4OGS

Crystal structure of GFP S205A/T203V at 2.2 A resolution

Summary for 4OGS

• Entry DOI: 10.2210/pdb4ogs/pdb
• Related: 2QLE
• Descriptor: Green fluorescent protein, CHLORIDE ION (3 entities in total)
• Functional Keywords: beta-can, fluorescent protein
• Biological source: Aequorea victoria (Jellyfish)
• Total number of polymer chains: 2
• Total formula weight: 53890.27

Authors

Remington, S.J.,Saif, M. (deposition date: 2014-01-16, release date: 2014-05-14, Last modification date: 2024-11-27)

Primary citation

Wineman-Fisher, V.,Simkovitch, R.,Shomer, S.,Gepshtein, R.,Huppert, D.,Saif, M.,Kallio, K.,Remington, S.J.,Miller, Y.
Insight into the structure and the mechanism of the slow proton transfer in the GFP double mutant T203V/S205A.
Phys Chem Chem Phys, 16:11211-11223, 2014

PubMed Abstract: Mutations near the fluorescing chromophore of the green fluorescent protein (GFP) have direct effects on the absorption and emission spectra. Some mutants have significant band shifts and most of the mutants exhibit a loss of fluorescence intensity. In this study we continue our investigation of the factors controlling the excited state proton transfer (PT) process of GFP, in particular to study the effects of modifications to the key side chain Ser205 in wt-GFP, proposed to participate in the proton wire. To this aim we combined mutagenesis, X-ray crystallography, steady-state spectroscopy, time-resolved emission spectroscopy and all-atom explicit molecular dynamics (MD) simulations to study the double mutant T203V/S205A. Our results show that while in the previously described GFP double mutant T203V/S205V the PT process does not occur, in the T203V/S205A mutant the PT process does occur, but with a 350 times slower rate than in wild-type GFP (wt-GFP). Furthermore, the kinetic isotope effect in the GFP double mutant T203V/S205A is twice smaller than in the wt-GFP and in the GFP single mutant S205V, which forms a novel PT pathway. On the other hand, the crystal structure of GFP T203V/S205A does not reveal a viable proton transfer pathway. To explain PT in GFP T203V/S205A, we argue on the basis of the MD simulations for an alternative, novel proton-wire pathway which involves the phenol group of the chromophore and water molecules infrequently entering from the bulk. This alternative pathway may explain the dramatically slow PT in the GFP double mutant T203V/S205A compared to wt-GFP.

PubMed: 24776960
DOI: 10.1039/c4cp00311j

Experimental method

X-RAY DIFFRACTION (2.21 Å)