Table IV.

NBD fluorescence lifetimes in various environments^a

NBD-labeled speciesb	Membranes	Probe-PTC separationc	NBD location	τ1	f1d	τ2	f2d	χ2	<τ>e
				ns		ns			ns
εNBD-Lysf	−	−	Solvent	1.2 ± 0.2	0.91	2.8 ± 0.2	0.09	1	1.3 ± 0.2
2TML12K2126g	−	−	Solvent	2.0 ± 0.1	0.78	8.8 ± 0.3	0.22	3	3.5 ± 0.2
2TML12K2126	−	20	Tunnel	2.2 ± 0.3	0.52	8.6 ± 0.2	0.48	3	5.3 ± 0.3
2TML12K2126	+	20	Tunnel	1.9 ± 0.3	0.49	8.6 ± 0.2	0.51	6	5.3 ± 0.3
2TML12K2281h	+	−	Bilayer	2.8 ± 0.5	0.35	10.0 ± 0.3	0.65	1	7.5 ± 0.4

^aFor each RTC, data from three or more independent experiments were combined and analyzed together as described in Materials and methods.

^bNBD is located at residue 108 of 2TM_L12K2 in these experiments.

^cProbe–PTC separation, nascent chain residues between nascent chain εNBD-Lys and the PTC.

^dMolar fraction.

^eAverage lifetime based on molar fractions.

^fThis RTC sample was treated with puromycin, EDTA, and RNase to release the nascent chain from the ribosome into the solvent, and then with proteinase K to digest the nascent chains.

^gThis RTC sample was treated with puromycin, EDTA, and RNase to release the nascent chain from the ribosome into the solvent.

^hFull-length 2TM_L12K2 proteins were translated and integrated into the ER membrane. The TMS2 and its NBD probe were located in the nonpolar core of the bilayer after their release from the translocon.