Fluorescence lifetimes of NBD at C terminus of TMS in various 2TML12K2 speciesa
| NBD-labeled species | Membranes | Donor–PTC separationb | NBD location | τ1 | f1c | τ2 | f2c | χ2 | < τ >d |
| nsec | nsec | nsec | |||||||
| 2TML12DA2122 | + | 2 | Tunnel | 2.8 ± 0.1 | 0.70 | 9.1 ± 0.3 | 0.30 | 3 | 4.7 ± 0.2 |
| 2TML12DA2125 | + | 5 | Tunnel | 2.9 ± 0.2 | 0.68 | 9.6 ± 0.5 | 0.32 | 1 | 5.0 ± 0.3 |
| 2TML12DA2126 | + | 6 | Tunnel | 2.5 ± 0.2 | 0.64 | 8.5 ± 0.3 | 0.36 | 3 | 4.7 ± 0.2 |
| 2TML12DA2130 | + | 10 | Tunnel | 3.1 ± 0.1 | 0.76 | 10.1 ± 0.4 | 0.24 | 3 | 4.8 ± 0.2 |
| 2TML12DA2FLe | + | − | Membrane | 3.0 ± 0.2 | 0.67 | 10.1 ± 0.4 | 0.33 | 2 | 5.3 ± 0.3 |
| 2TML12DA2130f | − | 10 | Tunnel | 2.9 ± 0.2 | 0.71 | 9.9 ± 0.5 | 0.29 | 5 | 4.9 ± 0.3 |
| 2TML12DA2180f | − | 60 | Solvent | 1.5 ± 0.1 | 0.77 | 8.3 ± 0.2 | 0.23 | 2 | 3.1 ± 0.1 |
| 2TML12DA2130g | − | − | Solvent | 1.7 ± 0.1 | 0.88 | 8.8 ± 0.4 | 0.12 | 1 | 2.6 ± 0.1 |
| 2TML12DA2130h | − | − | Solvent | 1.9 ± 0.1 | 0.98 | 7.4 ± 2.0 | 0.02 | 2 | 2.0 ± 0.1 |
| NBD-labeled species | Membranes | Donor–PTC separationb | NBD location | τ1 | f1c | τ2 | f2c | χ2 | < τ >d |
| nsec | nsec | nsec | |||||||
| 2TML12DA2122 | + | 2 | Tunnel | 2.8 ± 0.1 | 0.70 | 9.1 ± 0.3 | 0.30 | 3 | 4.7 ± 0.2 |
| 2TML12DA2125 | + | 5 | Tunnel | 2.9 ± 0.2 | 0.68 | 9.6 ± 0.5 | 0.32 | 1 | 5.0 ± 0.3 |
| 2TML12DA2126 | + | 6 | Tunnel | 2.5 ± 0.2 | 0.64 | 8.5 ± 0.3 | 0.36 | 3 | 4.7 ± 0.2 |
| 2TML12DA2130 | + | 10 | Tunnel | 3.1 ± 0.1 | 0.76 | 10.1 ± 0.4 | 0.24 | 3 | 4.8 ± 0.2 |
| 2TML12DA2FLe | + | − | Membrane | 3.0 ± 0.2 | 0.67 | 10.1 ± 0.4 | 0.33 | 2 | 5.3 ± 0.3 |
| 2TML12DA2130f | − | 10 | Tunnel | 2.9 ± 0.2 | 0.71 | 9.9 ± 0.5 | 0.29 | 5 | 4.9 ± 0.3 |
| 2TML12DA2180f | − | 60 | Solvent | 1.5 ± 0.1 | 0.77 | 8.3 ± 0.2 | 0.23 | 2 | 3.1 ± 0.1 |
| 2TML12DA2130g | − | − | Solvent | 1.7 ± 0.1 | 0.88 | 8.8 ± 0.4 | 0.12 | 1 | 2.6 ± 0.1 |
| 2TML12DA2130h | − | − | Solvent | 1.9 ± 0.1 | 0.98 | 7.4 ± 2.0 | 0.02 | 2 | 2.0 ± 0.1 |
For each RTC, data from three or more independent experiments were combined and analyzed together as described in Materials and methods.
Donor–PTC separation = nascent chain residues between the PTC and the εNBD-Lys at residue 120 in the nascent chain; in these experiments, no acceptor dye was present.
Molar fraction
Average lifetime calculated from molar fractions.
Full-length 2TML12K2 proteins were translated, released from the translocon, and integrated into the ER membrane; note that the donor dye will be located in the membrane–lumen interface, not in the lipid core of the bilayer.
RNC sample prepared without microsomes or SRP.
RNC sample treated with puromycin, EDTA, and RNase to release the nascent chain from the ribosome into the solvent.
RNC sample treated with puromycin, EDTA, and RNase to release the nascent chain from the ribosome; proteinase K was added to digest the nascent chain.