期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2022
卷号：119
期号：1
DOI：10.1073/pnas.2109169119
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Significance
The conformational properties of denatured states of proteins affect their folding, turnover, translocation, and chaperone action. We find that the denatured state ensemble of a helical membrane protein is richer than depicted in the classic two-stage model of membrane protein folding. Some helices undergo topological changes and partial unfolding in the denatured state. In the bilayer, the ensemble is compact relative to the fully expanded state, indicating that the bilayer may not act as a good solvent. This behavior, however, varies depending on the properties of the system, including the local protein sequence, helical and linker lengths, and lipid composition. This study provides insights into the lipid bilayer as a solvent that mediates the assembly and function of membrane proteins.
Defining the denatured state ensemble (DSE) and disordered proteins is essential to understanding folding, chaperone action, degradation, and translocation. As compared with water-soluble proteins, the DSE of membrane proteins is much less characterized. Here, we measure the DSE of the helical membrane protein GlpG of
Escherichia coli (
E. coli) in native-like lipid bilayers. The DSE was obtained using our steric trapping method, which couples denaturation of doubly biotinylated GlpG to binding of two streptavidin molecules. The helices and loops are probed using limited proteolysis and mass spectrometry, while the dimensions are determined using our paramagnetic biotin derivative and double electron–electron resonance spectroscopy. These data, along with our
Upside simulations, identify the DSE as being highly dynamic, involving the topology changes and unfolding of some of the transmembrane (TM) helices. The DSE is expanded relative to the native state but only to 15 to 75% of the fully expanded condition. The degree of expansion depends on the local protein packing and the lipid composition.
E. coli’s lipid bilayer promotes the association of TM helices in the DSE and, probably in general, facilitates interhelical interactions. This tendency may be the outcome of a general lipophobic effect of proteins within the cell membranes.
关键词：endenatured statemembrane protein foldingGlpGsteric trappingUpsidesimulation
