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https://hdl.handle.net/2142/20052
Description
Title
NMR studies of bio-systems at high pressure
Author(s)
Samarasinghe, Shantha Dharmapriya
Issue Date
1993
Doctoral Committee Chair(s)
Jonas, Jiri
Department of Study
Chemistry
Discipline
Chemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Analytical
Chemistry, Physical
Language
eng
Abstract
Dipalmitoylphosphatidylcholine/cholesterol bilayer membranes were studied using $\sp2$H and $\sp{31}$P NMR spectroscopy over the pressure range from 1 bar to 5 kbar. The phase diagram was constructed, structure and dynamics of the bilayers were examined. The pressure dependence of the dynamic and conformational characteristics were studied using $\sp{31}$P NMR spectroscopy at 50$\sp\circ$C over the pressure range from 1 bar to 5 kbar. Cholesterol is able to maintain the mobility of the head groups despite the ordering of hydrocarbon chains in clear contrast to the same in pure DPPC bilayers where large changes in the head group motion was evident. Limited interdigitation of the chain region was evident in the presence of 20 mol% cholesterol. The spin lattice relaxation times cross over from fast to slow correlation time regime with the main phase transition from the liquid crystal to gel phase.
Pressure induced reversible unfolding of lysozyme was investigated by high resolution proton magnetic resonance spectroscopy by following the proton spectra of the following residues: His-15$\sp{\varepsilon1},$ Trp-28$\sp{\varepsilon3},$ Leu-17$\sp{\delta\sp2},$ Cys-64$\sp\alpha$ and Trp-108$\sp{\varepsilon3}.$ The experiments were performed at pH = 3.9 and 68.5$\sp\circ$C in the pressure range from 1 bar to 5 kbar both in the absence and presence tri-N-acetylglucosamine (tri-NAG). Small but statistically significant differences in $\Delta$V were found for residues located in different regions of the protein. The effect of tri-NAG binding to lysozyme was to change of $\Delta$V from $-$10.3 $\pm$ 0.6 to cm$\sp3$/mol to $-$18.1 $\pm$ 1.7 cm$\sp3$/mol for the Trp-108$\sp{\varepsilon3}$ residue which is located close to the active site. It is important to note that the Cys-64$\sp\alpha$ residue also senses the binding of the substrate analog. The ability to detect statistically significant differences for $\Delta$V of individual residues located in different regions of lysozyme represents the main result of these experiments.
The pressure induced unfolding of $\alpha$-helix, the helix-coil transition, of poly-N$\sp5$- (3-hydroxypropyl)-L-glutamine was investigated by high resolution $\sp1$H and $\sp{13}$C NMR spectroscopy and circular dichroism in aqueous medium and 7:3 methanol/water mixture at different temperatures. The line widths and spin lattice relaxation times were measured as a function of pressure over the range 1 bar to 5 kbar. Pressure induces an anomalous line width behavior facilitating exploration of the changes in H-bonding interactions associated with the helix-coil transition process. The current results provide reinforcing evidence for the proposed existence of an intermediate conformational state between the random coil and the $\alpha$-helix. A calibration curve for the estimation of the helix content in PHPG solutions was constructed.
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