Fluorophore Conformation in Green Fluorescent Protein: A Quantum Mechanics/Molecular Mechanics Study
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Fluorophore Conformation in Green Fluorescent Protein: A Quantum Mechanics/Molecular Mechanics Study Steven Trohalaki,1 Soumya S. Patnaik,1 and Ruth Pachter Air Force Research Laboratory, Materials & Manufacturing Directorate, Wright-Patterson Air Force Base, OH 45433-7702 1
The Anteon Corporation, 5100 Springfield Pike, Dayton, OH 45431-1231
ABSTRACT Green Fluorescent Protein (GFP) is a widely used fluorescent marker exhibiting two excitation peaks – a strong peak at 398 nm and a second at 475 nm, with the fluorescence at ca. 510 nm. Its molecular structure consists of a β-barrel composed of 11 β-strands and a central helix containing the fluorophore. Two different forms of the fluorophore – a protonated/neutral fluorophore and a de-protonated/anionic fluorophore – are thought to be responsible for the two distinct spectroscopic states. Notably, the isolated fluorophore in solution is efficiently quenched. Conformational flexibility within the protein cavity is an implicitly important factor that governs the photochemistry of GFP. However, the literature contains accounts of studies that reach conflicting conclusions, claiming that either the fluorophore’s barrier to internal rotation is negligibly small or that the protein cavity is not complementary to a planar fluorophore. In this work, we calculate the torsional potential of one of the two exocyclic bonds that connect the two rings in the fluorophore, taking into account its immediate environment by applying a quantum mechanics/molecular mechanics method, with the ultimate aim of evaluating the protein-environment effects on the fluorescence.
INTRODUCTION Green Fluorescent Protein (GFP), naturally occurring in the jellyfish Aequorea victoria, has found widespread application in biochemistry, cell biology, and molecular genetics as a fluorescent marker [1]. GFP can be used in living systems because the formation of the fluorophore by autocatalytic, post-translational cyclization of three consecutive internal residues in the primary structure does not require a cofactor [1]. GFP has also recently been shown to be a very efficient two-photon absorber [2,3]. GFP consists of 238 amino acid residues and has a fluorescence quantum yield of 0.8 [1]. The fluorophore (see figure 1) is a π-conjugated system. Crystallographic studies have shown that the fluorophore is embedded in a hydrogen-bonded network within a β-barrel, which is composed of 11 β-strands, with approximate dimensions of 40 Å in length and 30 Å in diameter [4]. Molecular dynamics (MD) simulations have been used to show that, although the cavity provided by the β-barrel is large, its shape is not complementary to the fluorophore in a planar conformation [5]. Denaturation of GFP results in rapid quenching of the fluorescence [6]. This is interpreted as evidence that the β-barrel enhances fluorescence by protecting the fluorophore
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from quenching by oxygen and attack by hydronium ions and by imposing conformational constraints on the fluorophore [1]. The absorption spectrum of GFP display
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