title: Structure and excited-state proton transfer in the GFP S205A mutant.
abstract: To further explore excited state proton transfer (ESPT) pathways within green fluorescent protein (GFP), mutagenesis, X-ray crystallography, and time-resolved and steady-state optical spectroscopy were employed to create and study the GFP mutant S205A. In wild type GFP (wt-GFP), the proton transfer pathway includes the hydroxyl group of the chromophore, a water molecule, Ser205, and Glu222. We found that the ESPT rate constant of S205A is smaller by a factor of 20 than that of wt-GFP and larger by a factor of 2 in comparison to the ESPT rate of S205V mutant which we previously characterized. (1)  High resolution crystal structures reveal that in both S205A and S205V mutants, an alternative proton transfer pathway is formed that involves the chromophore hydroxyl, a bridging water molecule, Thr203 and Glu222. The slow PT rate is explained by the long (∼3.2 Å and presumably weak) hydrogen bond between Thr203 and the water molecule, compared to the 2.7 Å normal hydrogen bond between the water molecule and Ser205 in wt-GFP. For data analysis of the experimental data from both GFP mutants, we used a two-rotamer kinetic model, assuming only one rotamer is capable of ESPT. Data analysis supports an agreement with the underlying assumption of this model.
authors: Raymond and Beverly Sackler Faculty of Exact Sciences, School of Chemistry Tel Aviv University, Tel Aviv 69978, Israel.
authors: Yuval
authors: Y
authors: Erez
authors: Rinat
authors: R
authors: Gepshtein
authors: Itay
authors: I
authors: Presiado
authors: Kristina
authors: K
authors: Trujillo
authors: Karen
authors: K
authors: Kallio
authors: S James
authors: SJ
authors: Remington
authors: Dan
authors: D
authors: Huppert
mesh_terms: D019943:Amino Acid Substitution; D018360:Crystallography, X-Ray; D041961:Deuterium Exchange Measurement; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D009154:Mutation; D017434:Protein Structure, Tertiary; D011522:Protons; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013696:Temperature
chemical_list: D011522:Protons; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins