Lipid bilayer induces contraction of the denatured state ensemble of a helical-bundle membrane protein.

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.

The rhomboid protease GlpG has weak interaction energies in its active site hydrogen bond network.

Intramembrane rhomboid proteases are of particular interest because of their function to hydrolyze a peptide bond of a substrate buried in the membrane. Crystal structures of the bacterial rhomboid protease GlpG have revealed a catalytic dyad (Ser201-His254) and oxyanion hole (His150/Asn154/the backbone amide of Ser201) surrounded by the protein matrix and contacting a narrow water channel. Although multiple crystal structures have been solved, the catalytic mechanism of GlpG is not completely understood. Because it is a serine protease, hydrogen bonding interactions between the active site residues are thought to play a critical role in the catalytic cycle. Here, we dissect the interaction energies among the active site residues His254, Ser201, and Asn154 of Escherichia coli GlpG, which form a hydrogen bonding network. We combine double mutant cycle analysis with stability measurements using steric trapping. In mild detergent, the active site residues are weakly coupled with interaction energies (ΔΔG Inter) of ‒1.4 kcal/mol between His254 and Ser201 and ‒0.2 kcal/mol between Ser201 and Asn154. Further, by analyzing the propagation of single mutations of the active site residues, we find that these residues are important not only for function but also for the folding cooperativity of GlpG. The weak interaction between Ser and His in the catalytic dyad may partly explain the unusually slow proteolysis by GlpG compared with other canonical serine proteases. Our result suggests that the weak hydrogen bonds in the active site are sufficient to carry out the proteolytic function of rhomboid proteases.

Folding-Degradation Relationship of a Membrane Protein Mediated by the Universally Conserved ATP-Dependent Protease FtsH.

ATP-dependent protein degradation mediated by AAA+ proteases is one of the major cellular pathways for protein quality control and regulation of functional networks. While a majority of studies of protein degradation have focused on water-soluble proteins, it is not well understood how membrane proteins with abnormal conformation are selectively degraded. The knowledge gap stems from the lack of an in vitro system in which detailed molecular mechanisms can be studied as well as difficulties in studying membrane protein folding in lipid bilayers. To quantitatively define the folding-degradation relationship of membrane proteins, we reconstituted the degradation using the conserved membrane-integrated AAA+ protease FtsH as a model degradation machine and the stable helical-bundle membrane protein GlpG as a model substrate in the lipid bilayer environment. We demonstrate that FtsH possesses a substantial ability to actively unfold GlpG, and the degradation significantly depends on the stability and hydrophobicity near the degradation marker. We find that FtsH hydrolyzes 380-550 ATP molecules to degrade one copy of GlpG. Remarkably, FtsH overcomes the dual-energetic burden of substrate unfolding and membrane dislocation with the ATP cost comparable to that for water-soluble substrates by robust ClpAP/XP proteases. The physical principles elucidated in this study provide general insights into membrane protein degradation mediated by ATP-dependent proteolytic systems.

Steric trapping reveals a cooperativity network in the intramembrane protease GlpG.

Membrane proteins are assembled through balanced interactions among proteins, lipids and water. Studying their folding while maintaining the native lipid environment is necessary but challenging. Here we present methods for analyzing key elements of membrane protein folding including thermodynamic stability, compactness of the unfolded state and folding cooperativity under native conditions. The methods are based on steric trapping, which couples the unfolding of a doubly biotinylated protein to the binding of monovalent streptavidin (mSA). We further advanced this technology for general application by developing versatile biotin probes possessing spectroscopic reporters that are sensitized by mSA binding or protein unfolding. By applying these methods to the Escherichia coli intramembrane protease GlpG, we elucidated a widely unraveled unfolded state, subglobal unfolding of the region encompassing the active site, and a network of cooperative and localized interactions to maintain stability. These findings provide crucial insights into the folding energy landscape of membrane proteins.