6h01

Publication Abstract from PubMed

Photoactivatable fluorescent proteins (PA-FPs) are a powerful non-invasive tool in high-resolution live-cell imaging. They can be converted from an inactive to an active form by light, enabling the spatial and temporal trafficking of proteins and cell dynamics. PA-FPs have been previously generated by mutating selected residues in the chromophore or in its close proximity. A new strategy to generate PA-FPs is the genetic incorporation of unnatural amino acids (UAAs) containing photocaged groups using unique suppressor tRNA/aminoacyl-tRNA synthetase pairs. We set out to develop a photoactivatable GFP variant suitable for time-resolved structural studies. Here, we report the crystal structure of superfolder GFP (sfGFP) containing the UAA ortho-nitrobenzyl-tyrosine (ONBY) at position 66 and its spectroscopic characterization. Surprisingly, the crystal structure (to 2.7 A resolution) reveals a dimeric domain-swapped arrangement of sfGFP66ONBY with residues 1-142 of one molecule associating with residues 148-234 from another molecule. This unusual domain-swapped structure supports a previously postulated GFP folding pathway that proceeds via an equilibrium intermediate.

Crystal structure of a domain-swapped photoactivatable sfGFP variant provides evidence for GFP folding pathway.,Kesgin-Schaefer S, Heidemann J, Puchert A, Koelbel K, Yorke BA, Huse N, Pearson AR, Uetrecht C, Tidow H FEBS J. 2019 Feb 28. doi: 10.1111/febs.14797. PMID:30817081^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.