title: Thermodynamic Protein Destabilization by GFP Tagging: A Case of Interdomain Allostery.
abstract: 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.
authors.affiliation: Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel.
authors.forename: Miri
authors.initials: M
authors.lastname: Sokolovski
authors.affiliation: Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel.
authors.forename: Arnab
authors.initials: A
authors.lastname: Bhattacherjee
authors.affiliation: Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel.
authors.forename: Naama
authors.initials: N
authors.lastname: Kessler
authors.affiliation: Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel. Electronic address: koby.levy@weizmann.ac.il.
authors.forename: Yaakov
authors.initials: Y
authors.lastname: Levy
authors.affiliation: Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel. Electronic address: amnon.horovitz@weizmann.ac.il.
authors.forename: Amnon
authors.initials: A
authors.lastname: Horovitz
mesh_terms: D000494:Allosteric Regulation; D000818:Animals; D002942:Circular Dichroism; D029721:Drosophila Proteins; D004331:Drosophila melanogaster; D004926:Escherichia coli; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D056004:Molecular Dynamics Simulation; D011489:Protein Denaturation; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D012996:Solutions; D013816:Thermodynamics; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D029721:Drosophila Proteins; C063459:En protein, Drosophila; D018398:Homeodomain Proteins; D012996:Solutions; D014157:Transcription Factors; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins