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  • br Acknowledgements br Data Hexahistidine tag His binding

    2018-10-25


    Acknowledgements
    Data Hexahistidine-tag (His6) binding to Nickel (Ni) chelated with nitrilotriacetic SCR7 (NTA) is a well-characterized process [2,3] and it is extensively used to reconstitute protein systems in giant unilamellar vesicles (GUVs) [4–6]. We made GUVs consisting of 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholin (DOPC) and 2, 3, 4 or 5mol% 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] nickel salt (DGS-NTA(Ni)), labeled with 0.05mol% ATTO-647N-DOPE. These GUVs were incubated with increasing amounts of His6-tagged enhanced green fluorescent protein (eGFP-His6) and point fluorescence correlation spectroscopy (FCS) was performed both at the top pole of the GUVs and in solution. From the obtained FCS auto-correlation functions the diffusion coefficient of both eGFP-His6 and ATTO-647N-DOPE as well as the dissociation constant of the NTA(Ni)/eGFP-His6 system were calculated.
    Experimental design, materials and methods The materials, the preparation of eGFP-His6 and GUVs, the optical setup used and the FCS data acquisition/analysis were described elsewhere [1].
    Experimental design, materials and methods
    Acknowledgments This research was funded by the NSF (1149387) (CP). The authors would like to thank Jourdan Howard, Alex Kula, Paul O’Malley, Matthew Putonti, Amy Rosenfeld, Daniel Searle, and Nick White for their assistance in collecting and processing samples.
    Value of the data
    Data H. pylori broth culture supernatants, soluble cellular fractions, and membrane fractions were analyzed by one dimensional LC-MS/MS (1D) or multidimensional protein identification technology (MudPIT). The numbers of assigned spectra were analyzed to calculate the proportional abundance of individual proteins in samples, the enrichment of proteins in the supernatant compared to soluble bacterial fraction, and the distribution of proteins between soluble and membrane fractions (membrane localization). Supplemental Table S1 shows all assigned spectra detected by single-dimensional LC-MS/MS analysis of supernatant and cellular fractions from five time points, collected in three independent experiments. Supplemental Table S2 shows an analysis of merged single-dimensional LC-MS/MS data from SCR7 Supplemental Table S1 to calculate enrichment of individual proteins in the supernatant compared to the soluble cellular fraction. Supplemental Table S3 shows an analysis of merged single-dimensional LC-MS/MS data from Supplemental Table S1 for 74 putative secreted proteins (identified as described in [1]). The table shows a calculation of the enrichment of individual proteins in the culture supernatant, as well as membrane localization calculations. Supplemental Table S4 shows all assigned spectra detected by MudPIT analysis in supernatant and cellular fractions from two time points, along with an analysis of the enrichment of individual proteins in the supernatant compared to the soluble cellular fraction. Supplemental Table S5 shows an analysis of the MudPIT data in Supplemental Table S4 for 33 putative secreted proteins (identified as described in [1]). The table shows the enrichment of individual proteins in the supernatant compared to the soluble cellular fraction, as well as membrane localization calculations at two time points.
    Experimental design, materials and methods
    Acknowledgments Supported by NIHAI039657, CA116087, and the Department of Veterans Merit Review Grant 2I01BX000627. Proteomics experiments were supported by the Vanderbilt Digestive Diseases Research Center (P30DK058404) and Vanderbilt-Ingram Cancer Center (P30 CA068485).
    Data
    Experimental design, materials and methods
    Acknowledgment This work was supported by Cancer Prevention and Research Institute of Texas (R1207) (K.D.W.), Welch Foundation (I1829) (K.D.W), V Foundation (K.D.W), and Pancreatic Cancer Action Network (J.C.H). Data shown in this report are was collected at Argonne National Laboratory, Structural Biology Center at the Advanced Photon Source. Argonne is operated by U Chicago Argonne, LLC, for the U.S. Department of Energy, Office of Biological and Environmental Research under Contract DEAC02-06CH11357.