Pinholin S2168 is an essential part of the phage Φ21 lytic protein system to release the virus progeny at the end of the infection cycle. It is known as the simplest natural timing system for its precise control of hole formation in the inner cytoplasmic membrane. Pinholin S2168 is a 68 amino acid integral membrane protein consisting of two transmembrane domains (TMDs) called TMD1 and TMD2. Despite its biological importance, structural and dynamic information of the S2168 protein in a membrane environment is not well understood. Systematic site-directed spin labeling and continuous wave electron paramagnetic resonance (CW-EPR) spectroscopic studies of pinholin S2168 in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) proteoliposomes are used to reveal the structural topology and dynamic properties in a native-like environment. CW-EPR spectral line-shape analysis of the R1 side chain for 39 residue positions of S2168 indicates that the TMDs have more restricted mobility when compared to the N- and C-termini. CW-EPR power saturation data indicate that 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. A tentative structural topology model of pinholin S2168 is also suggested based on EPR spectroscopic data reported in this study.

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.

The mechanism for the lysis pathway of double-stranded DNA bacteriophages involves a small hole-forming class of membrane proteins, the holins. This study focuses on a poorly characterized class of holins, the pinholin, of which the S 21 protein of phage φ21 is the prototype. Here we report the first in vitro synthesis of the wildtype form of the S21 pinholin, S 21 68, and negativedominant mutant form, S 21 IRS, both prepared using solid phase peptide synthesis and studied using biophysical techniques. Both forms of the pinholin were labeled with a nitroxide spin label and successfully incorporated into both bicelles and multilamellar vesicles which are membrane mimetic systems. Circular dichroism revealed the two forms were both >80% alpha helical, in agreement with the predictions based on the literature. The molar ellipticity ratio [θ] 222 / [θ] 208 for both forms of the pinholin was 1.4, suggesting a coiled-coil tertiary structure in the bilayer consistent with the proposed oligomerization step in models for the mechanism of hole formation. 31 P solid-state NMR spectroscopic data on pinholin indicate a strong interaction of both forms of the pinholin with the membrane headgroups. The 31 P NMR data has an axially symmetric line shape which is consistent with lamellar phase proteoliposomes lipid mimetics.

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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.

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Calcium (Ca2þ) dysregulation is a hallmark of heart failure and is characterized by impaired Ca 2þ sequestration into the sarcoplasmic reticulum (SR) by the SR-Ca 2þ -ATPase (SERCA). Regulins, single-pass membrane proteins, bind and inhibit SERCA by allosterically modulating the affinity of its Ca 2þ binding sites. DWarf Open Reading Frame (DWORF) is a 35 amino acid regulin that enhances SERCA2a activity in cardiomyocytes. As a first step to understanding DWORF regulation of SERCA, we used Oriented-Sample Solid-State NMR spectroscopy (OS-ssNMR) and computational methods to determine the structure and topology of DWORF in liquid crystalline lipid bilayers. We found that DWORF adopts a helical structure, with a pronounced kink at the N-terminus. Restrained molecular dynamics calculations using backbone dipolar couplings and chemical shift anisotropy show the dynamic topology of DWORF, with the N-terminal helix spanning tilt angles ranging from 50-60 degrees and C-terminal helix ranging from 30-40 degrees. The two helical domains are anchored to both membrane leaflets by polar residues. This topology differs from that of phospholamban, a main regulator of SERCA, and might explain the opposite effects on the ATPase's apparent affinity for Ca 2þ ions of these two regulins.