################################################################################ # Introduction This is the raw biochemical data for the paper titled: "The role of membrane destabilisation and protein dynamics in BAM catalysed OMP folding" by White et al. Structural data has been deposited in the relevant repositories and the reader is directed to the Data availability section of the paper for details. ################################################################################ # Useful software ######## # FIJI # ######## Used for quantification of gels by densitometry ################## # Igor Pro V8.04 # ################## Used for plotting and fitting of kinetic assay data (single exponential functions) Used for plotting Generalised polarisation from laurdan data and for calculating and plotting the first derivative of that data. ################# # MassLynx v4.2 # ################# Used for analysing the intact mass spectra obtained for BAM-LL labelled with maleimide-C5-fluorescein or N-ethyl-maleimide ################################################################################ # Organisation Data is divided into folders according to broad categories: ####### # DLS # ####### Dynamic light scattering data obtained for the proteoliposome preparations used in this paper. Data was collected on a Wyatt miniDawnTreos® system (equipped with an additional DLS detector) and has been analysed with the Astra 6.0.3® software to generate plots of hydrodynamic radius (Rh) vs intensity for each sample. All samples were measured 3 times. ############ # Kinetics # ############ Quantification of kinetics assays in which folding of the small, 8-stranded outer membrane proteins (OMPs) tOmpA or OmpX into proteoliposomes is monitored by band shift on SDS-PAGE gels (see paper methods). Proteoliposomes contain different BAM variants, allowing quantification of differences in their function. Raw kinetics are the fraction of folded OMP at each time point in the assay, arranged according to BAM variant used and any added redox agent. This data was plotted and fitted to a single exponential in Igor Pro to obtain the kinetics graphs used in the paper Initial rates_linear5 show calculation of initial rates for folding reactions by linear fit to the first 5% of the time course (also see paper methods) 24 hour folding yields are fraction of folded OMPs taken 24 hours after initiation of folding ########### # Laurdan # ########### Raw data obtained for laurdan membrane destabilisation assay (see paper methods). Fluorescence of Laurdan-doped DMPC liposomes at 440 and 490 nm was measured in a PTI QuantaMaster, following excitation at 340nm. Measurements were taken throughout a temperature ramp from 6 - 40 degrees. Generalised polarisation (GP) was calculated from the ratio of fluorescence intensity at 440 nm and 490 nm, averaged over the 10 second acquisition, using the formula GP = (I440-I490)/(I440+I490). Note that each set of measurements alternates between the samples present i.e S1 then S2 then S3 then S4 is measured, before moving back to measuring sample 1. The BAM variant in the DMPC-proteoliposomes for each sample is indicated at the top of each spreadsheet. #################### # OMP_Folding_Gels # #################### Raw gels from kinetics assays. Densitometry of these gels produced the data in the Kinetics folder. Gels are in folders according to what BAM variant and redox agent were included in the assay (e.g. all gels for assays of BAM-LL are in the BAM-LL folder). Within these folders, gels are further subdivided into tOmpA or OmpX folders according to which substrate was used in the folding assay. In all folders, a labels.txt file is included detailing lane contents for each gel. ########################## # Proteoliposome_QC_gels # ########################## Raw gels from the preparation process for the BAM containing proteoliposomes. A labels.txt file is included detailing lane contents for each gel. ################################# # Proteoliposome_trypsin_digest # ################################# Raw gels from the trypsin digestion of BAM containing proteoliposomes carried out to test the orientation of BAM (outside facing vs inside facing). A labels.txt file is included detailing lane contents for each gel. Proteoliposomes_trypsin_densitometry.xlsx contains the densitometry and calculations for percentage in/out based on these gels. #################################### # thiol_labelling_fluorescence_gel # #################################### thiol_labelling_fluorescence_gel - Raw gels for the thiol labelling gel of the disulphide locked BAM variants (BAM-LL and BAM-P5L). A labels.txt file is included detailing lane contents for each gel. Calculations of % disulphide in each sample are shown in thiol_labelling_quantification.xlsx ###################### # thiol_labelling_MS # ###################### Mass spectra obtained from the thiol labelling experiments with BAM-LL and either maleimide-C5-fluorescein or N-ethyl maleimide. The deconvoluted mass spectra plotted in the paper are shown in the excel spreadsheet, while the .raw files are the raw spectra for viewing in MassLynx. ################################################################################ # Data underlying figures is indicated below: Main text: ############ # Figure 1 # ############ Figure 1c - Kinetics/tOmpA raw kinetics.xlsx Figure 1d - Kinetics/OmpX raw Kinetics.xlsx Figure 1e - Kinetics/Initial rates_linear5_tOmpA.xlsx Figure 1f - Kinetics/Initial rates_linear5_OmpX.xlsx ############ # Figure 4 # ############ Figure 4d - Kinetics/tOmpA raw kinetics.xlsx Figure 4e - Kinetics/OmpX raw kinetics.xlsx Figure 4f - Kinetics/tOmpA raw kinetics.xlsx Figure 4g - Kinetics/OmpX raw kinetics.xlsx ############ # Figure 5 # ############ Figure 5a - Laurdan_raw.xlsx Figure 5b - first derivative of data in Laurdan_raw.xlsx Supplementary Information: ########################## # Supplementary Table 1 # ########################## Kinetics/Initial rates_linear5_tOmpA.xlsx Kinetics/Initial rates_linear5_OmpX.xlsx ########################## # Supplementary Table 1 # ########################## Kinetics/24 hour folding yields.xlsx ########################### # Supplementary Figure 1 # ########################### Supplementary Figure 1a - Proteoliposome_QC_gels/191031_LipoQC_WT-LL.Tif (lanes 2-6) Supplementary Figure 1b - Proteoliposome_QC_gels/BAM FAB proteoliposomes.Tif (lanes 5-7) Supplementary Figure 1c - Proteoliposome_QC_gels/BamA PLE DMPC liposomes normal gel.Tif (lanes 2-5) Supplementary Figure 1d - Proteoliposome_QC_gels/191031_LipoQC_P5L-E.Tif (lanes 2-6) Supplementary Figure 1e - Proteoliposome_QC_gels/191031_LipoQC_WT-LL.Tif (lanes 8-12) ########################### # Supplementary Figure 2 # ########################### DLS/bam_proteoliposomes_all_data_clean.csv ########################### # Supplementary Figure 3 # ########################### Supplementary Figure 3a - Proteoliposome_trypsin_digest/WT P5L LL BamA.tif Supplementary Figure 3b - Proteoliposome_trypsin_digest/WT-Fab1 P5L-Fab1 LL-Fab1.tif Supplementary Figure 3c - Proteoliposomes_trypsin_densitometry.xlsx ########################### # Supplementary Figure 4 # ########################### Supplementary Figure 3b - thiol_labelling_fluorescence_gel/Charlotte BAM Fluorescent PL LL Gel 101219.Tif Supplementary Figure 3c - thiol_labelling_fluorescence_gel/Charlotte Fluorescent Redox Gel Coomassie PL + LL 111219.Tif Supplementary Figure 3d - thiol_labelling_MS/Deconvoluted_Mass_Spectra.xlsx Supplementary Figure 3e - thiol_labelling_MS/Deconvoluted_Mass_Spectra.xlsx ########################### # Supplementary Figure 5 # ########################### Supplementary Figure 5 - tOmpA folders in OMP_Folding_Gels ########################### # Supplementary Figure 6 # ########################### Supplementary Figure 6 - OmpX folders in OMP_Folding_Gels ########################### # Supplementary Figure 7 # ########################### Supplementary Figure 7a - OMP_Folding_Gels/WT BAM + DTT/tOmpA Supplementary Figure 7b - OMP_Folding_Gels/Cys-free BAM/tOmpA Supplementary Figure 7c - OMP_Folding_Gels/Cys-free BAM + DTT/tOmpA Supplementary Figure 7d - Kinetics/tOmpA raw kinetics.xlsx Supplementary Figure 7e - Kinetics/Initial rates_linear5_tOmpA.xlsx ########################### # Supplementary Figure 14 # ########################### Supplementary Figure 14a - Proteoliposome_QC_gels/191213 QC gel LL-Fab.Tif (lanes 8-12) Supplementary Figure 14b - Proteoliposome_QC_gels/P5L-Fab1_lipo_generation_wash.Tif (lanes 1-5) ########################### # Supplementary Figure 15 # ########################### Supplementary Figure 15a - OMP_Folding_Gels/BAM LL + Fab1/tOmpA Supplementary Figure 15b - OMP_Folding_Gels/BAM LL + Fab1/OmpX Supplementary Figure 15c - OMP_Folding_Gels/BAM PL + Fab1/tOmpA Supplementary Figure 15d - OMP_Folding_Gels/BAM PL + Fab1/OmpX ########################### # Supplementary Figure 16 # ########################### Supplementary Figure 16a - Proteoliposome_QC_gels/BAM DMPC liposomes.Tif Supplementary Figure 16b - Proteoliposome_QC_gels/P5L_LL_Fab1_DMPC_liposomes_120820.Tif