Probing local conformation and dynamics of molecular complexes using phase-selective fluorescence correlation and coherence spectroscopy

• Main Author: Lott, Geoffrey Adam, 1980-
• Language: en_US
• Published: University of Oregon 2010
• Subjects: Local conformation; Molecular complexes; Fluorescence correlation; Phase selective signals; Dipolar coupling; Dynamical processes; Exciton behavior; Physical chemistry; Molecular physics; Optics; Spectroscopy
• Online Access: http://hdl.handle.net/1794/10914

xv, 177 p. : ill. (some col.) A print copy of this thesis is available through the UO Libraries. Search the library catalog for the location and call number. === When two or more fluorescent chromophores are closely spaced in a macromolecular complex, dipolar coupling leads to delocalization of the excited states, forming excitons. The relative transition frequencies and magnitudes are sensitive to conformation, which can then be studied with optical spectroscopy. Non-invasive fluorescence spectroscopy techniques are useful tools for the study of dilute concentrations of such naturally fluorescent or fluorescently labeled biological systems. This dissertation presents two phase-selective fluorescence spectroscopy techniques for the study of dynamical processes in bio-molecular systems across a wide range of timescales. Polarization-modulated Fourier imaging correlation spectroscopy (PM-FICS) is a novel phase-selective fluorescence spectroscopy for simultaneous study of translational and conformational dynamics. We utilize modulated polarization and intensity gratings with phase-sensitive signal collection to monitor the collective fluctuations of an ensemble of fluorescent molecules. The translational and conformational dynamics can be separated and analyzed separately to generate 2D spectral densities and joint probability distributions. We present results of PM-FICS experiments on DsRed, a fluorescent protein complex. Detailed information on thermally driven dipole-coupled optical switching pathways is found, for which we propose a conformation transition mechanism. 2D phase-modulation electronic coherence spectroscopy is a third-order nonlinear spectroscopy that uses collinear pulse geometry and acousto-optic phase modulation to isolate rephasing and nonrephasing contributions to the collected fluorescence signal. We generate 2D spectra, from which we are able to determine relative dipole orientations, and therefore structural conformation, in addition to detailed coupling information. We present results of experiments on magnesium tetraphenylporphyrin dimers in lipid vesicle bilayers. The 2D spectra show clearly resolved diagonal and off-diagonal features, evidence of exciton behavior. The amplitudes of the distinct spectral features change on a femtosecond timescale, revealing information on time-dependent energy transfer dynamics. This dissertation includes co-authored and previously published material. === Committee in charge: Hailin Wang, Chairperson, Physics; Andrew Marcus, Advisor, Chemistry; Stephen Gregory, Member, Physics; Michael Raymer, Member, Physics; Marina Guenza, Outside Member, Chemistry