Elsevier

Chemical Physics

Volume 303, Issues 1–2, 2 August 2004, Pages 7-11
Chemical Physics

Hula-twisting in green fluorescent protein

https://doi.org/10.1016/j.chemphys.2004.04.022Get rights and content

Abstract

In the hula-twist (HT) process two adjacent bonds twist concertedly to give a product from simultaneous conformational and configurational isomerization. Based on quantum mechanical (QM) calculations and an isoviscosity analysis of internal conversion in green fluorescent protein (GFP) it has been proposed, in the literature, that the GFP chromophore undergoes a HT upon excitation. A volume analysis of HTs and, τ and ϕ one-bond-flips (OBF) in the chromophore model, 4-hydroxybenzylidene-2,3-dimethylimidazolidinone (I), revealed that the τ OBF displaces a larger volume than the HT or the ϕ OBF. However both the HT and ϕ OBF processes displace the same volume. Therefore the volume conserving property of the HT is not a sufficient reason for the excited chromophore to undergo a HT. Since the chromophore is unsymmetric there are 4 different possible HTs. Molecular mechanics (MM) calculations have been used to show that the protein matrix of GFP forms a cavity around the chromophore that is complementary to an excited state conformation in which the phenol and imidazolidinone rings are perpendicular to each other – a conformation that was obtained by a concerted positive 45° HT of both chromophore dihedral angles. The twisted chromophore obtained by concertedly rotating both dihedral angles in a negative direction is sterically disfavored relative to the planar and positively rotated forms. Rotating the τ and ϕ bonds in opposite directions does not produce a large change in strain energy, as the two ring systems stay in the same plane.

Introduction

Hula-twist: Torsional relaxation has been the commonly accepted mechanism for photoisomerization in simple alkenes and visual pigments. In 1985 Liu and Asato [1] proposed the hula-twist (HT) process to account for the fact that the volume demanding isomerization can take place in the short time normally associated with molecular vibrations. In the HT process two adjacent bonds twist concertedly to give a product from simultaneous conformational and configurational isomerization [2], [3]. The ring-fused stilbene analogue, which is shown in Fig. 1 and has some structural similarities to the green fluorescent protein (GFP) chromophore, has been proposed to undergo a one-bond-flip (OBF) in solution and a HT in the solid state, see Fig. 1 [4].

Green fluorescent protein (GFP): In recent years GFP from the jellyfish Aequorea victoria gained widespread interest in biochemistry and cell biology owing to the fact that it can be used as a cloneable, noninvasive marker for gene expression and protein localization in intact cells and organisms [5], [6]. Unlike other bioluminescent reporters, GFP fluoresces in the absence of any other proteins, substrates, or cofactors.

Proteolytic treatment of GFP demonstrated that the chromophore was contained in a stable hexapeptide fragment, 64FSYGVQ69 [7]. In the intact GFP, the intrinsic chromophore is formed by autocatalytic internal cyclization of the tripeptide 65SYG67 and subsequent oxidation to form the p-hydroxybenzylidene chromophore, see Fig. 2.

The structure of GFP has been described as a light in a can; the chromophore is located in the center of a can consisting of 11 β sheets. The can is a nearly perfect cylinder with a height of 42 Å and a radius of 12 Å. Deletion mapping experiments have shown that nearly the entire structure (residues 2–232) is required for chromophore formation and/or fluorescence [8].

Denatured wild-type GFP absorbs at 384 nm at neutral or acidic pH. In basic environments it absorbs at 448 nm, but neither denatured GFP [9], synthetic model chromophores [10], [11], nor the chromophore containing hexapeptide fragment fluoresce [7], [12]. There is a 104-fold increase in the fluorescence quantum yield when the chromophore is embedded in the correctly folded GFP, furthermore the hexapeptide fragment and the denatured GFP do become fluorescent at 77 K. It has been suggested that the chromophore is fluorescent in the fully folded GFP because the protein inhibits rotation around the exo-methylene double bond (τ torsion in Fig. 2) of the chromophore, and thereby reduces the loss of fluorescence due to internal conversion [13], [14]. Quantum mechanical (QM) calculations indicate that the fast internal conversion occurs from a perpendicular, τ=90°, excited state intermediate of the chromophore [15]. A temperature dependence and isoviscosity analysis has revealed that the internal conversion of GFP chromophore model compounds is essentially barrierless at room temperature, while at low temperatures or high viscosities there is evidence of a small barrier [16], [17]. Since the viscocity dependence of the rate constant for internal conversion is very weak it is likely to occur by means of a volume conserving HT [16], [18]. It has been shown that the viscosity of the surrounding medium affects the fluorescence decay of the isolated chromophore, but not that of GFP [19]. Chromophore model compounds that are non- or little substituted emit minimal fluorescence, while sterically bulky substituents modify the equilibrium between radiative and nonradiative deexcitation pathways, therefore the sterically hindered compounds are more fluorescent [20].

A model for the photophysical behavior of GFP has been proposed [21], see Fig. 3. It is based on QM calculations of a small non-peptide model compound. According to the model both the neutral (A) and anionic (B) forms of the chromophore can under go HT mediated nonadiabatic crossing (NAC).

The fluorescence of the anionic form can also be quenched by NAC due to a one-bond rotation around the τ dihedral angle shown in Fig. 2. However molecular mechanics (MM) calculations of the chromophore within GFP have shown that the protein environment, especially residues Phe165 and Thr62 severely restricts rotation around the τ dihedral angle [22], and therefore NAC is unlikely to occur through this channel. Fluorescence quenching through the zwitterionic form, which may undergo NAC by one-bond rotation around the ϕ dihedral angle is much more likely as rotations around the ϕ are not hindered by the protein [22].

In this paper we would like to examine the energetics, and the volume conserving properties of the concerted HT dihedral rotation of the chromophore within GFP.

Section snippets

Experimental

The coordinates of the wild-type GFP solid state structure (1GFL) [23]. were obtained from the Protein Data Bank, hydrogen atoms were added to protein and solvent atoms as required. The AMBER* force field of MacroModel v8.0 [24] was used for all calculations. A “hot” area with a radius of 8 Å from the chromophore was used in all calculations. It was held in place with three subsequent sub-shells each extending an additional 2.00 Å with increasing atomic restraints of 100, 200 and 400 kJ/Å. The

Results and discussion

Our earlier calculations have shown that the GFP cavity does not have a shape that is complementary with a planar chromophore and that the chromophore in GFP has more dihedral freedom around the ϕ torsion than around the τ torsion [22]. However, we did not examine hula-twisting of the chromophore within GFP.

Is the HT of p-hydroxybenzylidene imidazolidinone volume conserving? The possibility that a HT is involved in the photochemistry of GFP has been suggested on the basis of its volume

Conclusion

A volume analysis of the τ and ϕ OBF and HTs in the chromophore model compound I, revealed that the τ OBF displaces a larger volume than both the HT and the ϕ OBF. However, the HT and ϕ OBF processes displace the same volume, and therefore the volume conserving property of the HT is not a sufficient reason for the excited chromophore to undergo a HT. MM calculations have shown that the protein matrix of GFP forms a cavity around the chromophore that is complementary to an excited state

Acknowledgements

MZ is a Henry Dreyfus Teacher-Scholar. We thank the NIH (Grant GM59108-02) and the Research Corporation for financial support.

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