Denaturation and partial renaturation of a tightly tetramerized DsRed protein under mildly acidic conditions

The red fluorescent protein, DsRed, recently cloned from coral Discosoma sp . has one of the longest fluorescence waves and one of the most complex abs orbance spectra among the family of fluorescent proteins. In this work we f ound that with time DsRed fluorescence decreases under mildly acidic condit ions (pH 4.0-4.8) in a pH-dependent manner, and this fluorescence inactivat ion could be partially recovered by subsequent re-alkalization. The DsRed a bsorbance and circular dichroism spectra under these conditions revealed th at the fluorescence changes were caused by denaturation followed by partial renaturation of the protein. Further, analytical ultracentrifugation deter mined that native DsRed formed a tight tetramer under various native condit ions, Quantitative analysis of the data showed that several distinct states of protein exist during the fluorescence inactivation and recovery, and th e inactivation of fluorescence can be caused by protonation of a single ion ogenic group in each monomer of DsRed tetramer. (C) 2000 Federation of Euro pean Biochemical Societies. Published by Elsevier Science B.V. Alt rights r eserved.