Two-step folding of recombinant mitochondrial porin in detergent

¹ University of Manitoba

^* To whom correspondence should be addressed. E-mail: dcourt{at}cc.umanitoba.ca.

Submitted on June 14, 2007
Revised on July 6, 2007
Accepted on 4 September 2007

	Abstract

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 (SDS) and dodecyl--D-maltopyranoside (DDM) in a 1:6 molar ratio supports a -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 SDS/DDM 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.

Key Words: UV absorption, VDAC, circular dichroism, detergent, fluorescence, mitochondrial porin