Abstract
The enhanced green fluorescent protein (EGFP) is broadly used as an intracellular fluorescent marker. Its numerous applications require a comprehensive understanding of EGFP folding properties, as well as the properties of EGFP-derived mutants.
Herein, we report on the spectroscopic characterization of two single-cysteine mutants, C48S-EGFP and C70S-EGFP, which were prepared to monitor the protein folding dynamics by EPR, specifically in the close vicinity of both cysteine residues.
Chromophore absorption, excitation and emission spectra for these mutants were similar to those for EGFP. Both single-cysteine mutants were more susceptible to aggregation than EGFP in E. coli. C48S-EGFP exhibited similar resistance to chemical denaturation as EGFP. In contrast, C70S-EGFP was less resistant to denaturation in guanidine hydrochloride and its folding was less efficient. Hence, C48S-EGFP seems to be a more suitable probe for EPR measurements than C70S-EGFP.
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