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. 2015 Jan 6;108(1):126-32.
doi: 10.1016/j.bpj.2014.11.1859.

Estimating Orientation Factors in the FRET Theory of Fluorescent Proteins: The TagRFP-KFP Pair and Beyond

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Free PMC article

Estimating Orientation Factors in the FRET Theory of Fluorescent Proteins: The TagRFP-KFP Pair and Beyond

Maria Khrenova et al. Biophys J. .
Free PMC article

Abstract

The orientation factor κ(2), one of the key parameters defining Förster resonance energy transfer efficiency, is determined by the transition dipole moment orientations of the donor and acceptor species. Using the results of quantum chemical and quantum mechanical/molecular mechanical calculations for the chromophore-containing pockets in selected colored proteins of the green fluorescent protein family, we derived transition dipole moments corresponding to the S0,min → S1 excitation for green fluorescent protein, red fluorescent protein (TagRFP), and kindling fluorescent protein, and the S1,min → S0 emission for TagRFP. These data allowed us to estimate κ(2) values for the TagRFP-linker-kindling fluorescent protein tetrameric complex required for constructing novel sensors.

Figures

Figure 1
Figure 1
Chemical structures of the chromophores from the fluorescent proteins: (a) GFP, (b) TagRFP, (c) KFP. Only the oxygen and nitrogen atoms are designated. To see this figure in color, go online.
Figure 2
Figure 2
A part of the molecular cluster for the chromophore-containing pocket of TagRFP. Selected distances between heavy atoms (in Å) are arranged as follows: QC cluster model (top), QM/MM model (middle), and crystal structure PDB ID 3M22 (bottom). In this and forthcoming figures, carbon atoms are colored in green, oxygen in red, and nitrogen in blue. To see this figure in color, go online.
Figure 3
Figure 3
Structures of the TagRFP chromophore and TDM orientations. Upper graph refers to the S0 minimum and the S0,min → S1 transition; bottom graph refers to the S1 minimum and the S1,min → S0 transition. Distances (in Å) illustrate the bond-length changes at the methylene bridge upon excitation. TDM vectors connecting the phenolic O and N3 are indicated by double-arrow lines. The inset in the upper-right corner illustrates that the chromophore is essentially nonplanar. To see this figure in color, go online.
Figure 4
Figure 4
A part of the molecular cluster that includes the chromophore-containing domain of KFP. Selected distances between heavy atoms are given in Å. The TDM direction for the S0,min → S1 excitation is shown as a solid, double-arrow line. To see this figure in color, go online.
Figure 5
Figure 5
Computed TDM for GFP with the anionic chromophore. The result of our approach is shown as a solid black vector. The vector estimated in (18) is indicated by a dashed, double-arrow line. To see this figure in color, go online.
Figure 6
Figure 6
Structure of the TagRFP-linker-KFP (tetramer) complex. The chromophores of the donor, D (from TagRFP), and four acceptors, Ai (from KFP), are represented by the solid balls. To see this figure in color, go online.
Figure 7
Figure 7
Dimeric structure of the Val-44-Ala mutant of KillerRed by motifs of PDB ID 4B30 (38). TDM directions are indicated by solid, double-arrow lines. The inset at the bottom shows the orientation angles of the TDM vectors of the crystal geometry. To see this figure in color, go online.

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