The Broken Ring: Reduced Aromaticity in Lys-Trp Cations and High pH Tautomer Correlates with Lower Quantum Yield and Shorter Lifetimes

Abstract

Several nonradiative processes compete with tryptophan fluorescence emission. The difficulty in spectral interpretation lies in associating specific molecular environmental features with these processes and thereby utilizing the fluorescence spectral data to identify the local environment of tryptophan. Here, spectroscopic and molecular modeling study of Lys-Trp dipeptide charged species shows that backbone-ring interactions are undistinguished. Instead, quantum mechanical ground state isosurfaces reveal variations in indole π electron distribution and density that parallel charge (as a function of pK1, pK2, and pKR) on the backbone and residues. A pattern of aromaticity-associated quantum yield and fluorescence lifetime changes emerges. Where quantum yield is high, isosurfaces have a charge distribution similar to the highest occupied molecular orbital (HOMO) of indole, which is the dominant fluorescent ground state of the 1La transition dipole moment. Where quantum yield is low, isosurface charge distribution over the ring is uneven, diminished, and even found off ring. At pH 13, the indole amine is deprotonated, and Lys-Trp quantum yield is extremely low due to tautomer structure that concentrates charge on the indole amine; the isosurface charge distribution bears scant resemblance to the indole HOMO. Such greatly diminished fluorescence has been observed for proteins where the indole nitrogen is hydrogen bonded, lending credence to the association of aromaticity changes with diminished quantum yield in proteins as well. Thus tryptophan ground state isosurfaces are an indicator of indole aromaticity, signaling the partition of excitation energy between radiative and nonradiative processes.

Excited state electron distribution and role of the terminal amine in acidic and basic tryptophan dipeptide fluorescence

“…Additional details about the Ti-Sapphire system can be found in Eisenberg and Juszczak [28]. Excitation was achieved at 280 nm while emission was monitored at 340 nm on both instruments.…”

“…Details of our method are given in Eisenberg and Juszczak [28–29] with the following addition and change. Time-dependent density functional theory (TD-DFT) excited state calculations were carried out with the B3LYP method and the 6-31++g(df,p) basis set to produce excited state orbitals as well as oscillator strengths for the electronic transitions; oscillator strengths are included in the Supporting information.…”

“…GaussView 5.0 software [36] was used for visualization of the dipeptide species and their isosurfaces. Distance between an isosurface and the nuclei of the indole ring are not directly proportional, but can be estimated with this empirically derived formula [28]: d = y 0 + Ae (−i/t) , where d is the distance in Å between the nucleus and the isosurface, y 0 = 0.370, A = 1.105, t = 0.07228, and i = the isosurface value.…”

“…In our previous molecular dynamics study of the Lys-Trp dipeptide in different charge states [28], optimized molecular dynamics conformations did not show the juxtaposition of the terminal amine cation next to the indole ring. Changes in ground state isosurface charge density on the indole ring, resulting from pH-induced changes in off-ring charge state, were shown to correlate with reduced quantum yield and weighted average lifetime, suggesting a static quenching mechanism as an explanation.…”

“…In the absence of excited state isosurfaces, molecular orbital calculations for Trp-Lys and Trp-Glu species absorption transition are also carried out. We are especially interested in the HOMO and LUMO for the fluorescent 1 L a transition because similarities between the isosurfaces of higher quantum yield Lys-Trp species and the HOMO have been observed [28]. We examine the LUMOs from most intense absorption transitions to distinguish between fluorescence, a vertical transition between excited and ground state, and dark state processes, where the excited state electron density can be found off-ring.…”