On the Interpretation of Force-Induced Unfolding Studies of Membrane Proteins Using Fast Simulations

Proc. Natl. Acad. Sci. U.S.A.

Bridges

et al. 2021

Self Cite

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.

Section: Resultssupporting

confidence: 86%

ln(frequency).…”

Section: Resultsmentioning

confidence: 99%

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

Gaffney

Proc. Natl. Acad. Sci. U.S.A.

Bridges

et al. 2021

Self Cite

“…The energy function used here, FF1.5, has been improved over the original function FF1.0 through the use of an updated contrastive divergence training procedure that includes more extensive sampling (SI text). We added a new membrane burial potential that dynamically accounts for the level of side-chain exposure to lipids and includes unfavorable energies for unsatisfied H-bond donors and acceptors in the membrane to allow helices to fold and unfold within the bilayer (59,60). Side-chain burial energies are determined from the statistics of a training set of proteins and accounts for the burial depth.…”

Section: Simulations Of Dses Of Glpgmentioning

confidence: 99%

“…Our membrane-burial potential includes knowledge-based, depth-dependent energies for side-chain burial and backbone hydrogen bonding within the bilayer. Helices are allowed to unfold and refold in the bilayer during the simulations (59,60). At every MD step, side-chain burial is recalculated to account for the exchange of protein-lipid interactions for protein-protein interactions as helices come into contact with each other.…”

Section: Dse Simulationsmentioning

confidence: 99%

Lipid Bilayer Induces Contraction of the Denatured State Ensemble of a Helical-Bundle Membrane Protein

Ka¹,

Bridges

et al. 2021

Preprint

Self Cite

Defining the denatured state ensemble (DSE) and intrinsically disordered proteins is essential to understanding protein folding, chaperone action, degradation, translocation and cell signaling. While a majority of studies have focused on water-soluble proteins, the DSE of membrane proteins is much less characterized. Here, we reconstituted the DSE of a helical bundle membrane protein GlpG of Escherichia coli in native lipid bilayers and measured its conformation and compactness. The DSE was obtained using steric trapping, which couples spontaneous denaturation of a doubly biotinylated GlpG to binding of two bulky monovalent streptavidin molecules. Using limited proteolysis and mass spectrometry, we mapped the flexible regions in the DSE. Using our paramagnetic biotin derivative and double electron-electron resonance spectroscopy, we determined the dimensions of the DSE. Finally, we employed our Upside model for molecular dynamics simulations to generate the DSE including the collapsed and fully expanded states in a bilayer. We find that the DSE is highly dynamic involving the topology changes of transmembrane segments and their unfolding. The DSE is expanded relative to the native state, but only to 55-90% of the fully expanded condition. The degree of expansion depends on the chemical potential with regards to local packing and the lipid composition. Our result suggests that the native lipid bilayer promotes the association of helices in the DSE of membrane proteins and, probably in general, facilitating interhelical interactions. This tendency may be the outcome of a general lipophobic effect of proteins within the cell membranes.

“…Extensive enzyme kinetic and structural studies have been carried out to elucidate the proteolytic mechanisms (27)(28)(29)(30)(31)(32)(33)(34)(35)(36). Recently, GlpG has emerged as an important model for studying membrane protein folding (37)(38)(39)(40)(41)(42).…”

Section: Significancementioning

confidence: 99%

Structural cavities are critical to balancing stability and activity of a membrane-integral enzyme

Proc. Natl. Acad. Sci. U.S.A.

Cang

Yao

et al. 2020

Packing interaction is a critical driving force in the folding of helical membrane proteins. Despite the importance, packing defects (i.e., cavities including voids, pockets, and pores) are prevalent in membrane-integral enzymes, channels, transporters, and receptors, playing essential roles in function. Then, a question arises regarding how the two competing requirements, packing for stability vs. cavities for function, are reconciled in membrane protein structures. Here, using the intramembrane protease GlpG of Escherichiacoli as a model and cavity-filling mutation as a probe, we tested the impacts of native cavities on the thermodynamic stability and function of a membrane protein. We find several stabilizing mutations which induce substantial activity reduction without distorting the active site. Notably, these mutations are all mapped onto the regions of conformational flexibility and functional importance, indicating that the cavities facilitate functional movement of GlpG while compromising the stability. Experiment and molecular dynamics simulation suggest that the stabilization is induced by the coupling between enhanced protein packing and weakly unfavorable lipid desolvation, or solely by favorable lipid solvation on the cavities. Our result suggests that, stabilized by the relatively weak interactions with lipids, cavities are accommodated in membrane proteins without severe energetic cost, which, in turn, serve as a platform to fine-tune the balance between stability and flexibility for optimal activity.