Biophysical characterization of Gir2, a highly acidic protein of Saccharomyces cerevisiae with anomalous electrophoretic behavior

Abstract

Gir2 is an uncharacterized protein of Saccharomyces cerevisiae, containing a RWD/GI domain. In this work, we report the biophysical characterization of Gir2. His-tagged Gir2, expressed and purified from Escherichia coli, showed an abnormally slow migration on SDS–PAGE. The yeast expressed protein behaves similarly. Using mass spectrometry and peptide mass fingerprinting we demonstrated that the protein has the expected molecular mass (34 kDa). EDC modification of carboxylate groups reverted the anomalous migration on SDS–PAGE. Size exclusion chromatography showed that Gir2 has a Stokes radius larger than expected. Gir2 is thermostable and lacks extensive structure, as determined by CD analysis. Based on these findings, we suggest that Gir2 is a representative of the growing group of “natively unfolded” proteins.