The influence of sterols on the conformation of recombinant mitochondrial porin in detergent

Bay, Denice C.; O'Neil, Joe D.; Court, Deborah A.
December 2008
Biochemistry & Cell Biology;Dec2008, Vol. 86 Issue 6, p539
Academic Journal
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. Les porines mitochondriales (VDAC) sont des acteurs clés du métabolisme cellulaire. Lorsque isolées à partir des mitochondries, les porines sont purifiées avec les stérols et quelques formes isolées de protéines nécessitent la présence de stérols afin d’être insérées dans des membranes artificielles. Néanmoins, la contribution des stérols à l’état replié des porines mitochondriales n’est pas comprise. Récemment, dans le cadre d’études structurales à haute résolution, plusieurs laboratoires ont développé des méthodes pour replier des porines recombinantes à haute concentration dans un détergent. Dans la présente étude, une porine recombinante de Neurospora crassa solubilisée dans des mélanges de stérols et de détergents a été examinée. Les stérols ne modifient pas de façon significative la structure secondaire de la porine dans l’oxyde de lauryl diméthylamine, ni dans un mélange de sodium dodécylsulfate et de dodécylmaltopyranoside. Cependant, tel que détecté par spectropolarimétrie en dichroïsme circulaire dans le proche UV et par spectroscopie à fluorescence, l’environnement des acides aminés aromatiques de la protéine solubilisée dans un mélange stérols-détergents est différent de manière mesurable à celui des protéines solubilisées dans un détergent seul. De plus, les effets sont différents en présence d’ergostérol, un stérol naturel des mitochondries fongiques, et de cholestérol. Alors que ces influences sur l’arrangement tertiaire des porines solubilisées dans un détergent sont subtiles, elles peuvent contribuer à l’obtention d’une protéine capable d’être insérée dans une bicouche artificielle pour des analyses électrophysiologiques et devraient être prises en compte lors d’études structurales futures des porines.


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