Djavani-Tabrizi Iden, Yuan Ziqi, Lindkvist Thomas Toft, Xing Jiamin, Chen Li, Nielsen Steen Bro Ndsted
Department of Physics and Astronomy, Aarhus University, Aarhus 8000, Denmark.
State Key Laboratory and Institute of Elemento-Organic Chemistry, College of Chemistry, Nankai University, Tianjin 300071, People's Republic of China.
J Am Soc Mass Spectrom. 2025 Feb 5;36(2):409-415. doi: 10.1021/jasms.4c00469. Epub 2025 Jan 6.
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), -leucine (-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--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.
Förster共振能量转移（FRET）正成为气相结构生物学中一种有价值的技术，用于识别生物分子（如肽和蛋白质）的局部结构基序和构象。该方法涉及用两种染料标记生物分子，一种供体染料和一种受体染料，通常是带电荷的罗丹明。在这里，我们使用低温离子荧光光谱法研究了通过酰胺键与染料相连的不同氨基酸（AA）甲酯如何影响染料跃迁能量，进而影响能量转移效率。通过在奥胡斯的LUNA2装置上进行荧光激发实验，记录了罗丹明B标记的AA酯（RB-AA）的吸收光谱，该装置在低温（低至约100K）下运行。研究的氨基酸包括脂肪族氨基酸（丙氨酸（A）、亮氨酸（L）、异亮氨酸（-L）和甲硫氨酸（M））、芳香族氨基酸（苯丙氨酸（F）和色氨酸（W））以及两种带有极性侧链的氨基酸（丝氨酸（S）和苏氨酸（T））。结果表明，与RB相比，最大吸收峰要么保持不变，要么在RB-M和RB-F的情况下红移超过3nm。虽然RB-A和RB-L的光谱与RB的光谱非常相似，但RB--L显示出约1.4nm的明显红移。光谱变化似乎受芳香族氨基酸侧链存在的影响并不比其他类型更大，因为脂肪族氨基酸之间观察到的差异与三组之间的差异相当。相反，这些变化似乎源于不同的构象，其中呫吨部分与侧链苯基之间的二面角因相连的氨基酸侧链的影响而变化。该角度决定了两个芳香部分之间的π重叠，根据TD-DFT计算，大于90°的角度很容易解释由于π电子云更大的离域导致的红移。另一个因素是侧链的极化率，它也可能导致红移。RB-F和RB-W光谱表现出红移、更窄的吸收谱，这可能与限制了贡献结构构型数量的大芳香侧链有关。
