Deletion Variants of Neurospora Mitochondrial Porin: Electrophysiological and Spectroscopic Analysis

Mitochondrial porins are predicted to traverse the outer membrane as a series of β-strands, but the precise structure of the resulting β-barrel has remained elusive. Toward determining the positions of the membrane-spanning segments, a series of small deletions was introduced into several of the predicted β-strands of the Neurospora crassa porin. Overall, three classes of porin variants were identified: i), those producing large, stable pores, indicating deletions likely outside of β-strands; ii), those with minimal pore-forming ability, indicating disruptions in key β-strands or β-turns; and iii), those that formed small unstable pores with a variety of gating and ion-selectivity properties. The latter class presumably results from a subset of proteins that adopt an alternative barrel structure upon the loss of stabilizing residues. Some variants were not sufficiently stable in detergent for structural analysis; circular dichroism spectropolarimetry of those that were did not reveal significant differences in the overall structural composition among the detergent-solubilized porin variants and the wild-type protein. Several of the variants displayed altered tryptophan fluorescence profiles, indicative of differing microenvironments surrounding these residues. Based on these results, modifications to the existing models for porin structure are proposed.