The bacteriophage infection cycle plays a crucial role in recycling the world's biomass. Bacteriophages devise various cell lysis systems to strictly control the length of the infection cycle for an efficient phage life cycle. Phages evolved with lysis protein systems, which can control and fine-tune the length of this infection cycle depending on the host and growing environment. Among these lysis proteins, holin controls the first and ratelimiting step of host cell lysis by permeabilizing the inner membrane at an allele-specific time and concentration hence known as the simplest molecular clock. Pinholin S 21 is the holin from phage Φ21, which defines the cell lysis time through a predefined ratio of active pinholin and antipinholin (inactive form of pinholin). Active pinholin and antipinholin fine-tune the lysis timing through structural dynamics and conformational changes. Previously we reported the structural dynamics and topology of active pinholin S 21 68. Currently, there is no detailed structural study of the antipinholin using biophysical techniques. In this study, the structural dynamics and topology of antipinholin S 21 68 IRS in DMPC proteoliposomes is investigated using electron paramagnetic resonance (EPR) spectroscopic techniques. Continuous-wave (CW) EPR line shape analysis experiments of 35 different R1 side chains of S 21 68 IRS indicated restricted mobility of the transmembrane domains (TMDs), which were predicted to be inside the lipid bilayer when compared to the N-and C-termini R1 side chains. In addition, the R1 accessibility test performed on 24 residues using the CW-EPR power saturation experiment indicated that TMD1 and TMD2 of S 21 68 IRS were incorporated into the lipid bilayer where N-and C-termini were located outside of the lipid bilayer. Based on this study, a tentative model of S 21 68 IRS is proposed where both TMDs remain incorporated into the lipid bilayer and N-and C-termini are located outside of the lipid bilayer. This work will pave the way for the further studies of other holins using biophysical techniques and will give structural insights into these biological clocks in molecular detail.

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Bacteriophages have evolved with an efficient host cell lysis mechanism to terminate the infection cycle and release the new progeny virions at the optimum time, allowing adaptation with the changing host and environment. Among the lytic proteins, holin controls the first and rate-limiting step of host cell lysis by permeabilizing the inner membrane at an allele-specific time known as "holin triggering". Pinholin S 21 is a prototype holin of phage Φ21 which makes many nanoscale holes and destroys the proton motive force, which in turn activates the signal anchor release (SAR) endolysin system to degrade the peptidoglycan layer of the host cell and destruction of the outer membrane by the spanin complex. Like many others, phage Φ21 has two holin proteins: active pinholin and antipinholin. The antipinholin form differs only by three extra amino acids at the N-terminus; however, it has a different structural topology and conformation with respect to the membrane. Predefined combinations of active pinholin and antipinholin fine-tune the lysis timing through structural dynamics and conformational changes. Previously, the dynamics and topology of active pinholin and antipinholin were investigated (Ahammad et al. JPCB 2019, 2020) using continuous wave electron paramagnetic resonance (CW-EPR) spectroscopy. However, detailed structural studies and direct comparison of these two forms of pinholin S 21 are absent in the literature. In this study, the structural topology and conformations of active pinholin (S 21 68) and inactive antipinholin (S 21 68 IRS ) in DMPC (1,2-dimyristoyl-snglycero-3-phosphocholine) proteoliposomes were investigated using the four-pulse double electron−electron resonance (DEER) EPR spectroscopic technique to measure distances between transmembrane domains 1 and 2 (TMD1 and TMD2). Five sets of interlabel distances were measured via DEER spectroscopy for both the active and inactive forms of pinholin S 21 . Structural models of the active pinholin and inactive antipinholin forms in DMPC proteoliposomes were obtained using the experimental DEER distances coupled with the simulated annealing software package Xplor-NIH. TMD2 of S 21 68 remains in the lipid bilayer, and TMD1 is partially externalized from the bilayer with some residues located on the surface. However, both TMDs remain incorporated in the lipid bilayer for the inactive S 21 68 IRS form. This study demonstrates, for the first time, clear structural topology and conformational differences between the two forms of pinholin S 21 . This work will pave the way for further studies of other holin systems using the DEER spectroscopic technique and will give structural insight into these biological clocks in molecular detail.

surfaces. Through molecular dynamics simulations, we have identified fourteen basic residues on the human Slp-4 C2A domain that contribute directly to membrane binding. These include a cluster of three lysines that are known to comprise the central PIP2-binding site conserved among PIP2-binding C2 domains, while the remaining eleven are spread over a large electropositive surface. We hypothesize that this surface provides a nonspecific electrostatic anchor to PS and that therefore that the net charge on the surface is likely to be more strongly evolutionarily conserved than the individual residues. To test this hypothesis, we are using primary sequence alignments to assess the level of conservation at individual residues, along with electrostatic calculations coupled with high-throughput structure prediction to compare the electropositive surface among Slp C2 domains from a wide range of animals. Current results from this ongoing study will be shown. This approach may be of broad interest for characterizing nonspecific yet potentially strong electrostatic interactions between proteins and charged surfaces.

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