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. 2014 Jun 24;9(6):e99136.
doi: 10.1371/journal.pone.0099136. eCollection 2014.

Orange fluorescent proteins: structural studies of LSSmOrange, PSmOrange and PSmOrange2

Sergei Pletnev  1 , Daria M Shcherbakova  2 , Oksana M Subach  3 , Nadya V Pletneva  4 , Vladimir N Malashkevich  5 , Steven C Almo  5 , Zbigniew Dauter  6 , Vladislav V Verkhusha  2
Affiliations

Orange fluorescent proteins: structural studies of LSSmOrange, PSmOrange and PSmOrange2

Sergei Pletnev et al. PLoS One. .

Abstract

A structural analysis of the recently developed orange fluorescent proteins with novel phenotypes, LSSmOrange (λex/λem at 437/572 nm), PSmOrange (λex/λem at 548/565 nm and for photoconverted form at 636/662 nm) and PSmOrange2 (λex/λem at 546/561 nm and for photoconverted form at 619/651 nm), is presented. The obtained crystallographic structures provide an understanding of how the ensemble of a few key mutations enabled special properties of the orange FPs. While only a single Ile161Asp mutation, enabling excited state proton transfer, is critical for LSSmOrange, other substitutions provide refinement of its special properties and an exceptional 120 nm large Stokes shift. Similarly, a single Gln64Leu mutation was sufficient to cause structural changes resulting in photoswitchability of PSmOrange, and only one additional substitution (Phe65Ile), yielding PSmOrange2, was enough to greatly decrease the energy of photoconversion and increase its efficiency of photoswitching. Fluorescence of photoconverted PSmOrange and PSmOrange2 demonstrated an unexpected bathochromic shift relative to the fluorescence of classic red FPs, such as DsRed, eqFP578 and zFP574. The structural changes associated with this fluorescence shift are of considerable value for the design of advanced far-red FPs. For this reason the chromophore transformations accompanying photoconversion of the orange FPs are discussed.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Evolution of the subfamily of orange fluorescent proteins.
(A) Phylogenic tree showing the history of the development of different orange fluorescent proteins. (B) Chemical structures of the chromophores found in orange fluorescent proteins.
Figure 2
Figure 2. Amino acid alignment of mOrange, LSSmOrange, PSmOrange and PSmOrange2.
The chromophore-forming tri-peptides are highlighted in yellow.
Figure 3
Figure 3. Amino acid differences between the parental and successor proteins in 3D.
(A) The transformation of mOrange into LSSmOrange. (B) The transformation of mOrange into PSmOrange. (C) The transformation of PSmOrange into PSmOrange2.
Figure 4
Figure 4. The differences in the immediate chromophore environment between the parental and successor proteins.
(A) The difference between mOrange and LSSmOrange. (B) The difference between mOrange and PSmOrange. (C) The difference between PSmOrange and PSmOrange2.
Figure 5
Figure 5. The structures of the PSmOrange and DsRed chromophores.
(A) The structure of the orange form of the PSmOrange chromophore. (B) The modeled structure of the photoconverted far-red form of the PSmOrange chromophore. (C) The structure of the DsRed chromophore.
Figure 6
Figure 6. An evolution of orange fluorescent proteins derived from KO, DsRed and zFP538 with mutations critical to the phenotype of each variant.
Figure 7
Figure 7. Possibilities for the future design of advanced orange and far-red fluorescent proteins.
See this image and copyright information in PMC

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