Voltage-dependent anion-selective channels (VDAC), or mitochondrial porins,regulate the flow of metabolites across the mitochondrial outer membrane. They presumably span the membrane as β-barrels, but the residues forming the individual β-strands are unknown. This information is essential for understanding the structure and function of the protein. Using Neurospora VDAC as a template, published data were reassessed to delineate a unified model for porin structure Bay and Court 2002, which was subsequently refined in collaboration with Greg Runke Runke et al. 2006. The focus of this work was the development and analysis of systems for maintaining high levels of folded porin for the acquisition of high resolution data needed for model testing. The conformation of hexahistidinyl-tagged Neurospora porin in detergent was probed by fluorescence, near-UV circular dichroism and ultraviolet absorption spectroscopy. Derivatives of tryptophan and tyrosine were also examined by fluorescence spectroscopy and UV absorbance spectroscopy to model the interactions between the detergents and the amino acid side chains in the protein. Detergent-specific levels of β-strand and tyrosine exposure were observed. In all cases, the two tryptophan residues reside in weakly asymmetric, hydrophobic environments, suggesting transient tertiary interactions. Porin solubilized in these detergents forms functional channels in liposomes and membrane insertion is accompanied by increased levels of β-strand and loss of protease sensitivity. These data were used to develop mixed detergent folding systems. A mixture of SDS and dodecyl-β-D-maltopyranoside (DDM)supports a β-strand rich conformation at high protein concentrations. The tertiary contacts and protease resistance of the SDS/DDM solubilized porin are very similar to those of the protein following reconstitution into liposomes. Finally, the role of sterols in porin folding was examined, as the addition of sterols to detergent-solubilized VDAC is required for channel formation in artificial membranes. Sterols do not alter the secondary structure of VDAC, and subtle alterations to tertiary interactions were detected, suggesting that sterols do not promote an insertion-competent structure, but rather facilitate insertion into artificial bilayers. In summary, this analysis of the folded states of detergent-solubilized porin has revealed a system that maintains high concentrations of mitochondrial porin in a state that is very promising for structural studies. / February 2007

Voltage-dependent anion-selective channels (VDAC), or mitochondrial porins,regulate the flow of metabolites across the mitochondrial outer membrane. They presumably span the membrane as β-barrels, but the residues forming the individual β-strands are unknown. This information is essential for understanding the structure and function of the protein. Using Neurospora VDAC as a template, published data were reassessed to delineate a unified model for porin structure Bay and Court 2002, which was subsequently refined in collaboration with Greg Runke Runke et al. 2006. The focus of this work was the development and analysis of systems for maintaining high levels of folded porin for the acquisition of high resolution data needed for model testing. The conformation of hexahistidinyl-tagged Neurospora porin in detergent was probed by fluorescence, near-UV circular dichroism and ultraviolet absorption spectroscopy. Derivatives of tryptophan and tyrosine were also examined by fluorescence spectroscopy and UV absorbance spectroscopy to model the interactions between the detergents and the amino acid side chains in the protein. Detergent-specific levels of β-strand and tyrosine exposure were observed. In all cases, the two tryptophan residues reside in weakly asymmetric, hydrophobic environments, suggesting transient tertiary interactions. Porin solubilized in these detergents forms functional channels in liposomes and membrane insertion is accompanied by increased levels of β-strand and loss of protease sensitivity. These data were used to develop mixed detergent folding systems. A mixture of SDS and dodecyl-β-D-maltopyranoside (DDM)supports a β-strand rich conformation at high protein concentrations. The tertiary contacts and protease resistance of the SDS/DDM solubilized porin are very similar to those of the protein following reconstitution into liposomes. Finally, the role of sterols in porin folding was examined, as the addition of sterols to detergent-solubilized VDAC is required for channel formation in artificial membranes. Sterols do not alter the secondary structure of VDAC, and subtle alterations to tertiary interactions were detected, suggesting that sterols do not promote an insertion-competent structure, but rather facilitate insertion into artificial bilayers. In summary, this analysis of the folded states of detergent-solubilized porin has revealed a system that maintains high concentrations of mitochondrial porin in a state that is very promising for structural studies.