Theoretical Computer-Aided Mutagenic Study on the Triple Green Fluorescent Protein Mutant S65T/H148D/Y145F.
Green fluorescent protein (GFP) mutant S65T/H148D has been proposed to host a photocycle that involves an excited-state proton transfer between the chromophore (Cro) and the Asp148 residue and takes place in less than 50 fs without a measurable kinetic isotope effect. It has been suggested that the interaction between the unsuspected Tyr145 residue and the chromophore is needed for the ultrafast sub-50 fs rise in fluorescence. To verify this, we have performed a computer-aided mutagenic study to introduce the additional mutation Y145F, which eliminates this interaction. By means of QM/MM molecular dynamics simulations and time-dependent density functional theory studies, we have assessed the importance of the Cro-Tyr145 interaction and the solvation of Asp148 and shown that in the triple mutant S65T/H148D/Y145F a significant loss in the ultrafast rise of the Stokes-shifted fluorescence should be expected.
Department de Química, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona (Spain).
Pau
P
Armengol
Department de Química, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona (Spain). ricard.gelabert@uab.cat.
Ricard
R
Gelabert
Department de Química, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona (Spain).
Miquel
M
Moreno
Department de Química, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona (Spain).|Institut de Bioquímica i de Biomedicina, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona (Spain).
José M
JM
Lluch
D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics*; D056004:Molecular Dynamics Simulation; D050505:Mutant Proteins / Q000737:chemistry*; D009154:Mutation; D011789:Quantum Theory; D013997:Time Factors
D050505:Mutant Proteins; D049452:Green Fluorescent Proteins
density functional calculations; molecular mechanics; protein structures; quantum mechanics; time-resolved fluorescence spectrum