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 pK(1), pK(2), and pK(R)) 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 (1)L(a) 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.

Figure 1

Electrostatic potential surfaces of the indole plane for all lowest energy Lys-Trp species. All conformers are arranged in order of increasing chi 1 angle from left to right: 60°, 180°, and 300°. The probability, P, of each conformer is indicated. (a–c) (Lys-Trp)²⁺, (d–f) (Lys-Trp)⁺, (g–i) (Lys-Trp)⁰, (j–l) (Lys-Trp)⁻. Significant electrostatic interactions (<4 Å) are given in Table S1 of the Supporting Information. The electrostatic potential scale at the bottom of each conformer is in atomic units, with red indicating negative charge and blue, positive charge.

Figure 2

Atomic labeling of the Lys-Trp dipeptide in the zwitterion state.

Figure 3

Charge density isosurfaces for the indole of each Lys-Trp charged dipeptide. The color-coded electrostatic potential scale, with zero potential marker, is given above each figure. The inset figures are models for the isosurface of conformers not represented by the color images. Solid contour lines represent ring position of high electron density (−10^–1) while dashed lines represent ring position of weak election density (less than −10^–2). Where no contour line is present, the isosurface show zero or positive charge. (a) (Lys-Trp)²⁺, chi 1 = 60°, 0.080 isosurface; also represents the 0.050 isosurface of the 300° conformer. Inset: chi 1 = 180°, 0.060 isosurface (b) (Lys-Trp)⁺, chi 1 = 180° and 300°, 0.020 isosurface. Inset: chi 1 = 60°, 0.020 isosurface. (c) (Lys-Trp)⁰, chi 1 = 60° and 180°, 0.020 isosurface. Inset: chi 1 = 300°, 0.02 isosurface. (d) (Lys-Trp)⁻ chi 1 = 60°, 0.020 isosurface; represents 0.020 isosurface of all anionic conformers. (e) (Lys-Trp)⁻/Ind N conj base, chi 1 = 60°, 0.02 isosurface. Inset: tautomer structure. (f) Highest occupied molecular orbital (HOMO) for indole in vacuum calculated via an ab initio method (MP2/6–31G*) image adapted from ref (44). This is the major ground state orbital for the ¹L_a transition, the primary fluorescing transition in indole.

Figure 4

Weighted average lifetime (ns, sets 1 and 2, Table 2) plotted as a function of quantum yield (Table 1) for Lys-Trp species at pH 1.5–13.0. Linear fit plotted with y intercept at (x,y) = (0,0), R² = 0.894.