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. 2022 Jan 4;119(1):e2109169119.
doi: 10.1073/pnas.2109169119.

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

Kristen A Gaffney  1 , Ruiqiong Guo  2 , Michael D Bridges  3   4 , Shaima Muhammednazaar  2 , Daoyang Chen  2 , Miyeon Kim  2 , Zhongyu Yang  5 , Anthony L Schilmiller  6 , Nabil F Faruk  7 , Xiangda Peng  8   9 , A Daniel Jones  1   6 , Kelly H Kim  1 , Liangliang Sun  2 , Wayne L Hubbell  3   4 , Tobin R Sosnick  10   9 , Heedeok Hong  11   2
Affiliations

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

Kristen A Gaffney et al. Proc Natl Acad Sci U S A. .

Erratum in

Abstract

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.

Keywords: GlpG; Upside simulation; denatured state; membrane protein folding; steric trapping.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
Steric trapping strategy to reconstitute denatured GlpG in the lipid bilayers. (A, Left) Doubly biotinylated GlpG was first denatured using steric trapping by the addition of excess mSA in micelles. Sterically denatured GlpG was transferred to bicelles or liposomes. (Center) Cryo-EM images of the bicelles and liposomes. In the top bicelle panel (with GlpG and mSA), the particle types were assigned as follows. White arrowheads (circular and ellipsoidal objects): flat or tilted bicellar disk planes; black arrowheads (the dark rod-shaped objects): bicellar rims; blue arrowheads (the dark small particles with diameters of 40 to 50 Å): mSA molecules. The bottom liposome panel (without the proteins) largely contains unilamellar liposomes with a minor fraction of multilamellar liposomes. (Right) The histograms for the diameter size distributions of the bicelles and liposomes. (B) Double cysteine variants employed for the steric trapping of the denatured state of GlpG. In each variant, designated cysteine residues were labeled with a thiol-reactive biotin derivative with a spectroscopic reporter group as shown in C. The regions of the backbone colored in cyan and orange indicate the N- and C-subdomains, respectively. (C) Thiol-reactive biotin derivatives with a paramagnetic spin label (Left, BtnRG‒TP) and fluorescent pyrene (Right, BtnPyr‒IA). Reproduced with permission from ref. . TP: thiopyridine; IA: iodoacetamide.
Fig. 2.
Fig. 2.
Reconstitution of denatured GlpG in the lipid bilayers. (A, Top) Schematic description of fluorescence-quenching assay to measure incorporation of native and sterically denatured GlpG into bicelles. GlpG was doubly labeled with fluorescent BtnPyr, while the bicelles contained quencher-labeled lipids, DOPE‒dabcyl. Native (“N”) and denatured (“D⋅mSA2”) GlpG solubilized in micelles were injected into bicellar solution. Negative control (“Unbound”): water-soluble pyrene-labeled mSA was injected into the bicellar solution. Positive control (“Bound”): native GlpG was first reconstituted in DMPC:DMPG:DOPE‒dabcyl liposomes and then solubilized by CHAPS to form bicelles. (Bottom) Assay results for the three doubly biotinylated GlpG variants. Error bars denote ± SEM (n = 3). Based on Student’s t test, significance of the difference between a data pair is marked with single (P < 0.05), double (P < 0.01), and triple asterisks (P < 0.001) or “NS” (not significant, P > 0.05). (B) Liposome-flotation assay in a sucrose gradient indicates a near-complete membrane association of native and denatured GlpG doubly labeled with BtnPyr. Liposomes contained fluorescent DPPE‒rhodamine. (C) Proteolytic activity of GlpG as a measure of denaturation and refolding efficiency. GlpG doubly labeled with BtnRG was first denatured upon addition of excess mSA in DDM and then transferred to bicelles or liposomes. The addition of DTT, which breaks the disulfide linkage between GlpG and the biotin label bound with mSA, enables refolding (“+DTT”). The activity was normalized relative to that of native GlpG without mSA in each hydrophobic phase. Error bars denote ± SEM (n = 3). Significance of the difference between a data pair is marked as in A. (D, first row) The schematic description of limited proteolysis of native (N) and sterically denatured GlpG (D⋅mSA2) by ProK. The time-dependent proteolysis in DDM micelles (second row), bicelles (third row), and liposomes (fourth row) was measured by SDS-PAGE. After quenching of proteolysis at each time point, DTT was added to release bound mSA from GlpG. The extent of GlpG proteolysis was quantified from the GlpG band intensities with and without ProK (asterisks). The proteolytic peptide fragment larger than 10 kDa is marked with a symbol (open circles, 17 kDa; open squares, 13 kDa; open triangles, 11 kDa) on the right side of each band. For mass spectrometry (see Fig. 3), the samples were further solubilized with DDM and the cysteine residues were alkylated with iodoacetamide (IA).
Fig. 3.
Fig. 3.
Mapping the flexible regions in native and denatured GlpG using limited proteolysis by ProK and mass spectrometry. (A, Left) The proteolysis products of native and denatured GlpG (the double biotin variant 172M267C) in liposomes (Top) and micelles (Bottom) were analyzed using CZE- (cyan vertical bars) and RPLC- (red vertical bars) MS/MS. The peptide identification number (peptide ID, lower x-axes) denotes a unique fragment identified under each designated condition in the ascending order of the N-terminal residue number of the fragment. Each vertical bar spans the range from the N- to the carboxyl-terminal residue number of each fragment and is mapped onto the primary and secondary structures (y-axes). The traces in green represent both the accuracy and frequency (the cumulative score, upper x-axes) of each cleavage site (y-axes) (SI Appendix, Supplementary Text). (Right) The cumulative score of each site was mapped onto the tertiary structure and color coded according to the heat map. The catalytic dyad (Ser201–His254) is shown in spheres in each structure. (B) Hydropathy plot of the TM domain of GlpG using the Wimley-White (WW) water–octanol (88), Hessa-von Heijne (HvH) translocon–membrane (17), Tian-Lin-Liang (TLL) membrane depth-dependent (44, 89), and Moon-Fleming (MF) water–membrane (74) hydrophobicity scales. For the WW and HvH scales, a 19-residue window was slid along the sequence of GlpG summing ΔGtransfer of each residue. For the TLL and MF scales, the ΔGtransfer values only for the residues in each TM helix from the structure were summed. (C) Possible modes of the membrane-topology dynamics and unfolding in the DSE of GlpG in E. coli liposomes. The models were based on limited proteolysis, MS/MS, MD simulations, and the charge distributions in the membrane-water interfacial regions of GlpG.
Fig. 4.
Fig. 4.
The physical dimension of the denatured states of GlpG measured by DEER. (A) Time-dependent dipolar evolution data for native (N, gray) and sterically denatured GlpG (D·mSA2, black) were fit using the model-free, nonnegative Tikhonov regularization algorithm (red). The data for 95N172M and 172M267C in micelles were adapted from ref. . (B) Corresponding distance distributions between the spin labels at the designated residues. For each, the mean distances (rMean) are shown as vertical solid lines for the native (gray) and denatured (black) states. The error bar at each distance corresponds to ± SD of the fitted probability from the model-free analysis. The “DEER limit” (dashed black line) denotes the maximal nominal interspin distance detectible by DEER.
Fig. 5.
Fig. 5.
Upside MD simulation under the statistical membrane-burial potential. (A) Modeling of GlpG doubly bound with mSA. Each mSA molecule was attached to the designated residue on GlpG via a stiff 4-Å virtual spring (spring constant = 30 kcal⋅mol−1⋅Å−1). (B) The Cα-RMSD and radius of gyration (Rg) of GlpG from temperature-dependent simulations. Each value is the average of 20 independent simulations. Each error bar denotes ± SD. The horizontal guidelines (also in C) indicate the average values for the simulation runs under the fully expanded protocol. At T > 343 K, a more diverse set of molecular configurations is observed, such as the complete unfolding of individual TM helices and the formation of β-strands within the membrane (SI Appendix, Fig. S13 D and E). (C) The Cα–Cα distances between each designated residue pair were monitored at an increasing temperature. When the distances were monitored between a specific residue pair (“Distance between”), mSA molecules are bound to the same or either of the other two residue pairs (“mSA bound at”). (D) Distribution of the Cα–Cα distances between the designated residue pair with or without bound mSA. The simulations were carried out with all energy terms (“native-like274K,” “collapsed308K,” and “partially expanded DSE343K”) or missing attractive side-chain interactions (“fully expanded DSEOFF”). A representative structural snapshot under each simulation condition is shown as an inset.
Fig. 6.
Fig. 6.
Comparison of the dimensions of the DSEs measured by DEER and Upside simulations. (A) Comparison of the distance distributions obtained from DEER and the simulated DSEs doubly bound with mSA. To mimic the interspin distances measured from DEER, the distances from the simulations were monitored between the Asn49 Cα’s on bound mSA molecules, which were the attachment sites to GlpG. The rMean values for the simulated “collpased308K” (green), the experimentally obtained “DSE (DEER)” (black), and the simulated “fully expanded DSEOFF” (red) are shown as vertical lines. (B) Expansion ratio (RExpansion, Eq. 1) of the DSE measured between each residue pair in the micelles, bicelles, and liposomes. The RExpansion values of the simulated DSEs at an increasing temperature are overlaid.
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References

    1. Dill K. A., Shortle D., Denatured states of proteins. Annu. Rev. Biochem. 60, 795–825 (1991). - PubMed
    1. Matouschek A., Protein unfolding–An important process in vivo? Curr. Opin. Struct. Biol. 13, 98–109 (2003). - PubMed
    1. Saibil H., Chaperone machines for protein folding, unfolding and disaggregation. Nat. Rev. Mol. Cell Biol. 14, 630–642 (2013). - PMC - PubMed
    1. Wright P. E., Dyson H. J., Intrinsically disordered proteins in cellular signalling and regulation. Nat. Rev. Mol. Cell Biol. 16, 18–29 (2015). - PMC - PubMed
    1. Sosnick T. R., Barrick D., The folding of single domain proteins–Have we reached a consensus? Curr. Opin. Struct. Biol. 21, 12–24 (2011). - PMC - PubMed

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