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

In this paper, 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 ~ 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. Additionally, 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. Beyondmore » 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.« less
Authors:
 [1] ;  [2] ;  [3] ; ORCiD logo [4] ;  [4] ;  [4] ; ORCiD logo [4] ;  [5] ;  [3] ; ORCiD logo [4]
  1. Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Univ. of New Mexico, Albuquerque, NM (United States)
  2. Los Alamos National Lab. (LANL), Los Alamos, NM (United States); ARUP Laboratories Institute for Clinical and Experimental Pathology, Salt Lake City, UT (United States)
  3. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
  4. Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
  5. Univ. of New Mexico, Albuquerque, NM (United States)
Publication Date:
Report Number(s):
LA-UR-15-29326
Journal ID: ISSN 0022-2836
Grant/Contract Number:
AC52-06NA25396
Type:
Accepted Manuscript
Journal Name:
Journal of Molecular Biology
Additional Journal Information:
Journal Volume: 428; Journal Issue: 9; Journal ID: ISSN 0022-2836
Publisher:
Elsevier
Research Org:
Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
Sponsoring Org:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22); USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; chromoprotein; photoswitchable; pcFRET; neutron crystallography; ensemble refinement
OSTI Identifier:
1457271
Alternate Identifier(s):
OSTI ID: 1359355