Ultrafast Excited-State Dynamics in the Green Fluorescent Protein Variant S65T/H148D. 1. Mutagenesis and Structural Studies^,

Abstract

Wild type green fluorescent protein (wt-GFP) and the variant S65T/H148D each exhibit two absorption bands, A and B, which are associated with the protonated and deprotonated chromophores respectively. Excitation of either band leads to green emission. In wt-GFP, excitation of band A (~390 nm) leads to green emission with a rise time of 10-15 picoseconds, due to excited state proton transfer (ESPT) from the chromophore hydroxyl group to an acceptor. This process produces an anionic excited state intermediate I* that subsequently emits a green photon. In the variant S65T/ H148D, the A band absorbance maximum is red-shifted to ~415 nm and as detailed in the accompanying papers (1,2), when the A band is excited, green fluorescence appears with rise time shorter than the instrument time resolution (~170 fs). Based on steady state spectroscopy and high resolution crystal structures of several variants described herein, we propose that in S65T/H148D, the red shift of absorption band A and the ultrafast appearance of green fluorescence upon excitation of band A is due to a very short (≤ 2.4 Å), and possibly low barrier, hydrogen bond between the chromophore hydroxyl and introduced Asp148.Wt-GFP exhibits two bands in absorption spectra, peaked at 395 and 475 nm and designated A and B respectively [see reviews (3)(4)(5)]. While band A is attributed to absorption of the protonated chromophore, band B is due to absorption of the deprotonated chromophore (6,7). Excitation of either band leads to green emission; however upon excitation of band A, the intensity of green emission rises on a time scale of several tens of picoseconds. This process is dramatically slowed upon deuteration of the sample, suggesting that excited state proton transfer (ESPT) from the neutral form of the chromophore generates an intermediate anionic excited state, which subsequently emits a green photon (6-9). On the basis of the crystal structures, a pathway has been proposed to allow proton transfer between the chromophore phenol moiety, through a water molecule and Ser205, to Glu222 as the terminal proton acceptor (10-12). In wt-GFP, the intensities of absorbance bands A and B are largely insensitive to pH over the range 4-10.Point mutations can lead to increased sensitivity to pH and the formation of new ESPT pathways within GFP. For example, dual emission GFPs (deGFPs) switch between a form that emits blue light at low pH and a form that emits green light at high pH. Time resolved spectroscopy and structural studies of deGFPs suggested that the dual emission behavior results † This work was supported by grants from the NIH (R01 GM42618 to S.J.R. and GM 27738 to S.G.B.). The atomic coordinates and structure factors have been deposited in the Protein Data Bank (entry 2DUF and 2DUE for GFP S65T/H148D at pH 5.6 and 10.0 respectively, 2DUG and 2DUH for GFP S65T/H148N at pH 5.0 and 9.5, 2DUI for GFP H148D at pH 9.0). *To whom correspondence and reprint requests should be addressed. Tel: (541) . Mutations can also eliminate the fluorescence fro...

An Alternate Proton Acceptor for Excited-State Proton Transfer in Green Fluorescent Protein: Rewiring GFP

“…This interpretation of the dynamics and spectroscopy of the H148D mutants is in accord with the recent high-resolution structural data from Remington and co-workers, demonstrating the presence of a very short (e2.4 Å) hydrogen (H)-bond between the chromophore phenol oxygen and D148 in S65T/H148D. 23 The consequences of the H148D mutation for radiationless decay of the I* state are described. …”

“…Recent work by Remington and coworkers on the structure of S65T/H148D GFP provides concrete evidence of the presence of such a low-barrier H-bond in these mutants. This group has resolved the structure to 1.5 Å, revealing an extremely short (e2.4 Å) H-bond connecting the chromophore to D148 (S. J. Remington, ref 23).…”

Monomeric red fluorescent proteins with a large Stokes shift

“…Structural and spectroscopic studies of the GFP mutants exhibiting ESPT suggest that their brightness depends on the chromophore coplanarity and the efficiency of the proton transfer (26). In addition, the geometry of the hydrogen bonds plays an important role in the proton transfer.…”

Excited-state structural dynamics of a dual-emission calmodulin-green fluorescent protein sensor for calcium ion imaging

“…In addition, the wtGFP mutant S65T/H148E exhibited dual emission but with strong pH dependence (47), hinting the role played by a repositioned glutamate to the chromophore. Our mutagenesis results show that the Ca 2+ -bound E61H variant has reduced blue but enhanced green fluorescence, indicative of less ESPT disruption and reduced dual-emission capability (Fig.…”

“…Previous work explored pH-dependent dual-emission GFP variants (15,47) with mutations at positions 65, 148, and 203: blue fluorescence was attributed to decreased H-bond interactions and increased hydrophobicity of the protein pocket. As an analogy, although P377 in the Ca 2+ -bound protein cannot directly participate in the extensive H-bond network due to lack of an ionizable group and restricted sidechain conformation, this mutation likely repositions other CaM residues (e.g., M375 or M379; SI Text) such that they increase the hydrophobicity of chromophore environment, promote the neutral form, reduce the available H bonds, and impede ESPT pathways by increasing the reaction barrier.…”