Neptune spectra and sequence. (A) Absorbance spectra of oxyhemoglobin (red), deoxyhemoglobin (dotted red), mKate (green dashed), mKate S158C (orange), mKate2 (dotted black), Neptune (dashed blue), and mNeptune (light blue). Units are M^-1cm^-1 versus nm. Hemoglobin spectra are previously published (Stamatas et al., 2006). Inset: Purified mCherry, mKate, S158C, and Neptune proteins at 0.5 mg/mL in visible light. (B) Normalized excitation (left) and emission (right) spectra of mKate (green dashed), mKate S158C (orange), Neptune (dashed blue), and mNeptune (light blue). mKate2 excitation and emission spectra are identical to mKate, as described (Shcherbo et al., 2009). (C) Sequence of mNeptune aligned with its parent, mKate. Changes responsible for the red shift are highlighted in red. The additional monomerization mutation to create mNeptune from Neptune is highlighted in green.

A functionally monomeric variant of Neptune for protein fusions. (A) Neptune exhibits more dimerization relative to mKate, while mNeptune exhibits less. Normalized elution profiles on size-exclusion HPLC (thin traces, axis on left) and estimated molecular weights upon elution by in-line light scattering (thick traces, axis on right) are shown for dTomato (orange solid), Neptune (dark blue dashed), mNeptune (light blue solid), mKate (green dashed), mKate2 (red dashed), and mCherry (maroon solid). Dots show estimated molecular weights upon elution by in-line light scattering. Proteins were analyzed sequentially in the same day at 5 μM in PBS. mCherry and dTomato serve as monomeric and dimeric controls, respectively. (B) mNeptune-actin and mNeptune-tubulin are correctly localized upon expression in HeLa cells, similarly to mKate2 fusions. Scale bar, 10 μm.

Changes in Arg-197 conformation and interactions in Neptune. (A) In mKate (upper), the Arg-197 and Ser-158 side chains (in space-fill representation) fill a space adjacent to the chromophore (stick), and the two rings of the chromophore are non-coplanar. A water molecule occupies a space above the methylidene bridge. Atoms are colored as in Figure 4A. In Neptune (lower), only Cys-158 is adjacent to the chromophore, while Arg-197 extends along the chromophore axis with its guanidinium group above the methylidene bridge. A water molecule occupies the former location of the guanidinium group. (B) In mKate (left), Arg-197 is hydrogen-bonded to water molecules. In Neptune (right), it also participates in a hydrogen-bond network involving Lys-67, Tyr-178, and Glu-145, including an unusual Arg-Lys hydrogen bond. Possible hydrogen bonds (interatomic distances < 3.5 Å) are shown as dotted lines. Colors are as in Figure 4A, except water molecules are in turquoise. (C) Detailed model of the hydrogen-bonding network with proposed donor-acceptor relationships. Hydrogen atoms are attached to donors with solid lines and to acceptors with dotted lines. Water molecules are omitted for clarity.

Performance of Neptune in cells and animals. (A) Design of a monocistronic adenovirus vector for coexpression of GFP and Neptune (Ad-Neptune) or (Ad-mKate) in hepatocytes. (B) Sections of liver were removed from a mouse injected with Ad-Neptune or Ad-mKate and imaged at red or far-red wavelengths on a confocal microscope. GFP was also imaged to confirm expression of the cistron. Fluorescence intensities are shown in the chart. Scale bar, 50 μm. (C) Epifluorescence of Neptune in the liver of a living mouse acquired on a Maestro imaging system (CRI) with excitation and emission at 610-630 nm and 660-700 nm, respectively. Scale bar, 5 mm. (D) Dual fluorescence imaging in the optical window is possible with Neptune and infrared fluorescent protein (IFP). Images of live mice expressing Neptune or IFP1.1 in liver were acquired with Cy5 and Cy5.5 channels. Excitation/emission wavelengths used were 610-630/660-700 nm for Cy5 and 625-675/700-730 nm for Cy5.5. In the right column, spectral unmixing was used to correct for Neptune cross-excitation and -detection in the Cy5.5 channel. Restricting IFP excitation to above 650 nm with custom filters would produce similar results.

Structural basis for red-shifting in Neptune. (A) Crystal structures of mKate at pH 7 (Pletnev et al., 2008) and Neptune at pH 7 were aligned, with mKate colored pink and Neptune light blue. The conjugated π system of the chromophore and side chain changes involved in red-shifting are shown in stick representation with nitrogen in blue, oxygen in red, and sulfur in yellow. Thin lines depict other side chains. (B) Neptune contains an additional hydrogen bond to the chromophore. Structures of mKate and Neptune cut away to the level of the chromophore are shown. The conjugated system of the chromophore and the Met-41 side chain are shown in stick representation colored as in (A), and the van der Waals surfaces of water oxygen atoms depicted as dotted spheres colored pink in mKate and light blue in Neptune. In Neptune, a water molecule occupies the space created by the M41G mutation and is in hydrogen bond distance of Ser-28 and the chromophore acylimine. Ser-28 is located beneath Met-41 in mKate and is not visible.