Proteins respond to electrostatic perturbations through complex reorganizations of their charged and polar groups, as well as those of the surrounding media. These solvation responses occur both in the protein interior and on its surface, though the exact mechanisms of solvation are not well understood, in part because of limited data on the solvation responses for any given protein. Here, we characterize the solvation kinetics at sites throughout the sequence of a small globular protein, the B1 domain of streptococcal protein G (GB1), using the synthetic fluorescent amino acid Aladan. Aladan was incorporated into seven different GB1 sites, and the time-dependent Stokes shift was measured over the femtosecond to nanosecond time scales by fluorescence upconversion and time-correlated single photon counting. The seven sites range from buried within the protein core to fully solvent-exposed on the protein surface, and are located on different protein secondary structures including beta-sheets, helices, and loops. The dynamics in the protein sites were compared against the free fluorophore in buffer. All protein sites exhibited an initial, ultrafast Stokes shift on the subpicosecond time scale similar to that observed for the free fluorophore, but smaller in magnitude. As the probe is moved from the surface to more buried sites, the dynamics of the solvation response become slower, while no clear correlation between dynamics and secondary structure is observed. We suggest that restricted movements of the surrounding protein residues give rise to the observed long time dynamics and that such movements comprise a large portion of the protein's solvation response. The proper treatment of dynamic Stokes shift data when the time scale for solvation is comparable to the fluorescence lifetime is discussed.

Solvent reorganization around the excited state of a chromophore leads to an emission shift to longer wavelengths during the excited-state lifetime. This solvation response is absent in wildtype green fluorescent protein, and this has been attributed to rigidity in the chromophore's environment necessary to exclude nonradiative transitions to the ground state. The fluorescent protein mPlum was developed via directed evolution by selection for red emission, and we use time-resolved fluorescence to study the dynamic Stokes shift through its evolutionary history. The far-red emission of mPlum is attributed to a picosecond solvation response that is observed at all temperatures above the glass transition. This time-dependent shift in emission is not observed in its evolutionary ancestors, suggesting that selective pressure has produced a chromophore environment that allows solvent reorganization. The evolutionary pathway and structures of related fluorescent proteins suggest the role of a single residue in close proximity to the chromophore as the primary cause of the solvation response.GFP ͉ solvation response ͉ ultrafast T he Stokes shift between the absorption and emission of a chromophore reflects the displacement in potential surface between the ground and excited states and loss of vibrational energy in the excited state. For chromophores that have a large increase in dipole moment between the ground and excited state, the fluorescence emission maximum often depends strongly on solvent polarity in simple fluid solvents and the emission is observed to shift to longer wavelengths during the excited-state lifetime. Such dynamic Stokes shifts have been extensively studied as a probe of solvent polarity and dynamics (1-3). For a chromophore in a protein, the solvent is much more organized and constrained than in a simple solvent, so the capacity for solvation is expected to be quite different, yet important for function, from that in a simple solvent. There are relatively few studies of dynamic Stokes shifts in proteins: a few dye-protein complexes (4-7), antibodies bound to fluorescein (8), surface tryptophan residues as a probe of hydration (9-11), unnatural amino acids (12, 13), studies on cytochrome c (14, 15), and photosynthetic antenna complexes (16). In contrast to smallmolecule solvents, it should in principle be possible to dissect the contributions of individual amino acids to the solvation response of a protein and even its evolutionary history, although such an analysis has not to our knowledge been reported.GFPs would seem to be ideal candidates for measurements of the dynamic Stokes shift because the chromophore is intrinsic to the protein and structurally well characterized (17), and the 6-to 7-debye change in dipole moment upon excitation of the chromophore (18) is as large as for most dyes used to probe solvation dynamics in simple fluid solvents. Furthermore, recent studies confirmed that, like conventional solvation probes, the emission of synthetic GFP chromophores shifts substantially as a func...

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...