Date on Master's Thesis/Doctoral Dissertation

5-2012

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Chemistry

Committee Chair

Wittebort, Richard J.

Author's Keywords

Solid state NMR; Structural hypothesis; Cardiolipin; Biochemistry; Elastin; NMR

Subject

Nuclear magnetic resonance; Elastin; Cardiolipin

Abstract

This dissertation has five chapters. Following an introduction, chapter two describes an investigation of the hypothesis that cardiolipin has a proton storage function in mitochondrial membranes. Chapters three and four present a novel NMR study of the elastic mechanism in elastin. Directions for the future research are given in the last chapter. To investigate the hypothesis that cardiolipin has a proton storage function, we have used 31p, 1H and 13C NMR. A single axially symmetric 31p pattern with reversed anisotropy compared to the static pattern is observed from pH 4 to 9 for tetraoleoyl cardiolipin (TOCL). This confirms that (a) the samples studied are in the biologically relevant liquid crystalline phase, (b) the phosphatidic moieties are equivalent, and (c) TOCL molecules reorient axially about the bilayer normal. Well resolved 13C and 31p MAS NMR spectra show no pH dependent chemical shifts. Based on a large number of 1H, 13C and 31p NMR experiments, we conclude that there is no evidence that lamellar multibilayers of TOCL possess a titratable group with a pKa in the range of 4 to 9. We also discuss why the proton storage hypothesis is not energetically realistic. The mechanism of elastin's recoil is addressed in chapters three and four. The conventional view based on conformational entropy is not supported by preliminary 13C NMR studies, which show no observable increase in backbone ordering with stretch. To investigate the alternative hypothesis that the hydrophobic effect drives elastic recoil, novel 2H double quantum NMR experiments were implemented. NMR pulse sequences, phase cycles, and methods of analysis were devised to determine the amount of ordered water in elastin and how it changes with stretch, temperature and hydration. Based on the data, which shows a significant increase in the amount of ordered water in all three cases, we conclude that the hydrophobic effect has a significant role in the mechanism of elastin's reversible recoil. Consequently, elastic recoil exploits the increase of the solvent ordering on the hydrophobic surface of the protein when it is stretched, and recoil is driven by a favorable increase of the solvent entropy.

Download

Share

COinS