4ka9
Crystal structure analysis of single amino acid deletion mutations in EGFP
Structural highlights
FunctionGFP_AEQVI Energy-transfer acceptor. Its role is to transduce the blue chemiluminescence of the protein aequorin into green fluorescent light by energy transfer. Fluoresces in vivo upon receiving energy from the Ca(2+)-activated photoprotein aequorin. Publication Abstract from PubMedAltering a protein's backbone through amino acid deletion is a common evolutionary mutational mechanism, but is generally ignored during protein engineering primarily because its effect on the folding-structure-function relationship is difficult to predict. Using directed evolution, enhanced green fluorescent protein (EGFP) was observed to tolerate residue deletion across the breadth of the protein, particularly within short and long loops, helical elements, and at the termini of strands. A variant with G4 removed from a helix (EGFP(G4Delta)) conferred significantly higher cellular fluorescence. Folding analysis revealed that EGFP(G4Delta) retained more structure upon unfolding and refolded with almost 100% efficiency but at the expense of thermodynamic stability. The EGFP(G4Delta) structure revealed that G4 deletion caused a beneficial helical registry shift resulting in a new polar interaction network, which potentially stabilizes a cis proline peptide bond and links secondary structure elements. Thus, deletion mutations and registry shifts can enhance proteins through structural rearrangements not possible by substitution mutations alone. Random single amino acid deletion sampling unveils structural tolerance and the benefits of helical registry shift on GFP folding and structure.,Arpino JA, Reddington SC, Halliwell LM, Rizkallah PJ, Jones DD Structure. 2014 Jun 10;22(6):889-98. doi: 10.1016/j.str.2014.03.014. Epub 2014, May 22. PMID:24856363[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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