Following that, SS-NMR spectroscopy was utilized to probe the differences in the way the active and inactive forms of pinholin interact with the membrane and gives the first piece of direct quantitative evidence indicating TMD1 interacts with the lipid headgroups of the bilayer. After sample optimization, electron spin echo envelope modulation (ESEEM) spectroscopy was used to determine the local α-helical secondary structure of the inhibitory and functional helices of pinholin. First, we report the first in vitro synthesis of both the active S2168 and inactive S21IRS pinholin using solid phase peptide synthesis (SPPS) and reveals the first experimental data indicating the global α-helical structure of pinholin using circular dichroism (CD) spectroscopy.
The work in this dissertation will use a variety of powerful biophysical techniques to study these structure and dynamic properties of pinholin. Despite the biological importance of pinholin the structure and dynamic properties have not been well characterized. Recently a new family of holins have been discovered, the pinholin, which have been shown to depolarize the cytosolic membrane leading to the release of membrane-bound signal-anchor-released (SAR) endolysin from the bilayer. Holins are a family of lytic membrane proteins responsible for the lysis of the cytosolic membrane in host cells of double stranded DNA bacteriophages.
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