Evolution and characterization of a new reversibly photoswitching chromogenic protein, Dathail

JOURNAL OF MOLECULAR BIOLOGY

卷:428

Article

Langan, Patricia S.^1,6  Close, Devin W.^1,2,3  Coates, Leighton⁴  Rocha, Reginaldo C.²  Ghosh, Koushik²  Kiss, Csaba¹  Waldo, Geoff¹  Freyer, James⁵  Kovalevsky, Andrey⁴  Bradbury, Andrew R. M.¹ 

[1] Los Alamos Natl Lab, Biosci Div, POB 1663, Los Alamos, NM 87545 USA

[2] Los Alamos Natl Lab, Ctr Integrated Nanotechnol, POB 1663, Los Alamos, NM 87545 USA

[3] ARUP Labs Inst Clin & Expt Pathol, Salt Lake City, UT USA

[4] Oak Ridge Natl Lab, Biol & Soft Matter Div, Oak Ridge, TN 37831 USA

[5] Univ New Mexico, Ctr Biomed Engn, Albuquerque, NM 87131 USA

[6] Univ New Mexico, Nanosci & Microsyst Engn, Albuquerque, NM 87131 USA

关键词: chromoprotein;    photoswitchable;    pcFRET;    neutron crystallography;    ensemble refinement;   

DOI  :  10.1016/j.jmb.2016.02.029

来源: Elsevier

【 摘 要 】

We report the engineering of a new reversibly switching chromogenic protein, Dathail. Dathail was evolved from the extremely thermostable fluorescent proteins thermal green protein (TGP) and eCGP123 using directed evolution and ratiometric sorting. Dathail has two spectrally distinct chromogenic states with low quantum yields, corresponding to absorbance in a ground state with a maximum at 389 nm, and a photo-induced metastable state with a maximum at 497 nm. In contrast to all previously described photoswitchable proteins, both spectral states of Dathail are non-fluorescent. The photo-induced chromogenic state of Dathail has a lifetime of similar to 50 min at 293 K and pH 7.5 as measured by UV-Vis spectrophotometry, returning to the ground state through thermal relaxation. X-ray crystallography provided structural insights supporting a change in conformation and coordination in the chromophore pocket as being responsible for Dathail's photoswitching. Neutron crystallography, carried out for the first time on a protein from the green fluorescent protein family, showed a distribution of hydrogen atoms revealing protonation of the chromophore 4-hydroxybenzyl group in the ground state. The neutron structure also supports the hypothesis that the photo-induced proton transfer from the chromophore occurs through water-mediated proton relay into the bulk solvent. Beyond its spectroscopic curiosity, Dathail has several characteristics that are improvements for applications, including low background fluorescence, large spectral separation, rapid switching time, and the ability to switch many times. Therefore, Dathail is likely to be extremely useful in the quickly developing fields of imaging and biosensors, including photochromic Forster resonance energy transfer, high-resolution microscopy, and live tracking within the cell. (C) 2016 Elsevier Ltd. All rights reserved.

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