The nearly degenerate asymmetric stretch vibrations near 1600 cm −1 of the guanidinium cation in D-glycerol/D 2 O mixtures having different viscosity were studied by 2D IR photon echo spectroscopy. The polarization dependent photon echo signal shows two separate frequency distributions in the 2D spectrum in D 2 O, even though only one band is evident from inspection of the linear FTIR spectrum. The split components are more clearly seen at higher viscosity. The interactions with solvent induce energy transfer between the degenerate component modes on the time scale of 0.5 ps. The energy transfer between modes is directly observed in 2D IR and distinguished by the waiting time dependence of the cross peaks from the transfers between threefold symmetric configurations of the distorted ion and solvent. The 2D IR analysis carried out for various polarization conditions required specification of frequency-frequency auto-and cross-correlation functions for the degenerate components.

The effect of the guanidinium cation on the hydrogen bonding strength of water was analyzed using temperature-excursion Fourier transform infrared spectra of the OH stretching vibration in 5% H 2 O / 95% D 2 O solutions containing a range of different guanidine-HCl and guanidine-HBr concentrations. Our findings indicate that the guanidinium cation causes the water H-bonds in solution to become more linear than those found in bulk water, and that it also inhibits the response of the H-bond network to increased temperature. Quantum chemical calculations also reveal that guanidinium affects both the charge distribution on water molecules directly H-bonded to it as well as the OH stretch frequency of H-bonds in which that water molecule is the donor. The implications of our findings to hydrophobic solvation and protein denaturation are discussed.

Nearly degenerate asymmetric stretches with perpendicular transition dipole moments of the deuterated guanidinium cation (DGdm + ) in D 2 O and D-glycerol/D 2 O mixtures at 1600 cm −1 were investigated by linear FTIR spectroscopy and polarization dependent femtosecond pump-probe spectroscopy. The vibrational coupling of the asymmetric stretches of guanidinium occurs within 0.5 ps and leads to fast decay of the anisotropy to a level of 0.1. A systematic study of the influence of the coherence transfer on pump-probe signals is given. Following this decay the anisotropy decays with a time constant of 4.1 ps in D 2 O by rotational diffusion about an axis perpendicular to the DGdm + mean plane. The presence of aggregation was demonstrated for concentrations higher than 0.2 M.

Genetically encoded probes with red-shifted absorption and fluorescence are highly desirable for imaging applications because they can report from deeper tissue layers with lower background and because they provide additional colors for multicolor imaging. Unfortunately, red and especially far-red fluorescent proteins have very low quantum yields, which undermines their other advantages. Elucidating the mechanism of nonradiative relaxation in red fluorescent proteins (RFPs) could help developing ones with higher quantum yields. Here we consider two possible mechanisms of fast nonradiative relaxation of electronic excitation in RFPs. The first, known as the energy gap law, predicts a steep exponential drop of fluorescence quantum yield with a systematic red shift of fluorescence frequency. In this case the relaxation of excitation occurs in the chromophore without any significant changes of its geometry. The second mechanism is related to a twisted intramolecular charge transfer in the excited state, followed by an ultrafast internal conversion. The chromophore twisting can strongly depend on the local electric field because the field can affect the activation energy. We present a spectroscopic method of evaluating local electric fields experienced by the chromophore in the protein environment. The method is based on linear and two-photon absorption spectroscopy, as well as on quantum-mechanically calculated parameters of the isolated chromophore. Using this method, which is substantiated by our molecular dynamics simulations, we obtain the components of electric field in the chromophore plane for seven different RFPs with the same chromophore structure. We find that in five of these RFPs, the nonradiative relaxation rate increases with the strength of the field along the chromophore axis directed from the center of imidazolinone ring to the center of phenolate ring. Furthermore, this rate depends on the corresponding electrostatic energy change (calculated from the known fields and charge displacements), in quantitative agreement with the Marcus theory of charge transfer. This result supports the dominant role of the twisted intramolecular charge transfer mechanism over the energy gap law for most of the studied RFPs. It provides important guidelines of how to shift the absorption wavelength of an RFP to the red, while keeping its brightness reasonably high.

The steady state fluorescence spectral maximum (λmax) for tryptophan 140 of Staphylococcal nuclease remains virtually unchanged when nearby charged groups are removed by mutation, even though large electrostatic effects on λmax might be expected. To help understand the underlying mechanism of this curious result, we have modeled λmax with three sets of 50-ns molecular dynamics simulations in explicit water, equilibrated with excited state and with ground state charges. Semiempirical quantum mechanics and independent electrostatic analysis for the wild-type protein and four charge-altering mutants were performed on the chromophore using the coordinates from the simulations. Electrostatic contributions from the nearby charged lysines by themselves contribute 30-90 nm red shifts relative to the gas phase, but in each case, contributions from water create compensating blue shifts that bring the predicted λmax within 2 nm of the experimental value, 332 ± 0.5 nm for all five proteins. Although long-range collective interactions from ordered water make large blue shifts, crucial for determining the steady state λmax for absorption and fluorescence, such blue shifts do not contribute to the amplitude of the time dependent Stokes shift following excitation, which comes from nearby charges and only ∼6 waters tightly networked with those charges. We therefore conclude that for STNase, water and protein effects on the Stokes shift are not separable.