A systematic study of phosphorescent probes in cryosolvents, amorphous solids, and proteins

A systematic comparison was made of four different phosphorescent probes: erythrosine, tryptophan, tyrosine, and vanillin. Over a large temperature range, phosphorescence decays were collected for each in a 3:1 glycerol-water (v/v) mixture, a well characterized glass-forming solvent. The molecular mobility results were compared to fluctuations in the solvent as determined by dielectric relaxation spectroscopy. The rate of non-radiative decay of each probe followed the solvent fluctuations at different temperature ranges and with differing degrees of efficiency. Phosphorescence of two single-tryptophan proteins, human serum albumin (HSA) and a 20-amino acid tryptophan cage mini-protein, was collected in glycerol-water solvent. The results are consistent with the solvent slaving model in that the activation energy for the observed rate of non-radiative transition for the tryptophan residue matches that of specific solvent motions. In the glassy state, Arrhenius-like behavior reveals that the probe mobility is dominated by a low-energy vibrational process. As temperature is raised above Tg, the probe motions become increasingly driven first by β-fluctuations of the protein hydration shell, and eventually by α fluctuations of the bulk solvent. The effect on protein dynamics of an amorphous solid matrix was investigated. Free tryptophan and HSA were embedded in glucose, sucrose, maltose, and trehalose glasses. Tryptophan phosphorescence decays were collected over a wide temperature range. Temperature correlations between protein mobility and bulk mobility were generated. In the glassy state, despite a significantly faster process in the protein than in the bulk, Arrhenius-like behavior revealed similar energetics. Both values climb in similar fashion until the Tg of the bulk matrix is approached, where there is a decoupling of matrix and protein dynamics. The fluorescence of pyranine bound to HSA probes the water in the protein’s hydration shell. The sugars are seen to dehydrate the hydration shell to different degrees.Ph. D.Includes bibliographical referencesby Andrew R Dragansk