Dynamics of Trp residues in crystals of human α1-acid glycoprotein (orosomucoid) followed by red-edge excitation spectra

The present work reports for the first time the development of a method that allowed us to obtain crystals of orosomucoid complexed to progesterone. Then we investigated the dynamics of the microenvironments of the two buried Trp residues in the crystals of protein, by the red-edge excitation spectra method. The fluorescence excitation spectrum of the crystals is characteristic of that known for Trp residues (lambda(max) = 290 nm and bandwidth = 38 +/- 1 nm), indicating that the Trp residues are responsible for the fluorescence of the protein in the crystals. The position of the maximum and the bandwidth of the steady-state emission spectrum of the crystals (331 +/- 1 and 43 +/- 1 nm, respectively) are equal to those obtained in aqueous buffer for the orosomucoid-progesterone complex (330 +/- 1 and 43 +/- 1 nm) (lambda(ex), 295 nm). Thus, the fluorescence of the crystals occurs from the Trp residues buried in the protein core. The red-edge excitation spectra studies indicate that the Trp residues are surrounded by microenvironments that display motions, a result identical to that observed in solution. Thus, the crystallization process does not modify the structure or the dynamics of orosomucoid core. The fluorescence intensities depend on the angular orientation of the crystals with respect to the polarization of the incident beam. The general feature of this dependence is identical at the three excitation wavelengths used (295, 300, and 305 nm). Our results confirm the fact that the local structure and dynamics are the key for any interpretation of tryptophan fluorescence parameters of orosomucoid.