Two-step folding of recombinant mitochondrial porin in detergent.
Precise information regarding the transmembrane topology of mitochondrial porin is essential for understanding the mechanisms by which this protein functions. Porin acts as a channel in the outer membrane and interacts with small solutes and proteins to regulate mitochondrial function. The acquisition of high-resolution structural data requires a method of maintaining high concentrations of unaggregated, properly folded porin. In the current studies, several mixed detergent systems were analyzed for their ability to fold Neurospora mitochondrial porin expressed in and isolated from Escherichia coli. A mixture of sodium dodecyl sulfate and dodecyl-beta-D-maltopyranoside in a 1:6 molar ratio supports a beta-strand-rich conformation. In this state, the two tryptophan residues in the protein reside in hydrophobic environments, and about half of the nine tyrosines are solvent exposed. Most importantly, heat-labile tertiary contacts, as detected by near-UV circular dichroism spectropolarimetry, in the sodium dodecyl sulfate/dodecyl-beta-D-maltopyranoside-solubilized porin are very similar to those of the protein following functional reconstitution into liposomes. Similarly, both forms are protease resistant. Thus, a method has been identified with the potential to solubilize high concentrations of mitochondrial porin in a state virtually indistinguishable from the membrane-embedded form.
Bay
Denice C
DC
Department of Microbiology, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada.
O'Neil
Joe D
JD
Court
Deborah A
DA
D002942:Circular Dichroism; D003902:Detergents / Q000378:metabolism* / Q000494:pharmacology; D005453:Fluorescence; D006639:Histidine; D008081:Liposomes / Q000378:metabolism; D008933:Mitochondrial Swelling / Q000187:drug effects; D009492:Neurospora crassa / Q000378:metabolism*; D009842:Oligopeptides; D010447:Peptide Hydrolases / Q000378:metabolism; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism*; D012995:Solubility / Q000187:drug effects; D013056:Spectrophotometry, Ultraviolet; D014364:Tryptophan; D014443:Tyrosine; D050994:Voltage-Dependent Anion Channels / Q000737:chemistry* / Q000378:metabolism*
D003902:Detergents; C471213:His-His-His-His-His-His; D008081:Liposomes; D009842:Oligopeptides; D011994:Recombinant Proteins; D050994:Voltage-Dependent Anion Channels; D014443:Tyrosine; D006639:Histidine; D014364:Tryptophan; D010447:Peptide Hydrolases