Fig. 2: Comparative SEC, enzymatic activities, HDX MS profiles, and membrane interaction analyses of wild-type VLCAD protein and two proline-mutant variants observed in human VLCAD deficiency.
From: Structural basis for defective membrane targeting of mutant enzyme in human VLCAD deficiency
a SDS-PAGE and Coomassie stain of expressed and purified full-length VLCAD protein and its A450P and L462P mutants. The experiment was repeated twice using independent preparations of VLCAD proteins with similar results. b SEC elution profiles of wild-type, A450P, and L462P VLCAD proteins. c Comparative enzymatic activities of wild-type, A450P, and L462P VLCAD proteins, as assessed using a series of long-chain substrates and ferrocenium hexafluorophosphate as the electron acceptor. Data are mean ± s.e.m. for experiments performed in technical quadruplicate and repeated twice with independent preparations of assay reagents with similar results. d, e Deuterium difference plots showing the relative deuterium incorporation in solution of VLCAD A450P (d) and L462P (e) minus the relative deuterium incorporation of wild-type VLCAD, as measured after 10 s, 1 m, 10 m, and 1 h of deuteration. Regions of deprotection and protection above 0.5 Da (dotted line) are considered meaningful. HDX MS experiments were performed twice with independent preparations of VLCAD proteins. See Supplementary Data 1 for the HDX MS data used to create this figure. f Comparative liposomal translocation of wild-type, A450P, and L462P VLCAD proteins, as monitored by VLCAD western analysis of SEC fractions. The experiment was repeated twice with independent preparations of VLCAD proteins with similar results. g Quantitation of VLCAD observed in liposomal translocation assay fractions (f) by densitometry using ImageJ software.
