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

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

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

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

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

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

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

Crystal Structure of the Marburg Virus GP2 Core Domain in Its Postfusion Conformation

Marburg virus (MARV) and Ebola virus (EBOV) are members of the family <i>Filoviridae</i> (“filoviruses”) and cause severe hemorrhagic fever with human case fatality rates of up to 90%. Filovirus infection requires fusion of the host cell and virus membranes, a process that is mediated by the envelope glycoprotein (GP). GP contains two subunits, the surface subunit (GP1), which is responsible for cell attachment, and the transmembrane subunit (GP2), which catalyzes membrane fusion. The GP2 ectodomain contains two heptad repeat regions, N-terminal and C-terminal (NHR and CHR, respectively), that adopt a six-helix bundle during the fusion process. The refolding of this six-helix bundle provides the thermodynamic driving force to overcome barriers associated with membrane fusion. Here we report the crystal structure of the MARV GP2 core domain in its postfusion (six-helix bundle) conformation at 1.9 Å resolution. The MARV GP2 core domain backbone conformation is virtually identical to that of EBOV GP2 (reported previously), and consists of a central NHR core trimeric coiled coil packed against peripheral CHR α-helices and an intervening loop and helix–turn–helix segments. We previously reported that the stability of the MARV GP2 postfusion structure is highly pH-dependent, with increasing stability at lower pH [Harrison, J. S., Koellhoffer, J. K., Chandran, K., and Lai, J. R. (2012) <i>Biochemistry</i> <i>51</i>, 2515–2525]. We hypothesized that this pH-dependent stability provides a mechanism for conformational control such that the postfusion six-helix bundle is promoted in the environments of appropriately mature endosomes. In this report, a structural rationale for this pH-dependent stability is described and involves a high-density array of core and surface acidic side chains at the midsection of the structure, termed the “anion stripe”. In addition, many surface-exposed salt bridges likely contribute to the stabilization of the postfusion structure at low pH. These results provide structural insights into the mechanism of MARV GP2-mediated membrane fusion

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

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

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