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. 2012 Jan 1;392(1):143-148.
doi: 10.1016/j.chemphys.2011.11.001. Epub 2011 Nov 19.

Water Diffusion In And Out Of The β-Barrel Of GFP and The Fast Maturing Fluorescent Protein, TurboGFP

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Water Diffusion In And Out Of The β-Barrel Of GFP and The Fast Maturing Fluorescent Protein, TurboGFP

Binsen Li et al. Chem Phys. .
Free PMC article

Abstract

The chromophore of fluorescent proteins is formed by an internal cyclization of the tripeptide 65SYG67 fragment and a subsequent oxidation. The oxidation is slow - the kinetics of this step is presumably improved in fast maturing GFPs. Water molecules can aid in the chromophore formation. We have used 50ns molecular dynamics simulations of the mature and immature forms of avGFP and TurboGFP to examine the diffusion of water molecules in-and-out of the protein β-barrel. Most crystal structures of GFPs have well-structured waters within hydrogen-bonding distance of Glu222 and Arg96. It has been proposed that they have an important role in chromophore formation. Stable waters are found in similar positions in all simulations conducted. The simulations confirm the existence of a pore that leads to the chromophore in the rapidly maturing TurboGFP; decreased water diffusion upon chromophore formation; and increased water diffusion due to the pore formation.

Figures

Figure 1
Figure 1
The cyclization-oxidation-dehydration mechanism proposed by Wachter(18) (right) and the cyclization-dehydration-oxidation mechanism proposed by Getzoff(19) (left).
Figure 2
Figure 2
Possible roles played by the structurally conserved waters in the chromophore formation(1,21). The cyclization-oxidation-dehydration mechanism proposed by Wachter(18,22) (top and bottom left) and the cyclization-dehydration-oxidation mechanism proposed by Getzoff(19) (bottom right).
Figure 3
Figure 3
Anionic (A) and neutral (B) forms of the chromophore. Neutral immature form (C).
Figure 4
Figure 4
Highest ranked channel MolAxis(40,41) pores and channels for each of the structures. The lower the channel number the higher flux score (shorter and wider). A) avGFP channel 1: Bottleneck radius 0.79 Å B) avGFP channel 2: Bottleneck radius 0.34 Å C) TurboGFP channel 1: Bottleneck radius 1.23 Å. (Note how channel 2 in avGFP and channel 1 in TurboGFP exit at the same point, however avGFP has a much smaller bottleneck.) D) TurboGFP V197L mutant channel 1: Bottleneck radius 1.00 Å E) Immature 1GFL channel 1: Bottleneck radius 0.35 Å F) immature TurboGFP channel 1: Bottleneck radius 1.03 Å.
Figure 5
Figure 5
Residues surrounding bifurcated pore in avGFP are indicated by red CPK spheres (left). The residues surrounding the pore purportedly responsible for TurboGFP’s rapid chromophore formation are shown as red CPK spheres (right). The chromophore, tube representation, is visible through the pore. The pores surrounded by the residues colored blue and yellow are located on the lid-edges and are less permanent.
Figure 6
Figure 6
700 snapshots taken during the 3.6ns it takes one of the water molecules (red oxygen and white hydrogen) to exit through the primary pore of the TurboGFP β-barrel. Water positions were superimposed on the average TurboGFP structure using VMD. (43)
Figure 7
Figure 7
Left: An Isostar overlay plot of all the crystal structures of GFP and GFP-like proteins with fully cyclized chromophores found with Relibase+ in the Protein Databank. The imidazolone rings of all the structures were overlapped in order to show the orientation of the glutamic acid residues (Glu222 in GFP numbering) and the water molecule discussed in the text relative to the position of the chromophoric imidazolone ring. Right: 10,416 snapshots taken during the 50 ns simulation of the anionic form of TurboGFP. Water positions (white dots) were superimposed on the average TurboGFP structure using VMD. Only the chromophore and Glu210 of average TurboGFP shown. (43)
Figure 7
Figure 7
Left: An Isostar overlay plot of all the crystal structures of GFP and GFP-like proteins with fully cyclized chromophores found with Relibase+ in the Protein Databank. The imidazolone rings of all the structures were overlapped in order to show the orientation of the glutamic acid residues (Glu222 in GFP numbering) and the water molecule discussed in the text relative to the position of the chromophoric imidazolone ring. Right: 10,416 snapshots taken during the 50 ns simulation of the anionic form of TurboGFP. Water positions (white dots) were superimposed on the average TurboGFP structure using VMD. Only the chromophore and Glu210 of average TurboGFP shown. (43)

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