Structure Changes of a Membrane Polypeptide under an Applied Voltage Observed with Surface-Enhanced 2D IR Spectroscopy

The structures of many membrane-bound proteins and polypeptides depend on the membrane potential. However, spectroscopically studying their structures under an applied field is challenging, because a potential is difficult to generate across more than a few bilayers. We study the voltage-dependent structures of the membrane-bound polypeptide, alamethicin, using a spectroelectrochemical cell coated with a rough, gold film to create surface plasmons. The plasmons sufficiently enhance the 2D IR signal to measure a single bilayer. The film is also thick enough to conduct current and thereby apply a potential. The 2D IR spectra resolve features from both 3(10)- and alpha-helical structures and cross-peaks connecting the two. We observe changes in the peak intensity, not their frequencies, upon applying a voltage. A similar change occurs with pH, which is known to alter the angle of alamethicin relative to the surface normal. The spectra are modeled using a vibrational exciton Hamiltonian, and the voltage-dependent spectra are consistent with a change in angle of the 3(10)- and alpha-helices in the membrane from 55 to 44 degrees and from 31 to 60 degrees, respectively. The 3(10)- and a-helices are coupled by approximately 10 cm(-1). These experiments provide new structural information about alamethicin under a potential difference and demonstrate a technique that might be applied to voltage-gated membrane proteins and compared to molecular dynamics structures.

Automated summary

The study demonstrates that voltage-dependent structures of membrane-bound polypeptide alamethicin can be measured using a spectroelectrochemical cell with a rough gold film, revealing changes in structure under different voltage and pH conditions. The 2D IR spectra can resolve features between different helical structures, providing new structural information for voltage-gated membrane proteins.