines the timing of host cell lysis through the function of pinholin (S2168) and antipinholin (S2171). The activity of pinholin or antipinholin directly depends on the function of two transmembrane domains (TMDs) within the membrane. For active pinholin, TMD1 externalizes and lies on the surface while TMD2 remains incorporated inside the membrane forming the lining of the small pinhole. In this study, spin labeled pinholin TMDs were incorporated separately into mechanically aligned POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine) lipid bilayers and investigated with electron paramagnetic resonance (EPR) spectroscopy to determine the topology of both TMD1 and TMD2 with respect to the lipid bilayer; the TOAC (2,2,6,6-tetramethyl-N-oxyl-4-amino-4-carboxylic acid) spin label was used here because it attaches to the backbone of a peptide and is very rigid. TMD2 was found to be nearly colinear with the bilayer normal (n) with a helical tilt angle of 16 ± 4° while TMD1 lies on or near the surface with a helical tilt angle of 84 ± 4°. The order parameters (~0.6 for both TMDs) obtained from our alignment study were reasonable, which indicates the samples incorporated inside the membrane were well aligned with respect to the magnetic field (B0). The data obtained from this study supports previous findings on pinholin: TMD1 partially externalizes from the lipid bilayer and interacts with the membrane surface, whereas TMD2 remains buried in the lipid bilayer in the active conformation of pinholin S2168. In this study, the helical tilt angle of TMD1 was measured for the first time. For TMD2 our experimental data corroborates the findings of the previously reported helical tilt angle by the Ulrich group.