Fig 1.
Comparison of photobleaching kinetics.
(A) FPs with a maximum emission wavelength of aprox. 670 nm. (B) Monomeric FPs with a maximum emission wavelength above 680 nm.
Table 1.
Characteristics of the near-infrared fluorescent protein variants obtained in this study.
Fig 2.
Gel filtration analysis and SDS-PAGE electrophoresis of FPs.
Size exclusion chromatography of FPs (approximate 3–4 mg/ml) in PBS using a Supdex 200 10/300GL column. The elution peaks of eqFP670 and mNeptune serve as monomeric and dimeric controls, respectively (see reference 3 and reference 7). The elution volume of eqFP670 (shown as solid blue line) and mNeptune (shown as solid cyan line) were used as the standard reference of dimer and monomer. (A) The variants of mNeptune, G41Q (solid green line), G41Q/C158M (short black dashed line) and C158M (short red dashed line), have the same oligomeric characteristics to that of mNeptune, suggesting that they are monomer. (B) mNeptune681 (solid gray line) and its several monomeric deriveatives, Q159A (solid black line), Q159P (solid pink line), Q159N (solid wine line) and Q159G (solid dark cyan line), with the maximum emission wavelength above 680 nm display the same monomeric property as that of mNeptune. (C) The variants of mNeptune681, Q159W (solid orange line), Q159I (solid magenta line), Q159L (solid dark yellow line), Q159M (short navy dashed line), Q159T (solid olive line), Q159V (solid yellow line) and Q159C (solid red line), with the maximum emission wavelength around 680 nm, display the weak dimeric characteristics. (D) Two independent mutations Q159Y (short pink dashed line) and Q159F (short dark yellow dashed line) in mNeptune681 cause proteins dimerization. (E)-(F) After gel filtration, SDS-Polyacrylamide electrophoresis of FP variants. (E) mNeptune (lane 1), mNeptune_G41Q (lane 2), mNeptune_C158M (lane 3), mNeptune_G41Q/C158M (lane 4). (F) mNeptune681 (lane 1) and its derivatives (lane 2 to 14), they are mNeptune681_Q159G (lane 2), mNeptune_Q159P (lane 3), mNeptune_Q159F (lane 4), mNeptune_Q159Y (lane 5), mNeptune_Q159C (lane 6), mNeptune_Q159V (lane 7), mNeptune_Q159T (lane 8), mNeptune_Q159I (lane 9), mNeptune_Q159L (lane 10), mNeptune_Q159A (lane 11), mNeptune_Q159M (lane 12), mNeptune_Q159N (lane 13), mNeptune_Q159W (lane 14).
Fig 3.
Sequence alignments and structures comparison of mNeptune’s two NIR mutant forms with NIR FP eqFP670.
(A) Amino-acids in the vicinity of chromophore are marked with blue rectangles, the chromophore is marked with a red rectangle. (B) The 3D structures of eqFP670 and mNeptune are marked with green ribbon using PyMol software, and the chromophore (pink color) and its surrounding different amino acids (yellow color) between two proteins are labeled with Sticks pattern. amino-acid differences adjacent to the chromophore are marked in blue showed in the crystal structure diagrams.
Table 2.
Contributions of individual mutations to red-shifting in mNeptune681.
Fig 4.
Immediate chromophore hydrogen bonds environment of (A) eqFP670 (PDB entry: 4EDS), (B) mNeptune (PDB entry: 3IP2).
Water molecules and hydrogen bonds measured in Å are shown as red spheres and dashed red lines, respectively.
Fig 5.
Spectral properties of mNeptune681.
(A) Absorbance spectra at pH 3.0–11.0. (B) Fluorescence excitation (dashed line) and emission (solid line) spectra at pH 6.5 (blue line) and pH 11.0 (red line). (C) Equilibrium pH dependence of fluorescence emission intensity for mNeptune681 and its monomeric variants. mNeptune681 (black line), mNeptune681_Q159A (red line), mNeptune681_Q159N (green line), mNeptune681_Q159P (blue line), mNeptune681_Q159G (Cyan line). (D) Fluorescence excitation (dashed line) and emission (solid line) spectra of mNeptune681.
Fig 6.
The extensive hydrogen-bond network resulting in the NIR emission of eqFP670 and simulated structures of mNeptune681 and its monomeric derivatives.
a and b in red color in the diagram stand for the majority conformation state and the minority conformation respectively. (A) From the crystal structure of eqFP670 (PDB entry: 4EDS). (B)-(F) From the simulated structures of mNeptune681 and its monomeric derivatives with the residue 159 mutations using homology modeling method based on the crystal structure of eqFP670. Immediate chromophore hydrogen-bond environment of (A) eqFP670, (B) mNeptune681, (C) mNeptune681_Q159A, (D) mNeptune684, (E) mNeptune681_Q159N and (F) mNeptune681_Q159G. Water molecules and hydrogen bonds measured in Å are shown as red spheres and red dashed lines, respectively. The hydrogen-bonds between residue 159 and Asn158 of these mutants are crucial to maintaining NIR fluorescence emission. Among these hydrogen-bonds, a distinctive hydrogen-bond was found in the variant Q159P, which is located between the alpha-amino nitrogen atom of Pro159 and the amide nitrogen atom of Asn158, and is the strongest and most stable hydrogen-bond due to its shortest length of 3.00 Å.