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Proc Natl Acad Sci U S A
Proc Natl Acad Sci U S A
PNAS
Proceedings of the National Academy of Sciences of the United States of America
0027-8424
1091-6490
National Academy of Sciences

202415554
10.1073/pnas.2415554121
correctCorrection408
438
Correction
Correction to Gaffney et al., Lipid bilayer induces contraction of the denatured state ensemble of a helical-bundle membrane protein
10 9 2024
17 9 2024
10 9 2024
121 38 e2415554121Copyright © 2024 the Author(s). Published by PNAS.
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).

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pmcBiophysics and Computational Biology Correction to “Lipid bilayer induces contraction of the denatured state ensemble of a helical-bundle membrane protein,” by Kristen A. Gaffney, Ruiqiong Guo, Michael D. Bridges, Shaima Muhammednazaar, Daoyang Chen, Miyeon Kim, Zhongyu Yang, Anthony L. Schilmiller, Nabil F. Faruk, Xiangda Peng, A. Daniel Jones, Kelly H. Kim, Liangliang Sun, Wayne L. Hubbell, Tobin R. Sosnick, and Heedeok Hong, which published December 28, 2021; 10.1073/pnas.2109169119 (Proc. Natl. Acad. Sci. U.S.A. 119, e2109169119).

The authors identified an error in converting simulation to real time that led to an overestimation of the total trajectory length. As a result, the maximum value of the time axis in SI Appendix, Fig. S13 A–F should be 40 μs rather than 4 ms, and for SI Appendix, Fig. S13, the legend should read “Data shown do not include a 4 μs equilibration period.” Also, on page 6 of the article, the last paragraph should read “A total of twenty 40 μs simulation were run” while on page 8 of the SI Appendix (lines 29–30), the legend should read “Using a value of 0.36 ps per Verlet step, each Upside time unit is 40 ps. In the simulations, every 50th conformation is stored giving a final output spacing of 2 ns/frame.” While these corrections do not affect the study’s conclusions or comparisons with experimental data, they are important for those trying to replicate the data or infer the timescale of dynamics.

Finally, the authors note that Fig. 2B in the article lacked annotations for a liposome flotation assay showing native and denatured GlpG incorporation into liposomes. Each subfigure now specifies the tested protein – the annotation “N” (native GlpG) has been added to each subfigure in the top row, and the annotation (“D·mSA_2” (denatured GlpG) has been added to each subfigure in the bottom row.

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).

The corrected figure and its legend appear below.

The online version and SI Appendix have been corrected online.
