Lee Hyun Kyung, Conrad Chelsie E, Magidson Valentin, Heinz William F, Pauly Gary, Yu Ping, Ramakrishnan Saminathan, Stagno Jason R, Wang Yun-Xing
Protein-Nucleic Acid Interaction Section, Center for Structural Biology, Center for Cancer Research, National Cancer Institute, Frederick, MD, United States.
Optical Microscopy and Analysis Laboratory, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, MD, United States.
Front Mol Biosci. 2022 Aug 16;9:964595. doi: 10.3389/fmolb.2022.964595. eCollection 2022.
Crystallographic observation of structural changes in real time requires that those changes be uniform both spatially and temporally. A primary challenge with time-resolved ligand-mixing diffraction experiments is asynchrony caused by variable factors, such as efficiency of mixing, rate of diffusion, crystal size, and subsequently, conformational heterogeneity. One method of minimizing such variability is use of a photolabile caged ligand, which can fully saturate the crystal environment (spatially), and whose photoactivation can rapidly (temporally) trigger the reaction in a controlled manner. Our recently published results on a ligand-mixing experiment using time-resolved X-ray crystallography (TRX) with an X-ray free electron laser (XFEL) demonstrated that large conformational changes upon ligand binding resulted in a solid-to-solid phase transition (SSPT), while maintaining Bragg diffraction. Here we investigate this SSPT by polarized video microscopy (PVM) after light-triggered release of a photo-caged adenine (pcADE). In general, the mean transition times and transition widths of the SSPT were less dependent on crystal size than what was observed in previous PVM studies with direct ADE mixing. Instead, the photo-induced transition appears to be heavily influenced by the equilibrium between caged and uncaged ADE due to relatively low sample exposure and uncaging efficiency. Nevertheless, we successfully demonstrate a method for the characterization of phase transitions in RNA crystals that are inducible with a photocaged ligand. The transition data for three crystals of different sizes were then applied to kinetic analysis by fitting to the known four-state model associated with ligand-induced conformational changes, revealing an apparent concentration of uncaged ADE in crystal of 0.43-0.46 mM. These results provide further insight into approaches to study time-resolved ligand-induced conformational changes in crystals, and in particular, highlight the feasibility of triggering phase transitions using a light-inducible system. Developing such approaches may be paramount for the rapidly emerging field of time-resolved crystallography.
实时观察结构变化的晶体学研究要求这些变化在空间和时间上都是均匀的。时间分辨配体混合衍射实验的一个主要挑战是由可变因素引起的异步性，这些因素包括混合效率、扩散速率、晶体大小，以及随之而来的构象异质性。最小化这种变异性的一种方法是使用光不稳定的笼形配体，它可以使晶体环境（在空间上）完全饱和，并且其光激活可以以可控的方式迅速（在时间上）触发反应。我们最近发表的关于使用时间分辨X射线晶体学（TRX）和X射线自由电子激光（XFEL）进行配体混合实验的结果表明，配体结合时的大构象变化导致了固-固相变（SSPT），同时保持布拉格衍射。在这里，我们通过光触发释放光笼腺嘌呤（pcADE）后的偏振视频显微镜（PVM）研究了这种SSPT。一般来说，SSPT的平均转变时间和转变宽度对晶体大小的依赖性比之前直接混合ADE的PVM研究中观察到的要小。相反，由于样品暴露和脱笼效率相对较低，光诱导转变似乎受到笼形和未笼形ADE之间平衡的严重影响。尽管如此，我们成功地证明了一种用于表征可由光笼配体诱导的RNA晶体相变的方法。然后，通过拟合与配体诱导的构象变化相关的已知四态模型，将三种不同大小晶体的转变数据应用于动力学分析，揭示了晶体中未笼形ADE的表观浓度为(0.43 - 0.46) mM。这些结果为研究晶体中时间分辨配体诱导的构象变化的方法提供了进一步的见解，特别是突出了使用光诱导系统触发相变的可行性。开发此类方法对于迅速兴起的时间分辨晶体学领域可能至关重要。
