Charge invariant protein-water relaxation in GB1 via ultrafast tryptophan fluorescence.

The protein-water interface is a critical determinant of protein structure and function, yet the precise nature of dynamics in this complex system remains elusive. Tryptophan fluorescence has become the probe of choice for such dynamics on the picosecond time scale (especially via fluorescence "upconversion"). In the absence of ultrafast ("quasi-static") quenching from nearby groups, the TDFSS (time-dependent fluorescence Stokes shift) for exposed Trp directly reports on dipolar relaxation near the interface (both water and polypeptide). The small protein GB1 contains a single Trp (W43) of this type, and its structure is refractory to pH above 3. Thus, it can be used to examine the dependence of dipolar relaxation upon charge reconfiguration with titration. Somewhat surprisingly, the dipolar dynamics in the 100 fs to 100 ps range were unchanged with pH, although nanosecond yield, rates, and access all changed. These results were rationalized with the help of molecular dynamics (including QM-MM) simulations that reveal a balancing, sometimes even countervailing influence of protein and water dipoles. Interestingly, these simulations also showed the dominant influence of water molecules which are associated with the protein interface for up to 30 ps yet free to rotate at approximately "bulk" water rates.

Solvent effects on the fluorescence quenching of tryptophan by amides via electron transfer. Experimental and computational studies.

Hybrid quantum mechanical/molecular mechanics (QM-MM) calculations [Callis and Liu, J. Phys. Chem. B 2004, 108, 4248-4259] make a strong case that the large variation in tryptophan (Trp) fluorescence yields in proteins is explained by ring-to-backbone amide electron transfer, as predicted decades ago. Quenching occurs in systems when the charge transfer (CT) state is brought below the fluorescing state (1L(a)) as a result of strong local electric fields. To further test this hypothesis, we have measured the fluorescence quantum yield in solvents of different polarity for the following systems: N-acetyl-L-tryptophanamide (NATA), an analogue for Trp in a protein; N-acetyl-L-tryptophan ethyl ester (NATE), wherein the Trp amide is replaced by an ester group, lowering the CT state energy; and 3-methylindole (3MI), a control wherein this quenching mechanism cannot take place. Experimental yields in water are 0.31, 0.13, and 0.057 for 3MI, NATA, and NATE, respectively, whereas, in the nonpolar aprotic solvent dioxane, all three have quantum yields near 0.35, indicating the absence of electron transfer. In alkyl alcohols the quantum yield for NATA and NATE is between that found for water and that found for dioxane, and it is surprisingly independent of chain length (varying from methanol to decanol), revealing that microscopic H-bonding, and not the bulk dielectric constant, dictates the electron transfer rate. QM-MM calculations indicate that, when averaged over the six rotamers, the greatly increased quenching found in water relative to dioxane can be attributed mainly to the larger fluctuations of the energy gap in water. These experiments and calculations are in complete accord with quenching by a solvent stabilized charge transfer from ring to amide state in proteins.

Ab initio prediction of tryptophan fluorescence quenching by protein electric field enabled electron transfer.

We report quantum mechanical-molecular mechanical (QM-MM) predictions of fluorescence quantum yields for 20 tryptophans in 17 proteins, whose yields span the range from 0.01 to 0.3, using ab initio computed coupling matrix elements for photoinduced electron transfer from the 1La excited indole ring to a local backbone amide. The average coupling elements span the range 140-1000 cm-1, depending on tryptophan rotamer conformation. The matrix elements were from the singles configuration interaction matrix, and were largely insensitive to which of the three basis sets was used. Large fluctuations were seen on the time scale of tens of femtoseconds, caused primarily by side chain and backbone torsional variations for 150 ps of dynamics at 300 K. The largest coupling occurs for the chi1 = -60 degrees rotamer and is purely through-bond. There is no apparent correlation between the coupling magnitude and quantum yield, which is still dominated by energy gap and reorganization energy. The source of error bars for predicted quenching rates using the weak coupling golden rule may be due to inaccurate averaged Franck-Condon weighted densities because of inadequate simulation times and parameters and/or to failure of the weak coupled golden rule used in these predictions because of the broad distribution of Landau-Zener probabilities arising from the large variable coupling.

Picosecond fluorescence dynamics of tryptophan and 5-fluorotryptophan in monellin: slow water-protein relaxation unmasked.

Time dependent fluorescence Stokes (emission wavelength) shifts (TDFSS) from tryptophan (Trp) following sub-picosecond excitation are increasingly used to investigate protein dynamics, most recently enabling active research interest into water dynamics near the surface of proteins. Unlike many fluorescence probes, both the efficiency and the wavelength of Trp fluorescence in proteins are highly sensitive to microenvironment, and Stokes shifts can be dominated by the well-known heterogeneous nature of protein structure, leading to what we call pseudo-TDFSS: shifts that arise from differential decay rates of subpopulations. Here we emphasize a novel, general method that obviates pseudo-TDFSS by replacing Trp by 5-fluorotryptophan (5Ftrp), a fluorescent analogue with higher ionization potential and greatly suppressed electron-transfer quenching. 5FTrp slows and suppresses pseudo-TDFSS, thereby providing a clearer view of genuine relaxation caused by solvent and protein response. This procedure is applied to the sweet-tasting protein monellin which has uniquely been the subject of ultrafast studies in two different laboratories (Peon, J.; et al. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 10964; Xu, J.; et al. J. Am. Chem. Soc. 2006, 128, 1214) that led to disparate interpretations of a 20 ps transient. They differed because of the pseudo-TDFSS present. The current study exploiting special properties of 5FTrp strongly supports the conclusion that both lifetime heterogeneity-based TDFSS and environment relaxation-based TDFSS are present in monellin and 5FTrp-monellin. The original experiments on monellin were most likely dominated by pseudo-TDFSS, whereas, in the present investigation of 5FTrp-monellin, the TDFSS is dominated by relaxation and any residual pseudo-TDFSS is overwhelmed and/or slowed to irrelevance.

Correlation of tryptophan fluorescence spectral shifts and lifetimes arising directly from heterogeneous environment.

Tryptophan (Trp) fluorescence is potentially a powerful probe for studying the conformational ensembles of proteins in solution, as it is highly sensitive to the local electrostatic environment of the indole side chain. However, interpretation of the wavelength-dependent complex fluorescence decays of proteins has been stymied by controversy about two plausible origins of the typical multiple fluorescence lifetimes: multiple ground-state populations or excited-state relaxation. The latter naturally predicts the commonly observed wavelength-lifetime correlation between decay components, which associates short lifetimes with blue-shifted emission spectra and long lifetimes with red-shifted spectra. Here we show how multiple conformational populations also lead to the same strong wavelength-lifetime correlation in cyclic hexapeptides containing a single Trp residue. Fluorescence quenching in these peptides is due to electron transfer. Quantum mechanics-molecular mechanics simulations with 150-ps trajectories were used to calculate fluorescence wavelengths and lifetimes for the six canonical rotamers of seven hexapeptides in aqueous solution at room temperature. The simulations capture most of the unexpected diversity of the fluorescence properties of the seven peptides and reveal that rotamers having blue-shifted emission spectra, i.e., higher average energy, have an increased probability for quenching, i.e., shorter average lifetime, during large fluctuations in environment that bring the nonfluorescent charge transfer state and the fluorescing state into resonance. This general mechanism should also be operative in proteins that exhibit multiexponential fluorescence decays, where myriad other sources of conformational heterogeneity besides rotamers are possible.

The OH* + CH3SH reaction: support for an addition-elimination mechanism from ab initio calculations.

Several intermediates for the CH(3)SH + OH(*) --> CH(3)S(*) + H(2)O reaction were identified using MP2(full) 6-311+g(2df,p) ab initio calculations. An adduct, CH(3)S(H)OH(*), I, with electronic energy 13.63 kJ mol(-1) lower than the reactants, and a transition state, II(double dagger), located 5.14 kJ mol(-1) above I, are identified as the entrance channel for an addition-elimination reaction mechanism. After adding zero-point and thermal energies, DeltaH(r,298) ( degrees )(reactants --> I) = -4.85 kJ mol(-1) and DeltaH(298) (double dagger)(I --> II(double dagger)) = +0.10 kJ mol(-1), which indicates that the potential energy surface is broad and flat near the transition state. The calculated imaginary vibrational frequency of the transition state, 62i cm(-1), is also consistent with an addition-elimination mechanism. These calculations are consistent with experimental observations of the OH(*) + CH(3)SH reaction that favored an addition-elimination mechanism rather than direct hydrogen atom abstraction. An alternative reaction, CH(3)SH + OH(*) --> CH(3)SOH + H(*), with DeltaH(r,298) ( degrees ) = +56.94 kJ mol(-1) was also studied, leading to a determination of DeltaH(f,298) ( degrees )(CH(3)SOH) = -149.8 kJ mol(-1).