Cofactor and tryptophan accessibility and unfolding of brain glutamate decarboxylase

Cofactor and tryptophan accessibility of the 65-kDa form of rat brain gluta mate decarboxylase (GAD) was investigated by fluorescence quenching measure ments using acrylamide, I-, and Cs+ as the quenchers. Trp residues were par tially exposed to solvent. I- was less able and Cs+ was more able to quench the fluorescence of Trp residues in the holoenzyme of GAD (holoGAD) than t he apoenzyme (apoGAD). The fraction of exposed Trp residues were in the ran ge of 30-49%. In contrast, pyridoxal-P bound to the active site of GAD was exposed to solvent. I- was more able and Cs' was less able to quench the fl uorescence of pyridoxal-P in holoGAD. The cofactor was present in a positiv ely charged microenvironment, making it accessible for interactions with an ions. A difference in the exposure of Trp residues and pyridoxal-P to these charged quenchers suggested that the exposed Trp residues were essentially located outside of the active site. Changes in the accessibility of Trp re sidues upon pyridoxal-P binding strongly supported a significant conformati onal change in GAD. Fluorescence intensity measurements were also carried o ut to investigate the unfolding of GAD using guanidine hydrochloride (GdnHC l) as the denaturant. At 0.8-1.5 M GdnHCl, an intermediate step was observe d during the unfolding of GAD from the native to the denatured state, and w as not found during the refolding of GAD from the denatured to native state , indicating that this intermediate step was not a reversible process. Howe ver, at > 1.5 M GdnHCl for holoGAD and >2.0 M GdnHCl for apoGAD, the transi tion leading to the denatured state was reversible. It was suggested that t he intermediate step involved the dissociation of native dimer of GAD into monomers and the change in the secondary structure of the protein. Circular dichroism revealed a decrease in the a-helix content of GAD from 36 to 28% . The unfolding pattern suggested that GAD may consist of at least two unfo lding domains. Unfolding of the lower GdnHCl-resisting domain occurred at a similar concentration of denaturant for apoGAD and holoGAD, while unfoldin g of the higher GdnHCl-resisting domain occurred at a higher concentration of GdnHCl for apoGAD than holoGAD. (C) 2001 Academic Press.