Department of Chemistry, Pohang University of Science and Technology (POSTECH), Pohang 37673, South Korea.
JILA, University of Colorado, and NIST, Boulder, Colorado 80309, United States.
J Phys Chem B. 2022 Mar 31;126(12):2337-2344. doi: 10.1021/acs.jpcb.1c10516. Epub 2022 Mar 17.
The development of bright fluorescent proteins (FPs) emitting beyond 600 nm continues to be of interest both from a fundamental perspective in understanding protein-chromophore interactions and from a practical perspective as these FPs would be valuable for cellular imaging. We previously reported ultrafast spectral observations of the excited-state dynamics in mPlum resulting from interconversion between direct hydrogen bonding and water-mediated hydrogen bonding between the chromophore acylimine carbonyl and the Glu16 side chain. Here, we report temperature-dependent steady-state and time-resolved fluorescence measurements of mPlum and its E16H variant, which does not contain a side-chain permitting hydrogen bonding with the acylimine carbonyl. Lowering the temperature of the system freezes interconversion between the hydrogen-bonding states, thus revealing the spectral signatures of the two states. Analysis of the temperature-dependent spectra assuming Boltzmann populations of the two states yields a 205 cm energy difference. This value agrees with the predictions from a quantum mechanics/molecular mechanics study of mPlum (198 cm). This study demonstrates the first use of cryogenic spectroscopy to quantify the energetics and timescales of FP chromophore structural states that were only previously obtained from computational methods and further confirms the importance of acylimine hydrogen-bonding dynamics to the fluorescence spectral shifts of red FPs.
研究人员之前曾报道过，通过超快光谱观察，mPlum 中发色团酰亚胺羰基和 Glu16 侧链之间的直接氢键和水分子介导的氢键之间的相互转换，揭示了激发态动力学。在此，我们报告了 mPlum 及其 E16H 变体的温度依赖稳态和时间分辨荧光测量结果，E16H 变体中不存在允许与酰亚胺羰基形成氢键的侧链。降低体系温度会冻结氢键状态之间的转换，从而揭示出两种状态的光谱特征。基于两种状态的玻尔兹曼分布对温度相关光谱进行分析，得出 205cm 的能量差。该值与对 mPlum 的量子力学/分子力学研究（198cm）的预测结果一致。该研究首次利用低温光谱学来量化 FP 发色团结构状态的能量和时间尺度，这些状态之前只能通过计算方法获得，进一步证实了酰亚胺氢键动力学对红色 FP 荧光光谱位移的重要性。
