TY - JOUR
T1 - Kinetics of an individual transmembrane helix during bacteriorhodopsin folding
AU - Compton, Emma L. R.
AU - Farmer, Nicola A.
AU - Lorch, Mark
AU - Mason, Jody M.
AU - Moreton, Kathleen M.
AU - Booth, Paula J.
PY - 2006/3/17
Y1 - 2006/3/17
N2 - The kinetics of an individual helix of bacteriorhodopsin have been monitored during folding of the protein into lipid bilayer vesicles. A fluorescence probe was introduced at individual sites throughout helix D of bacteriorhodopsin and the changes in the fluorescence of the label were time-resolved. Partially denatured, labelled bacteriorhodopsin in SDS was folded directly into phosphatidylcholine lipid vesicles. Stopped-flow mixing of the reactants allowed the folding kinetics to be monitored with millisecond time resolution by time-resolving changes in the label fluorescence, intrinsic protein fluorescence as well as in the absorption of the retinal chromophore. Monitoring specific positions on helix D showed that two kinetic phases were altered compared to those determined by monitoring the average protein behaviour. These two phases, of 6.7 s−1 and 0.33 s−1, were previously assigned to formation of a key apoprotein intermediate during bacteriorhodopsin folding. The faster 6.7 s−1 phase was missing when time-resolving fluorescence changes of labels attached to the middle of helix D. The amplitude of the 0.33 s−1 phase increased along the helix, as single labels were attached in turn from the cytoplasmic to the extracellular side. An interpretation of these results is that the 6.7 s−1 phase involves partitioning of helix D within the lipid headgroups of the bilayer vesicle, while the 0.33 s−1 phase could reflect transmembrane insertion of this helix. In addition, a single site on helix G was monitored during folding. The results indicate that, unlike helix D, the insertion of helix G cannot be differentiated from the average protein behaviour. The data show that, while folding of bacteriorhodopsin from SDS into lipids is a co-operative process, it is nevertheless possible to obtain information on specific regions of a membrane protein during folding in vitro.
AB - The kinetics of an individual helix of bacteriorhodopsin have been monitored during folding of the protein into lipid bilayer vesicles. A fluorescence probe was introduced at individual sites throughout helix D of bacteriorhodopsin and the changes in the fluorescence of the label were time-resolved. Partially denatured, labelled bacteriorhodopsin in SDS was folded directly into phosphatidylcholine lipid vesicles. Stopped-flow mixing of the reactants allowed the folding kinetics to be monitored with millisecond time resolution by time-resolving changes in the label fluorescence, intrinsic protein fluorescence as well as in the absorption of the retinal chromophore. Monitoring specific positions on helix D showed that two kinetic phases were altered compared to those determined by monitoring the average protein behaviour. These two phases, of 6.7 s−1 and 0.33 s−1, were previously assigned to formation of a key apoprotein intermediate during bacteriorhodopsin folding. The faster 6.7 s−1 phase was missing when time-resolving fluorescence changes of labels attached to the middle of helix D. The amplitude of the 0.33 s−1 phase increased along the helix, as single labels were attached in turn from the cytoplasmic to the extracellular side. An interpretation of these results is that the 6.7 s−1 phase involves partitioning of helix D within the lipid headgroups of the bilayer vesicle, while the 0.33 s−1 phase could reflect transmembrane insertion of this helix. In addition, a single site on helix G was monitored during folding. The results indicate that, unlike helix D, the insertion of helix G cannot be differentiated from the average protein behaviour. The data show that, while folding of bacteriorhodopsin from SDS into lipids is a co-operative process, it is nevertheless possible to obtain information on specific regions of a membrane protein during folding in vitro.
UR - http://dx.doi.org/10.1016/j.jmb.2005.12.042
U2 - 10.1016/j.jmb.2005.12.042
DO - 10.1016/j.jmb.2005.12.042
M3 - Article
VL - 357
SP - 325
EP - 338
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
SN - 0022-2836
IS - 1
ER -