Tracking Environment Sensitive Ultrafast Photophysics of Tryptophan with Sub-20-fs UV Pulses

The photophysics of tryptophan (Trp) is a subject of active investigation, because of its importance as a probe of protein structural dynamics [1]. The early time photophysics of Trp involve the interplay of the two lowest close-lying ππ* electronic states, La and Lb. Therefore, a complete explanation of the complex ultrafast behavior of tryptophan is a fundamental stepping stone to understand its role as a probe for monitoring local protein environment. In this work, we characterize the dynamics following photo-excitation of water solvated Trp in the sub-5-ps regime by combining transient absorption (TA) spectroscopy with state-of-the-art sub-30-fs temporal resolution and theoretical computations at CASPT2 level incorporating solvent effects within a hybrid QM/MM setup.<br/><br/>Using a 4.35 eV pump to excite the Lb state, at 50 fs delay we observe a transient spectrum composed of a photo-induced absorption (PA) peak at 3.4 eV and corresponding stimulated emission (SE) signal at higher energies. By comparison with computed transitions we can associate this spectrum with the Lb state. This initial spectrum then decays and gives rise to a new one with PA at 3.7 eV and SE below it, that we associate with the La state. Through global fitting we find two decay constants of 220 fs and 1.1 ps. Surprisingly for the 4.7 eV pump of the La state, we observe identical behavior. We explain it through steep and completely barrierless minimum energy path connecting the La state to the Lb state with conical intersection (CI), which happens before 50 fs. While populating the lower potential energy surface (PES), the wavepacket does not stay static in the Lb minimum, but due to low energy difference from the La region of the PES, it dynamically explores the surface, partially populating the La state. Because of high temporal resolution of our TA measurement, we also observe coherent vibrational fingerprints covering the whole probing region. After Fourier transform of the oscillations, we find a strong peak of 720 cm^-1 with a node and a π-phase jump located at 3.65 eV, characteristic of an SE band of the excited state, where the spectral position of the node is the central wavelength of the emitting band. Further analysis leads us to notice that the phase jump position is red-shifting over time. Comparison of this trend with the theoretical central emission wavelength of La and Lb states reveals that the emission of Lb is static, while the one of La is showing the expected shift. We conclude that not only the La state stays partially populated, but it is also going down in energy over time. This is due to the internal charge transfer character of the La state that continuously polarizes the solvent around the molecule, which in turn lowers the La minimum energy below the Lb minimum, and initiates the population transfer with the timescale of 220 fs. The increasing population of the La state further polarizes the solvent over 1.1 ps as the minimum settles down.<br/><br/>In conclusion, by combining ultrafast UV TA spectroscopy with high-level multiconfigurational methods within a hybrid QM/MM setup, we have demonstrated that the ultrafast dynamics of Trp are driven by two vibronically coupled lowest singlet states, and the response of the solvent modulates their electronic population. We find first an immediate CI with the Lb state happening before 50 fs, and then a 220 fs process of solvent mediated La and Lb minimum inversion that transfers population back to the La state, where it keeps relaxing on the picosecond timescale. This precise description of tryptophan behavior in the first picosecond after excitation and its sensitivity to the environment can be used to track ultrafast protein dynamics.<br/><br/>[1] D. Sharma, J. Leonard, and S. Haacke, “Ultrafast excited-state dynamics of tryptophan in water observed by transient absorption spectroscopy,” Phys. Chem. Chem. Phys. 12, 15744-15750 (2010).