Near-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes are widely used for structural and functional deep-tissue imaging in vivo. To fluoresce, NIR FPs covalently bind a chromophore, such as biliverdin IXa tetrapyrrole. The efficiency of biliverdin binding directly affects the fluorescence properties, rendering understanding of its molecular mechanism of major importance. miRFP proteins constitute a family of bright monomeric NIR FPs that comprise a Per-ARNT-Sim (PAS) and cGMP-specific phosphodiesterases - Adenylyl cyclases - FhlA (GAF) domain. Here, we structurally analyze biliverdin binding to miRFPs in real time using time-resolved stimulated Raman spectroscopy and quantum mechanics/molecular mechanics (QM/MM) calculations. Biliverdin undergoes isomerization, localization to its binding pocket, and pyrrolenine nitrogen protonation in <1 min, followed by hydrogen bond rearrangement in ~2 min. The covalent attachment to a cysteine in the GAF domain was detected in 4.3 min and 19 min in miRFP670 and its C20A mutant, respectively. In miRFP670, a second C–S covalent bond formation to a cysteine in the PAS domain occurred in 14 min, providing a rigid tetrapyrrole structure with high brightness. Our findings provide insights for the rational design of NIR FPs and a novel method to assess cofactor binding to light-sensitive proteins.

由细菌光敏色素改造而成的近红外荧光蛋白（NIR FP）广泛用于体内结构和功能性深组织成像。为了发出荧光，NIR FP 共价结合发色团，例如胆绿素 IXa 四吡咯。胆绿素结合的效率直接影响荧光特性，因此了解其分子机制非常重要。 miRFP 蛋白构成了一个明亮的单体 NIR FP 家族，其中包含 Per-ARNT-Sim (PAS) 和 cGMP 特异性磷酸二酯酶 - 腺苷酸环化酶 - FhlA (GAF) 结构域。在这里，我们使用时间分辨受激拉曼光谱和量子力学/分子力学 (QM/MM) 计算实时结构分析胆绿素与 miRFP 的结合。胆绿素在不到 1 分钟内经历异构化、定位到其结合袋和吡咯氮质子化，然后在约 2 分钟内发生氢键重排。在 miRFP670 及其 C20A 突变体中，分别在 4.3 分钟和 19 分钟内检测到与 GAF 结构域中半胱氨酸的共价连接。在 miRFP670 中，14 分钟内与 PAS 结构域中的半胱氨酸形成第二个 C-S 共价键，从而提供了具有高亮度的刚性四吡咯结构。我们的研究结果为 NIR FP 的合理设计和评估辅因子与光敏蛋白结合的新方法提供了见解。