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Review
. 2016 Aug:39:124-133.
doi: 10.1016/j.sbi.2016.07.010. Epub 2016 Jul 25.

Structure-guided wavelength tuning in far-red fluorescent proteins

Ho-Leung Ng  1 , Michael Z Lin  2
Affiliations
Review

Structure-guided wavelength tuning in far-red fluorescent proteins

Ho-Leung Ng et al. Curr Opin Struct Biol. 2016 Aug.

Abstract

In recent years, protein engineers have succeeded in tuning the excitation spectra of natural fluorescent proteins from green wavelengths into orange and red wavelengths, resulting in the creation of a series of fluorescent proteins with emission in the far-red portions of the optical spectrum. These results have arisen from the synergistic combination of structural knowledge of fluorescent proteins, chemical intuition, and high-throughput screening methods. Here we review structural features found in autocatalytic far-red fluorescent proteins, and discuss how they add to our understanding of the biophysical mechanisms of wavelength tuning in biological chromophores.

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Figures

Figure 1
Figure 1
Basic structural biology of RFPs. (A) Structures of GFP and DsRed chromophores. The GFP chromophore (left), shown with carbon in green, nitrogen blue, and oxygen red, is a π-conjugated system consisting of a phenolate ring (front) connected to a imidazolinone ring (rear) by a methylene bridge. The DsRed chromophore (right), shown with carbon in pink, consists of the same groups formed from cyclization of a Gln-Tyr-Gly sequence, but with conjugation extended through an acylimine group formed by additional oxidation of the backbone N-Cα bond of the Gln residue. (B) Altered electronic interactions of the chromophore with its environment in mCherry. In DsRed (left), the positive charges of Lys70 and Lys163 stabilize the ground state, where electron density preferentially resides in the phenol ring. In mCherry (right), movement of Lys70 away from the chromophore and mutation of Lys163 to Gln reduces this effect. Movement of Lys70 in mCherry is due to loss of electrostatic repulsion by Leu83. Side chains and alpha carbons of these important amino acids are shown a spheres, with carbon in white, nitrogen blue, and oxygen red.
Figure 2
Figure 2
Chromophore isomerization, reduced hydrogen bonding, and altered electrostatic interactions in mKate. The chromophore rings in TagRFP are in a trans conformation, where the phenolate oxygen makes two hydrogen bonds (cyan dotted lines) and the His197 engages in a cation-π interaction with the phenolate ring. In mKate, mutations Asn143Ser, Phe174Leu, and His197Arg cause the chromophore to assume a cis conformation, losing one hydrogen bond and the cation-π interaction.
Figure 3
Figure 3
Structural basis of red-shifted excitation in far-RFPs. (A) Increased chromophore planarity and a new hydrogen bond in Neptune and mCardinal. In mKate, the Arg197 side chain extends across and to the side of the phenolate ring of the chromophore, which is not coplanar with the imidazolinone ring. In Neptune, Cys158 pushes Arg197 to a new position above the chromophore, allowing for a more coplanar arrangement of the two chromophore rings. In addition, mutation Met41Gly creates a new cavity for a water molecule (red dotted sphere) to reside in near the acylimine. The white sphere labeled Gly41 is the Gly41 alpha carbon. Hydrogen bonding between the water molecule and the acylimine oxygen stabilizes the excited state and leads to an excitation red-shift. In mCardinal, the role of the water molecule is replaced by Gln41, which is held in position by Thr28. (B) Excited-state energetic relaxations near the acylimine in the excited state of mPlum. mPlum contains a new hydrogen bond from Glu16 to the acylimine oxygen of the chromophore. Conformational changes to the chromophore or to Glu16 occur in the excited state, causing a large Stokes shift.
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