Orange Fluorescent Proteins: Structural Studies of LSSmOrange, PSmOrange and PSmOrange2

<div><p>A structural analysis of the recently developed orange fluorescent proteins with novel phenotypes, LSSmOrange (λ_ex/λ_em at 437/572 nm), PSmOrange (λ_ex/λ_em at 548/565 nm and for photoconverted form at 636/662 nm) and PSmOrange2 (λ_ex/λ_em 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.</p></div

Possibilities for the future design of advanced orange and far-red fluorescent proteins.

<p>Possibilities for the future design of advanced orange and far-red fluorescent proteins.</p

The differences in the immediate chromophore environment between the parental and successor proteins.

<p>(<b>A</b>) The difference between mOrange and LSSmOrange. (<b>B</b>) The difference between mOrange and PSmOrange. (<b>C</b>) The difference between PSmOrange and PSmOrange2.</p

Amino acid differences between the parental and successor proteins in 3D.

<p>(<b>A</b>) The transformation of mOrange into LSSmOrange. (<b>B</b>) The transformation of mOrange into PSmOrange. (<b>C</b>) The transformation of PSmOrange into PSmOrange2.</p

Amino acid alignment of mOrange, LSSmOrange, PSmOrange and PSmOrange2.

<p>The chromophore-forming tri-peptides are highlighted in yellow.</p

The structures of the PSmOrange and DsRed chromophores.

<p>(<b>A</b>) The structure of the orange form of the PSmOrange chromophore. (<b>B</b>) The modeled structure of the photoconverted far-red form of the PSmOrange chromophore. (<b>C</b>) The structure of the DsRed chromophore.</p

Evolution of the subfamily of orange fluorescent proteins.

<p>(<b>A</b>) Phylogenic tree showing the history of the development of different orange fluorescent proteins. (<b>B</b>) Chemical structures of the chromophores found in orange fluorescent proteins.</p

Data collection and refinement statistics.

a<p>Data in parentheses are given for the outermost resolution shells: 1.98–1.95 Å for PSmOrange, 1.32–1.30 Å for PSmOrange2, and 1.45–1.40 Å for LSSmOrange.</p>b<p>R_merge  =  Σ_hklΣ_j |I_j(hkl) – |/Σ_hklΣ_j||, where I_j is the intensity measurement for reflection j and <i> is the mean intensity over j reflections.</i></p><i>c<p>R_work/(R_free)  =  Σ ||F_o(hkl)| – |F_c(hkl)||/Σ |F_o(hkl)|, where F_o and F_c are observed and calculated structure factors, respectively. No σ-cutoff was applied. 5% of the reflections were excluded from refinement and used to calculate R_free.</p></i

Spectral and evolutionary features of orange fluorescent proteins.

<p>*The values for EGFP are given for comparison.</p><p>**DsRed and DsRed-Express2 are red fluorescent proteins.</p>1<p>λ_ex^max/λ_em^max - excitation and emission maxima.</p>2<p>E_mol - extinction coefficient.</p>3<p>Φ_F -quantum yield.</p>4<p>B - brightness (E_mol × Φ_F)/1000.</p>5<p>B_rel^EGFP - brightness relative to EGFP (product of Φ_F and E_mol compared to the brightness of EGFP (53,000 M⁻¹ cm⁻¹×0.60) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099136#pone.0099136-Patterson1" target="_blank">[52]</a>).</p

An evolution of orange fluorescent proteins derived from KO, DsRed and zFP538 with mutations critical to the phenotype of each variant.

<p>An evolution of orange fluorescent proteins derived from KO, DsRed and zFP538 with mutations critical to the phenotype of each variant.</p