High pressure, micro spectroscopy, cells, polypeptide, lipids
Functional properties of living cells depend on the thermodynamic variables such as temperature and pressure. A unique tool to investigate volume effects on structure and metabolism of the cell and biomolecules is pressure perturbation. We have developed a new setup that enables micro-spectroscopy and optical imaging of individual live cells at variable pressure from 0.1 to 400 MPa. Following characterization of the setup, pressure and temperature effects on the secondary structure of the peptide Poly-L-glutamic acid (PGA) in deuterated water buffer solution were investigated. The amide I band of PGA is sensitive to pressure and temperature, and by spectral deconvolution, we determined the relative contributions due to the ?-helix and random coil conformations. The population of ?-helix increases with increasing pressure. Pressure effects on single red blood cells and the intracellular protein hemoglobin were studied by micro-Raman spectroscopy. In particular, we observed a shift in the frequency of the iron-histidine vibrational band in both the intracellular hemoglobin and hemoglobin in solutions. The iron-histidine mode is a sensitive structural marker of the crucial iron-protein linkage in heme proteins. The pressure dependent shift suggests a conformational change of the heme environment. This finding was further supported by micro-absorption measurements at variable pressure. In additional experiments, Raman spectroscopy was employed to probe molecular changes that occurred in hemoglobin in erythrocytes infected with the malaria parasite Plasmodium falciparum. The spectra of infected cells indicated that hemoglobin degradation can be correlated with the stages of the parasite multiplication cycle. The research was further extended towards probing size and shape changes of individual cells with pressure. The lateral diameter in yeast cells was observed to decrease with pressure in a reversible way. These results suggest that transport of the intra-cellular water may play a significant role for volume changes. In summary, pressure changes were shown to induce conformational changes in proteins and shape changes in yeast cells. A Raman technique was developed to monitor the states of Plasmodium falciparum multiplication cycle within a red blood cell.
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Doctor of Philosophy (Ph.D.)
College of Sciences
Length of Campus-only Access
Doctoral Dissertation (Open Access)
Dissertations, Academic -- Sciences; Sciences -- Dissertations, Academic
Park, Sang Hoon, "High Pressure Micro-Spectroscopy of Biological Assemblies and Cells" (2012). Electronic Theses and Dissertations. 4629.