Flourescent molecular reporter for the 3-D conformation of protein subdomains: The Mag-Indo system

In this study we investigate the use of the fluorescent probe Mag-Indo-1 as a molecular reporter for the three-dimensional (3-D) conformation of prote in subdomains. Magnesium complexation with this probe results in a shift of the emission fluorescence spectrum from 480 to 417 nm with an intensity pr oportional to the magnesium concentration in the range 0.6 to 30 mM. Althou gh designed as a specific magnesium chelator, Mag-Indo-1 is also able to bi nd calcium and zinc, the dissociation constants of the reactions being depe ndent of the nature of the cation. Furthermore, Mag-Indo-1 can also bind pr oteins through a specific interaction with some histidine residues. That in teraction induces a characteristic spectral shift of the emission fluoresce nce spectra from 480 to 457 nm. All these properties suggest that Mag-Indo- 1 could be used to study protein-cation binding. Emission and synchronous f luorescence techniques have been used to monitor the interaction of Mag-Ind o-1 with proteins such as bovine serum albumin, human serum albumin, and tu rkey egg white lysozyme. A method of resolution of complex fluorescence spe ctra was used to calculate the number of interaction sites and the correlat ive dissociation constants. Depending on the nature of the protein, a quenc hing of the natural fluorescence of the protein was observed, a process ass ociated with energy transfer from some tryptophan(s) to Mag-Indo-1. These d ata were tentatively correlated with the available information on the 3-D c onformation of the proteins investigated. The results of this study indicat e that Mag-Indo-1 can be used as an intramolecular fluorescent ruler to mon itor changes in the 3-D conformation of specific subdomains of proteins.