Probing the local secondary structure of bacteriophage S pinholin membrane protein using electron spin echo envelope modulation spectroscopy.

There have recently been advances in methods for detecting local secondary structures of membrane protein using electron paramagnetic resonance (EPR). A three pulsed electron spin echo envelope modulation (ESEEM) approach was used to determine the local helical secondary structure of the small hole forming membrane protein, S pinholin. This ESEEM approach uses a combination of site-directed spin labeling and H-labeled side chains. Pinholin S is responsible for the permeabilization of the inner cytosolic membrane of double stranded DNA bacteriophage host cells. In this study, we report on the overall global helical structure using circular dichroism (CD) spectroscopy for the active form and the negative-dominant inactive mutant form of S pinholin. The local helical secondary structure was confirmed for both transmembrane domains (TMDs) for the active and inactive S pinholin using the ESEEM spectroscopic technique. Comparison of the ESEEM normalized frequency domain intensity for each transmembrane domain gives an insight into the α-helical folding nature of these domains as opposed to a π or 3-helix which have been observed in other channel forming proteins.