Ultrafast Nonlinear Spectroscopy of Red Fluorescent Proteins

<p>Red-emitting homologues (RFPs) of the native Green Fluorescent Protein\&nbsp;<span style="line-height: 1.6em;">(GFP) with emission wavelengths beyond 650 nm are desirable probes for </span><em style="line-height: 1.6em;">in vivo</em><span style="line-height: 1.6em;">\&nbsp;</span><span style="line-height: 1.6em;">imaging experiments. They offer the potential for deeper tissue penetration and\&nbsp;</span><span style="line-height: 1.6em;">lower background scatter given a cleaner spectral window. However, bioimaging\&nbsp;</span><span style="line-height: 1.6em;">applications are hindered by poor photophysics (e.g., low fluorescence quantum\&nbsp;</span><span style="line-height: 1.6em;">yield, high photobleaching), which limits experimental resolution and represents a\&nbsp;</span><span style="line-height: 1.6em;">significant obstacle towards utilization for low copy-number, long-duration imaging\&nbsp;</span><span style="line-height: 1.6em;">applications. In this thesis, a variety of femtosecond nonlinear electronic\&nbsp;</span><span style="line-height: 1.6em;">spectroscopies were employed jointly with site-directed mutagenesis to investigate\&nbsp;</span><span style="line-height: 1.6em;">the photophysical properties of RFPs. In one study, the molecular mechanism of red\&nbsp;</span><span style="line-height: 1.6em;">emission was pursued in two notable RFPs, mPlum and TagRFP675. Solvation\&nbsp;</span><span style="line-height: 1.6em;">dynamics observed with time-resolved transient grating spectroscopy were\&nbsp;</span><span style="line-height: 1.6em;">interpreted with the aid of molecular dynamics simulations to indicate that their\&nbsp;</span><span style="line-height: 1.6em;">red-emission is correlated with the ability of specific chromophore-sidechain\&nbsp;</span><span style="line-height: 1.6em;">hydrogen-bonding interactions to interconvert between direct and water-mediated\&nbsp;</span><span style="line-height: 1.6em;">states. In a second set of studies, two-dimensional double quantum coherence\&nbsp;</span><span style="line-height: 1.6em;">spectroscopy was used to probe the electronic transitions of mPlum. It was\&nbsp;</span><span style="line-height: 1.6em;">discovered that it displayed a response distinctly different from an organic dye in\&nbsp;</span><span style="line-height: 1.6em;">bulk solvent. Modeling indicate of these spectra indicate the spectral features may\&nbsp;</span><span style="line-height: 1.6em;">be attributed to the existence of multiple high-lying (n\&gt;1) excited states. The results\&nbsp;</span><span style="line-height: 1.6em;">provide new insight into the electronic structure of these widely used fluorescent\&nbsp;</span><span style="line-height: 1.6em;">probes.</span></p>
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University of Colorado Boulder
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