Year of Award

2018

Document Type

Dissertation

Degree Type

Doctor of Philosophy (PhD)

Degree Name

Biochemistry & Biophysics

Department or School/College

Department of Chemistry and Biochemistry

Committee Chair

Bruce Bowler

Commitee Members

J. B. Alexander (Sandy) Ross, Stephen R. Sprang, Mike DeGrandpre, Travis Hughes

Keywords

Cardiolipin, Cytochrome c, lipid partitioning, lipid-protein binding, lipid quantification

Abstract

Recently, the electron transport protein Cytochrome c (Cytc) was shown to interact with the mitochondrial lipid cardiolipin (CL) during the initial stages of the intrinsic apoptotic pathway. This interaction is characterized by the protein binding and oxidation of cardiolipin on the concave surface of the outer leaflet of the inner mitochondrial membrane. To date, this interaction has been studied with a variety of methods and techniques reporting varied and often conflicting findings characterizing the nature and extent of the protein-lipid interaction. Cytc has been hypothesized to interact with cardiolipin electrostatically, hydrophobically and through hydrogen bonding and is thought to partially to completely unfold and imbed in the membrane during the interaction or remain folded on the membrane surface. This thesis aims to (1) quantitatively assess the electrostatic protein-lipid interaction, (2) characterize the cationic amino acid constituents that comprise the electrostatic protein binding site, (3) address the impact of membrane curvature on (a) lipid packing and (b) protein binding. Here, we present a complete analysis of electrostatic Cytc-CL binding to both yeast and human variants of the protein. Using a new technique for lipid quantification, we characterize the interaction of the protein with membrane surfaces with high reproducibility. Our findings strongly suggest a biphasic response of the protein when exposed to CL with attenuated unfolding of the human variant with respect to the yeast variant. Alanine scanning was used to elucidate the electrostatic constituents of the anionic site. Lysines 72, 73, 86 and 87 were shown to be involved in CL binding but do not completely characterize electrostatic binding site. We also show that CL preferentially partitions to concave surfaces and that this preferential localization attenuates the interaction of Cytc with the outer leaflet of mixed lipid vesicles. Lastly, we demonstrate the role of membrane curvature for electrostatically bound Cytc. Here, Cytc conformational rearrangements are attenuated on concave CL surfaces. The nature of the binding is intrinsically different with a concerted structural rearrangement observed on concave surfaces as opposed to the biphasic electrostatically-driven conformational rearrangement seen on convex outer leaflets.

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© Copyright 2018 Margaret Marie Elmer-Dixon