Figure 1.

Summary of isotopic labeling, sample preparation strategies, and sample compositions. The terms NA, UN, and UCN denote natural abundance, uniformly ¹⁵N-labeled, and uniformly ¹³C,¹⁵N-labeled media or samples, respectively. A sample of purified ASR reconstituted in DMPC/DMPA lipids (PL-ASR, leftmost column) was used as reference. Two types of ASR samples expressed in the E. coli membrane (EM-ASR) were prepared. For the first type, ASR and background proteins were labeled at the same level, and outer membrane and inner membrane fractions not containing ASR were minimized using a sucrose gradient and His-tag binding (41,42) (second left column). To reduce NMR signal from the background proteins, cells were grown to the exponential phase in NA media, and then resuspended at a high concentration in labeled media before protein expression was induced (denoted as rbUN or rbUCN, second right column). An additional control sample was produced by following the expression and isolation procedures for the rbUCN EM-ASR sample, but without induction (EM-NIC, rightmost column). Sample compositions are given at the bottom of the figure and are explained in the Supporting Material.