Understanding The Role of Arg96 in Structure and Stability of Green Fluorescent Protein

Authors

Olga V. Stepanenko, Institute of Cytology of the Russian Academy of Sciences
Vladislav V. Verkhusha, Albert Einstein College of Medicine
Michail M. Shavlovsky, Russian Academy of Medical Sciences
Irina M. Kuznetsova, Institute of Cytology of the Russian Academy of Sciences
Vladimir N. Uversky, Indiana University School of MedicineFollow
Konstantin K. Turoverov, Institute of Cytology of the Russian Academy of Sciences

Document Type

Article

Publication Date

2008

Digital Object Identifier (DOI)

https://doi.org/10.1002/prot.22089

Abstract

Arg96 is a highly conservative residue known to catalyze spontaneous green fluorescent protein (GFP) chromophore biosynthesis. To understand a role of Arg96 in conformational stability and structural behavior of EGFP, the properties of a series of the EGFP mutants bearing substitutions at this position were studied using circular dichroism, steady state fluorescence spectroscopy, fluorescence lifetime, kinetics and equilibrium unfolding analysis, and acrylamide-induced fluorescence quenching. During the protein production and purification, high yield was achieved for EGFP/Arg96Cys variant, whereas EGFP/Arg96Ser and EGFP/Arg96Ala were characterized by essentially lower yields and no protein was produced when Arg96 was substituted by Gly. We have also shown that only EGFP/Arg96Cys possessed relatively fast chromophore maturation, whereas it took EGFP/Arg96Ser and EGFP/Arg96Ala about a year to develop a noticeable green fluorescence. The intensity of the characteristic green fluorescence measured for the EGFP/Arg96Cys and EGFP/Arg96Ser (or EGFP/Arg96Ala) was 5- and 50-times lower than that of the nonmodified EGFP. Intriguingly, EGFP/Arg96Cys was shown to be more stable than EGFP toward the GdmCl-induced unfolding both in kinetics and in the quasi-equilibrium experiments. In comparison with EGFP, tryptophan residues of EGFP/Arg96Cys were more accessible to the solvent. These data taken together suggest that besides established earlier crucial catalytic role, Arg96 is important for the overall folding and conformational stability of GFP. Proteins 2008. © 2008 Wiley-Liss, Inc.

Was this content written or created while at USF?

Yes

Citation / Publisher Attribution

Proteins: Structure, Function, and Bioinformatics, v. 73, issue 3, p. 539-551

Scholar Commons Citation

Stepanenko, Olga V.; Verkhusha, Vladislav V.; Shavlovsky, Michail M.; Kuznetsova, Irina M.; Uversky, Vladimir N.; and Turoverov, Konstantin K., "Understanding The Role of Arg96 in Structure and Stability of Green Fluorescent Protein" (2008). Molecular Medicine Faculty Publications. 806.
https://digitalcommons.usf.edu/mme_facpub/806