Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Biochemistry

Supervisor

Stanley D. Dunn

Abstract

Stomatin-like protein 2 (SLP-2), a member of the SPFH superfamily, is a mitochondrial inner membrane protein required for optimal mitochondrial respiration. SLP-2 binds to the important mitochondrial phospholipid cardiolipin (CL) and has been proposed to mediate formation of CL-enriched microdomains that would foster respiratory chain supercomplex (RCS) formation and stability. However, little is known about how SLP-2 structure facilitates its cellular function. The goal of this thesis was to elucidate if and how SLP-2 oligomerizes and by what means does it bind CL.

Biophysical analysis of the expressed SLP-2 SPFH domain, either with or without flanking residues, indicates it to be monomeric and unlikely to mediate oligomer formation. These findings disagree with crystallographic studies of the mouse and bacterial stomatin SPFH domains which suggest that the domain forms a dimer and trimer, respectively. Additionally, lipid binding assays indicate that the SLP-2 SPFH domain does not recognize CL.

Using the biophysical techniques of Variable Field Sedimentation Velocity analytical ultracentrifugation and Size Exclusion Chromatography-Multi-Angle Light Scattering it is demonstrated that bacterially expressed SLP-2 forms variable sized complexes in the multi-MDa size range, similar to that observed for SLP-2 extracted from mitochondrial membranes. Circular dichroism analysis shows a predominantly α-helical protein likely to form concentration-dependent coiled-coil interactions, as determined by the [θ]222/[θ]208 ratio. Bioinformatic analysis revealed an 11-residue hendecad repeat pattern with characteristic small hydrophobic residues in the a, f, h, and j positions indicative of a right-handed coiled-coil, in the region C-terminal to the SPFH domain. To test the significance of the small size of residues at these positions, alanine residues at positions 251, 253, 255, and 257 were mutated to isoleucine. The mutant showed lower helicity than the wild-type and oligomerization was significantly diminished though not abolished. The results imply that SLP-2 oligomerization is mediated through right handed coil-coil interactions and occurs independently of CL binding.

Finally, the mechanism of SLP-2 CL binding was examined by identifying 10 candidate basic residues that are conserved in SLP-2 but not other stomatin family proteins. Mutating these 10 residues to glutamine abolished CL binding but did not impact protein secondary structure or oligomerization.

Available for download on Wednesday, April 01, 2020

Share

COinS