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

Patricia S Langan; Devin W Close; Leighton Coates; Reginaldo C Rocha; Koushik Ghosh; Csaba Kiss; Geoff Waldo; James Freyer; Andrey Kovalevsky; Andrew R M Bradbury

doi:10.1016/j.jmb.2016.02.029

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

J Mol Biol. 2016 May 8;428(9 Pt A):1776-89. doi: 10.1016/j.jmb.2016.02.029. Epub 2016 Mar 18.

Authors

Affiliations

¹ Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM 87545, USA; Nanoscience and Microsystems Engineering, University of New Mexico, Albuquerque, NM 87131, USA.
² Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM 87545, USA; Center for Integrated Nanotechnologies, Los Alamos National Laboratory, Los Alamos, NM 87545, USA; ARUP Laboratories Institute for Clinical and Experimental Pathology, Salt Lake City, UT, USA.
³ Biology and Soft Matter Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA.
⁴ Center for Integrated Nanotechnologies, Los Alamos National Laboratory, Los Alamos, NM 87545, USA.
⁵ Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM 87545, USA.
⁶ Center for Biomedical Engineering, University of New Mexico, Albuquerque, NM 87131, USA.
⁷ Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM 87545, USA. Electronic address: amb@lanl.gov.

PMID: 27000644
DOI: 10.1016/j.jmb.2016.02.029

Abstract

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 389nm, and a photo-induced metastable state with a maximum at 497nm. 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 ~50min at 293K and pH7.5 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 Förster resonance energy transfer, high-resolution microscopy, and live tracking within the cell.

Keywords: chromoprotein; ensemble refinement; neutron crystallography; pcFRET; photoswitchable.

Publication types

Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Color*
Crystallography, X-Ray
Directed Molecular Evolution
Models, Molecular
Photochemical Processes*
Protein Conformation
Recombinant Proteins / chemistry*
Recombinant Proteins / genetics
Recombinant Proteins / metabolism*
Spectrophotometry
Temperature
Time Factors

Substances

Recombinant Proteins