Thermodynamic Protein Destabilization by GFP Tagging: A Case of Interdomain Allostery

Miri Sokolovski, Arnab Bhattacherjee, Naama Kessler, Yaakov Levy, Amnon Horovitz

Research output: Contribution to journalArticlepeer-review


The Engrailed Homeodomain (EnHD) transcription factor of Drosophila melanogaster was fused to the enhanced green fluorescent protein (eGFP) either at its C- or N-terminus via three- or ten-residue flexible linkers. Here, we show that EnHD undergoes destabilization upon fusing it to eGFP regardless of the linker length used and whether the tethering is to its N- or C-terminus. The destabilization is reflected in melting points that are lower by up to 9°C. Thermodynamic analysis and coarse-grained molecular dynamic simulations indicate that this destabilization is due to eGFP-promoted entropic stabilization of the denatured state ensemble of EnHD. Our results provide, therefore, an example for destabilizing interdomain allostery. They are also important given the widespread use of eGFP tagging in cell biology, as they indicate that such tagging can cause unintended protein destabilization and concomitant effects.

Original languageEnglish
Pages (from-to)1157-1162
Number of pages6
JournalBiophysical Journal
Issue number6
StatePublished - 19 Sep 2015

All Science Journal Classification (ASJC) codes

  • Biophysics


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