The influence of sterols on the conformation of recombinant mitochondrial porin in detergent.
Mitochondrial porins (voltage-dependent anion-selective channels, VDAC) are key contributors to cellular metabolism. When isolated from mitochondria porins copurify with sterols, and some isolated forms of the protein require sterol for insertion into artificial membranes. Nonetheless, the contributions of sterols to the folded state of mitochondrial porin are not understood. Recently, with the goal of high-resolution structural studies, several laboratories have developed methods for folding recombinant porins at high concentration in detergent. In the present study, recombinant Neurospora crassa porin solubilized in detergent-sterol mixtures was examined. Sterols do not significantly alter the secondary structure of porin in lauryl dimethylamine oxide, nor in a mixture of sodium dodecylsulfate and dodecylmaltopyranoside. However, as detected by near-UV circular dichroism spectropolarimetry and fluorescence spectroscopy, the environments surrounding the aromatic amino acids in the detergent-sterol solubilized protein are measurably different from those in detergent alone. Furthermore, the effects are different in the presence of ergosterol, the native sterol in fungal mitochondria, and cholesterol. While these influences on the tertiary arrangement of detergent-solubilized porin are subtle, they may contribute to the generation of a form of the protein competent for insertion into the artificial bilayers used for electrophysiological analyses, and should be considered in future structural studies of porin.
Bay
Denice C
DC
Department of Microbiology, University of Manitoba, Winnipeg, MB R3T 2N2, Canada.
O'Neil
Joe D
JD
Court
Deborah A
DA
D003902:Detergents / Q000737:chemistry*; D015394:Molecular Structure; D009492:Neurospora crassa / Q000737:chemistry; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D013261:Sterols / Q000737:chemistry / Q000378:metabolism*; D050994:Voltage-Dependent Anion Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D003902:Detergents; D011994:Recombinant Proteins; D013261:Sterols; D050994:Voltage-Dependent Anion Channels