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|Title:||The Use of High Pressure Fluorescence Spectroscopy to Investigate Subunit Interactions in Oligomeric Proteins (Neurophysin, Lac Repressor)|
|Author(s):||Royer, Catherine Ann|
|Department / Program:||Biochemistry|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|Abstract:||The application of high hydrostatic pressures (1 atm - 3 kbar) has long been known to lead to the dissociation of oligomeric proteins due to the negative volume change accompanying this process. This technique coupled with fluorescence spectroscopy is used in this thesis in order to determine the thermodynamic constants, volume change and free energy, for dissociation equilibria of two oligomeric proteins both in absence and in presence of ligand. In this manner the effect of the ligand on the subunit interactions is determined. Two protein systems are explored using the techniques of high pressure fluorescence polarization and photon counting scanning fluorimetry.
The first oligomeric system discussed here is the Bovine Neurophysin II dimer. The polarization data from the intrinsic fluorescence and dansylated protein both yield a volume change upon association of 40 ml/mole and a free energy change upon dissociation of approximately 5 kcal/mole. Addition of the tripeptide ligand, Cys(SMe)-Phe-Ile, is shown to stabilize the dimer. In fact, these data indicate much greater affinity of the ligand for the dimer than previously reported. The volume change as calculated from the dansylated neurophysin polarization data and the center of spectral mass of the intrinsic fluorescence nearly tripled to approximately 120 ml/mole. The change in free energy doubled to over 10 kcal/mole, which corresponds to a free energy of coupling of approximately 5 kcal/mole.
The second oligomer presented is the lac repressor tetramer from E. Coli. While the polarization data from both the intrinsic fluorescence and the dansylated molecule indicate that high pressure induces dissociation, the relatively small changes in the values make polarization an inaccurate technique for the study of the dissociation process. The center of spectral mass of the unliganded tetramer is too low for the observation of any significant change upon dissociation. However, the large shift in the emission maximum upon ligation to lower wavelength allowed for the observation of large changes in the center of spectral mass of the liganded protein upon dissociation by high pressure. A study of the concentration dependence of the high pressure dissociation demonstrates that the dissociation transition is complete to monomer. The volume change for the association reaction is 178 ml/mole. The free energy change is approximately 30 kcal/mole.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1985.
|Date Available in IDEALS:||2014-12-15|