We have determined the lifetime of the OH-stretch vibration in pure liquid water as a function of temperature using femtosecond mid-infrared pump–probe spectroscopy. The lifetime T1 increases from 260±18 fs at T=298 K to 320±18 fs at T=358 K. The increase in lifetime with temperature can be quantitatively explained from the decrease in overlap resonance between the OH-stretch vibration and the overtone of the H–O–H bending mode.

We have studied the equilibration dynamics of liquid water and alcohols following a local deposition of energy using time-resolved femtosecond mid-infrared pump-probe spectroscopy. The equilibration dynamics is monitored via the spectral response of the OH-stretch vibration. It is found that the equilibration leads to complicated changes of the absorption band of the OH-stretch vibration including a shift of the absorption band and a decrease of the absorption cross section. Interestingly, these spectral changes do not occur simultaneously, which indicates that they are associated with the equilibration dynamics of different lowfrequency modes. For water, we find an equilibration time constant of 0.55 ( 0.05 ps. We observe that the equilibration time strongly increases going from water to alcohols such as methanol, ethanol, and propanol which means that water molecules can adapt much faster to a local deposition of energy than other hydrogenbonding liquids.

We demonstrate by using low-temperature high-resolution spectroscopy that red-shifted mutants of green fluorescent protein are photo-interconverted among three conformations and are, therefore, not photostable ''one-color'' systems as previously believed. From our experiments we have further derived the energy-level schemes governing the interconversion among the three forms. These results have significant implications for the molecular and cell biological applications of the green fluorescent protein family; for example, in fluorescence resonant energy transfer experiments, a change in "color" on irradiation may not necessarily be due to energy transfer but can also arise from a photo-induced conversion between conformers of the excited species. The Aequorea victoria green fluorescent protein (GFP) has become a favored marker in molecular and cell biology because of its strong intrinsic visible fluorescence and the feasibility of fusing it to other proteins without affecting their normal functions (1-5). For example, mutants of GFP with different absorption and fluorescence spectra (4-8) are presently used in fluorescence lifetime imaging microscopy and fluorescence resonance energy transfer experiments to study protein-protein interactions, signaling, and trafficking in cellular systems (refs. 3-5 and references therein; refs. 9-12). In all of these studies it is assumed that the color changes observed in the GFP emission are caused by dynamic processes involving the proteins to which GFP is attached and do not arise in the GFP itself. That is, GFP-mutants are generally assumed to be in one conformation (i.e., to emit light of ''one color'') and remain in that conformation under laser illumination (i.e., to be photostable), a supposition that we here prove to be incorrect.The recent determination of the crystal structure of wild-type (wt) GFP and its mutants (13-15) has facilitated a structurebased, rational design of further mutants (3,5,16) in which the amino acids exchanged are either directly involved in the cyclization of the chromophore (serine 65, tyrosine 66, and glycine 67) or are located in their vicinity (4, 6-8, 17, 18). Frequently, the purpose of these mutations is to obtain one-color GFPmutants with a single conformation, in contrast to wt-GFP, which exhibits absorption bands attributed to more than one conformation (3,4,(19)(20)(21)(22)(23).The photophysics of wt-GFP presents a complex problem, and that of its mutants has not been studied in detail. The roomtemperature spectra are broad and rather unstructured (3-7, 18, 19). To determine the energy-level schemes of GFPs it is necessary to go to low temperature, where the spectrum becomes more structured. This has the additional merit that many of the thermally induced conversions are blocked at low temperature and, therefore, discrimination among individual species is facilitated. Energy-level schemes derived from low-temperature experiments put constraints on the interpretation of roomtemperature results. A recent study by high-resolution spec...

We present femtosecond midinfrared pump-probe measurements of the molecular motion and energy-transfer dynamics of a water molecule that is enclosed by acetone molecules. These confined water molecules show hydrogen-bond and orientational dynamics that are much slower than in bulk liquid water. This behavior is surprising because the hydrogen bonds to the CAO groups of the acetone molecules are weaker than the hydrogen bonds in bulk water. The energy transfer between the OOH groups of the confined water molecules has a time constant of 1.3 ؎ 0.2 ps, which is >20 times slower than in bulk water. We find that this energy transfer is governed completely by the rate at which hydrogen bonds are broken and reformed, and we identify the short-lived molecular complex that forms the transition state of this process.hydrogen bonding ͉ infrared pump-probe spectroscopy ͉ energy transfer W ater plays an essential role in many chemical and biological processes. Over the last decades, this notion has motivated a lot of work on the dynamical properties of bulk liquid water (1-7). However, the role of water in (bio)chemical processes is often played by a limited number of water molecules in a strongly restricted molecular environment. For example, the stability, structure, and biological function of proteins are largely determined by only a few surrounding layers of water molecules (8). When the water molecules participate directly in a reaction, the number of involved water molecules is even smaller. For example, the proton-pumping function of bacteriorhodopsin involves changes of the hydrogen network that is formed by particular amino acids of the protein and only a few confined water molecules (9-12).Recently, the dynamics of water in restricted environments was studied by comparing the spectral dynamics of an optically excited probe molecule embedded in a hydrated (bio)molecule with the spectral dynamics of the same probe molecule in bulk water (13). The spectral dynamics reflect the collective rearrangement of the solvating water, and were found to be much slower within the hydrated (bio)molecule than in bulk water. In this article, we present a study of the hydrogen-bond and energy-transfer dynamics of individual H 2 O and 1 H 2 HO molecules in a confined environment. In this study, we probed the dynamics of the water molecules directly with femtosecond midinfrared laser pulses that are resonant with the OOH stretch vibrations. Experimental MethodsThe system of confined water molecules is prepared by dissolving water (0.4 mol͞liter) in a mixture of acetone (4.0 mol͞liter) and CCl 4 . The structures that are formed in this mixture have a polar internal part consisting of an enclosed water molecule forming hydrogen bonds to the CAO groups of a few surrounding acetone molecules, and an apolar external part formed by the methyl groups of the acetone molecules. The favorable interaction between the methyl groups and the CCl 4 molecules allows these structures to enter the apolar CCl 4 matrix. The molecular ratio of H 2 O͞aceton...

Nature Struct. Biol. 6, 557-560 (1999).A printer's error resulted in deletion of the last half of a sentence, which should have appeared as the last line on page 557. The fulltext and PDF versions on the web site are correct. The final sentence on page 557 of the print version should have read:For l exc ³ 435 nm, only the B form is excited and emits light, but no B* ® I* conversion takes place.In addition, in this paper, the legend of Fig. 4b should have read:b, Emission spectrum of I upon direct excitation into I. The photoinduced reaction between A and I is reversible.We regret any confusion these errors may have caused.