A protein molecule is an intricate system whose function is highly sensitive to small external perturbations. However, few examples that directly correlate protein function with continuous, progressive sub-angstrom structural perturbations have thus far been presented. In order to elucidate this relationship, we have investigated the Aequorea yellow fluorescent protein Citrine as a model system under high-pressure perturbation. Citrine has been compressed by high pressure to produce deformations of its ?-barrel scaffold and light absorbing and emitting center, the chromophore, by applying a novel high pressure cryo-cooling technique. A closely spaced series of high-pressure X-ray crystallographic structures of Citrine from 0.1 to 500 MPa reveal that the chromophore undergoes a progressive deformation of up to 0.8 ? at an applied pressure of 500 MPa. It is experimentally demonstrated that deformation of the chromophore is directly correlated with a progressive shift of the fluorescence peak of Citrine from yellow to green under these conditions. The re-orientation of the Citrine chromophore is actuated by the differential motion of two clusters of atoms that compose the ?-barrel scaffold of the molecule, resulting in a bending or buckling of the ?-barrel. The high-pressure structures also reveal a perturbation of the hydrogen bonding network stabilizing the excited state of the Citrine chromophore that is implicated in the reduction of fluorescence intensity of the molecule under high pressure. The blue-shift of the Citrine fluorescence spectrum resulting from the bending of the ?-barrel provides structural insight into the transient blue-shifting of isolated yellow fluorescent protein molecules under ambient conditions and suggests mechanisms to alter the time-dependent behavior of Citrine under ambient conditions. The results presented in this thesis demonstrate that the fluorescence spectrum of Citrine is highly sensitive to sub-angstrom deformations and its fluorescent function must be understood at the sub-angstrom level. These results provide important general lessons for the structure-function relationship of enzymes, and may have significant implications for protein function prediction and biomolecule design and engineering as they suggest methods to tune protein function by modification of the protein scaffold.