Gas-Phase Fluorescence Excitation Experiments on Cryogenically Cold Rhodamine B Cations Linked to Various Amino Acid Esters

Abstract

Förster resonance energy transfer (FRET) is becoming a valuable technique in gas-phase structural biology for identifying local structural motifs and conformations of biological molecules, such as peptides and proteins. This method involves labeling the biomolecule with two dyes, a donor dye and an acceptor dye, that are commonly charged rhodamines. Here we examine how different amino acid (AA) methyl esters linked to the dye via amide linkages can influence the dye transition energy and, consequently, the energy-transfer efficiency, using cryogenic ion fluorescence spectroscopy. Absorption spectra were recorded for rhodamine B⁺-labeled AA esters (RB⁺-AA) through fluorescence–excitation experiments at the LUNA2 setup in Aarhus, which operates at cryogenic temperatures (down to approximately 100 K). The AAs studied include aliphatic ones (alanine (A), leucine (L), tert-leucine (tert-L), and methionine (M)), aromatic ones (phenylalanine (F) and tryptophan (W)), and two with polar side chains (serine (S) and threonine (T)). Results show that the band maximum either remains unchanged compared to RB⁺ or red shifts by over 3 nm in the case of RB⁺-M and RB⁺-F. While the spectra of RB⁺-A and RB⁺-L closely resemble that of RB⁺, RB⁺-tert-L shows a distinct red shift of about 1.4 nm. Spectral variations do not appear to be more influenced by the presence of aromatic AA side chains than other types, as differences observed between aliphatic AAs are comparable to those between the three groups. Instead, these variations appear to arise from differing conformations where the dihedral angle between the xanthene moiety and the pendant phenyl group varies, as influenced by the linked AA side chain. The angle determines the π-overlap between the two aromatic moieties, and according to TD-DFT calculations, an angle larger than 90° can easily account for red shifts due to larger delocalization of the π-electron cloud. Another factor is the polarizability of the side chain that could also contribute to the red shift. RB⁺-F and RB⁺-W spectra exhibit red-shifted, narrower absorption profiles, which is likely associated with the large aromatic side chains that limit the number of contributing structural configurations.