Cellular membranes (biomembranes) provide both permeability barriers and multifunctional interfaces to cells. Because of their fundamental role in life, biomembranes have been the object of fascination and intensive study. However, understanding their structure in detail has proven challenging because they are intrinsically nanoscopic, with a transverse thickness of a few nanometers and lateral features up to tens of nanometers. Only probes of sufficiently short wavelength — X-rays, electrons and neutrons — can detect these features directly. The outstanding challenge has been creating contrast between the membrane and the cellular background. Here we describe the application of in-vivo neutron contrast variation to resolve both transverse and lateral structure in the plasma membrane in the Gram-positive bacterium Bacillus subtilis. As opposed to X-rays and electrons, neutrons can differentiate ordinary hydrogen (H) from its heavy isotope deuterium (D). Specifically, these isotopes have different neutron scattering lengths, and varying their ratio in a molecule changes its scattering length density (SLD). Contrast results from differences in SLD. We used a combination of classical and chemical genetics implement contrast variation in cells, first making them invisible by matching their SLDs to that of the aqueous medium, and second by introducing contrast into the membrane specifically (Figure). All common biomolecules can have their SLDs matched to that of heavy water (D2O) through deuteration of their carbon skeletons to the extent of about 75%. Subsequent introduction of all-H fatty acids introduced strong contrast into the membrane. Using small-angle neutron scattering (SANS), we directly observed the in-vivo lamellar structure of the B. subtilis plasma membrane and measured its transverse hydrophobic thickness as 24.3 ± 0.9 Å. Using a second set of contrast conditions, we were able to detect scattering signals indicative of nanoscopic lateral structure, possibly lipid domains.

In-vivo structure of the Gram-positive bacterial plasma membrane

255th National Meeting (2018)

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