In-situ spectroscopic detection of large-scale reorientations of transmembrane α-helices during viroporin channel opening

Abstract

Viroporins are small ion channels in membranes of enveloped viruses that play key roles during viral life cycles. To use viroporins as drug targets against viral infection requires in-depth mechanistic understanding and, with that, methods that enable investigations under in-situ conditions. Here, we apply surface-enhanced infrared absorption (SEIRA) spectroscopy to Influenza A M2 reconstituted within a solid-supported membrane, to shed light on the mechanics of its viroporin function. M2 is a paradigm of pH-activated proton channels and controls the proton flux into the viral interior during viral infection. We use SEIRA to track the large-scale reorientation of M2’s transmembrane α-helices in-situ during pH-activated channel opening. We quantify this event as a helical tilt from 26° to 40° by correlating the experimental results with solid-state NMR-informed computational spectroscopy. This mechanical motion is impeded upon addition of the inhibitor rimantadine, giving a direct spectroscopic marker to test antiviral activity. The presented approach provides a spectroscopic tool to quantify large-scale structural changes and to track the function and inhibition of the growing number of viroporins from pathogenic viruses in future studies.

Seeing double: the persistent dimer-of-dimers structure of drug resistant influenza A M2

“…The sample used for TREDOR (Figure 4) was expressed in D2O using 2 H, 13 C6-D-glucose and 15 N-ammonium chloride. Inclusion bodies were separated, then resuspended in 6 M guanidinium hydrochloride, purified through a nickel column and cleaved overnight using a 1000-fold excess of cyanogen bromide in 0.2 M HCl and 6 M guanidinium hydrochloride 38,39 to remove the TrpLE tag. Protein was then purified on a C4 HPLC column with gradient elution using a mixture of 3:2 isopropanol:acetonitrile (v/v).…”