Ultrafast Biophysical Studies of Proteins: Time-resolved WAXS Studies

时间分辨劳厄晶体学，顾名思义，只能在晶体样品上进行。维持晶体有序的分子间力限制了大幅度的构象运动，这种柔性的丧失可能会干扰甚至抑制蛋白质的功能。尽管如此，劳厄晶体学在超快时间尺度上获得近原子结构信息的能力是独一无二的。另一方面，X射线还可以从允许全范围构象运动的溶液中的分子提取结构信息。由于蛋白质溶液中没有长程有序，因此蛋白质的广角X射线散射（WAXS）是弥散的。然而，WAXS光谱中的衍射环受到蛋白质大小和形状的影响。虽然这种结构信息不是原子分辨率的，但它确实提供了一个指纹，可以通过模型与蛋白质结构相关联。因此，WAXS指纹的时间依赖性变化可以用于评估哪些模型最好地描述了反应途径。 当我们第一次开始用这种技术研究血红蛋白的四级结构转变时，我们在我们可以测量的最早时间观察到WAXS光谱的实质性变化，这表明WAXS光谱对三级结构变化以及四级结构变化敏感。为了证明这一点，我们记录了光解碳单氧肌红蛋白（MbCO）的WAXS光谱，并观察到类似的结果。根据MbCO和脱氧Mb的X射线结构，它们的原子位置的均方根差小于0.5埃。这种意想不到的能力，感觉到这种小，但系统的结构变化是相当令人鼓舞的，并促使我们继续努力，发展这种新的实验方法。 我们通过时间分辨WAXS表征四级结构转变的努力正在迅速推进。这些研究中面临的问题之一是激光光解后大量的成对配体再结合。即使我们可以用2 ns的泵浦脉冲光解大多数结合的配体，但在泵浦脉冲结束后，其中超过30%的配体重新结合，并且形成了连接状态的异质混合物。这些状态中的一些能够经历R到T的四级结构转变，但可能以不同的速率。例如，已知Hb（CO）2可以经历四级结构转变，条件是两个配体不都与α链结合或都与β链结合。人们会期望HbCO以更快的速率经历转变，并且Hb（没有结合的配体）将是最快的。为了产生更均匀分布的连接状态，我们牺牲了我们的时间分辨率通过光解的Hb（CO）4与100-150纳秒的激光脉冲。由于脉冲与33 ns的双生子再结合时间相比较长，因此激光脉冲有多个机会将CO从主要对接位点驱离，从而产生大量没有CO结合的Hb。WAXS光谱的光谱演化清楚地揭示了四元结构转变的特征时间常数。虽然我们仍在改进对这一过程的估计，但它似乎是以2微秒的时间常数发生的。这一速率明显快于纯光谱研究所报道的速率。这些研究通过光谱观测值探测局部特征，并不一定反映与血红蛋白四级跃迁相关的全局运动。另一方面，测量速率的差异可以反映各种连接状态经历四级结构转变的速度的差异。虽然我们在这一领域取得了很大进展，但还需要做更多的工作来合理化光谱和WAXS测量之间的差异。 从溶液中的蛋白质中提取结构信息的能力将有助于为研究参与信号传导的蛋白质铺平道路，其中通常推断出全局构象变化。由于WAXS光谱的形状可以从结构模型中计算出来，因此随时间演变的指纹将提供可用于验证反应途径的假定模型的深刻信息。通过与Gerhard Hummer博士的合作，我们的目标是进一步探索这种可能性。初步分析表明，他们的计算方法能够再现在光解MbCO的时间分辨WAXS研究中观察到的一些细微细节。

Time-resolved Laue crystallography, as implied by its name, can only be performed on crystalline samples. The intermolecular forces that maintain crystalline order constrain large amplitude conformational motion, and this loss of flexibility may perturb or even inhibit the function of a protein. Nonetheless, Laue crystallography stands alone in its ability to acquire near-atomic structural information on ultrafast time scales. On the other hand, X-rays can also extract structural information from molecules in solution where the full range of conformational motion is permitted. Because there is no long-range order in protein solutions, Wide-Angle-X-ray-Scattering (WAXS) from the protein is diffuse. However, diffraction rings in the WAXS spectrum are influenced by the size and shape of the protein. Though this structural information is not at atomic resolution, it does provide a fingerprint that can be correlated via models with the protein structure. Time-dependent changes of the WAXS fingerprint can therefore be used to assess which models best describe the reaction pathway. When we first set out to study the quaternary structure transition of hemoglobin with this technique, we observed a substantial change of the WAXS spectrum at the earliest time we could measure, which suggested that the WAXS spectrum is sensitive to tertiary structure changes as well as quaternary structure changes. To prove this point, we recorded the WAXS spectrum of photolyzed carbon monoxymyoglobin (MbCO) and observed a similar result. According to X-ray structures of MbCO and deoxy Mb, the rms difference in their atomic positions is less than 0.5 Angstroms. This unexpected ability to sense such small but systematic structure changes is quite encouraging, and has spurred us to continue our efforts to develop this new experimental methodology. Our efforts to characterize the quaternary structure transition by time-resolved WAXS are rapidly advancing. One of the problems faced in these studies is the substantial geminate ligand rebinding that follows laser photolysis. Even though we can photolyze the majority of the bound ligands with a 2-ns pump pulse, more than 30% of them rebind after the pump pulse is over, and a heterogeneous mixture of ligation states are formed. Some of these states are capable of undergoing the R to T quaternary structure transition, but perhaps at different rates. For example, it is known that Hb(CO)2 can undergo a quaternary structure transition, provided the two ligands are not both bound to alpha chains or both bound to beta chains. One would expect HbCO to undergo the transition at a faster rate, and Hb (no bound ligands) would be fastest. To produce a more homogeneous distribution of ligation states, we sacrificed our time resolution by photolyzing the Hb(CO)4 with a 100-150 ns duration laser pulse. Because the pulse was long compared to the 33 ns geminate rebinding time, the laser pulse had multiple opportunities to drive CO away from the primary docking site and thereby generated a high population of Hb with no CO bound. The spectral evolution of the WAXS spectrum reveals clearly the characteristic time constant for the quaternary structure transition. Though we are still in the process of refining our estimates for this process, it appears to occur with a time constant of 2 microseconds. This rate is significantly faster than the rates reported from purely spectroscopic studies. Those studies probe local features through spectroscopic observables, and do not necessarily reflect the global motion associated with the quaternary transition of hemoglobin. On the other hand, the differences in the measured rates may reflect differences in the speed at which various ligation states undergo the quaternary structure transition. Though we have made much progress in this area, more work is needed to rationalize the differences between the spectroscopic and WAXS measurements. The ability to extract structural information from proteins in solution will help pave the way to study proteins involved in signaling, where global conformational changes are usually inferred. Because the shape of the WAXS spectrum can be calculated from a structural model, the temporally evolving fingerprint will provide incisive information that can be used to validate putative models for the reaction pathway. Through our collaboration with the group of Dr. Gerhard Hummer, we aim to further explore this possibility. Preliminary analysis suggests that their computational methods are capable of reproducing some of the fine details observed in time-resolved WAXS studies of photolyzed MbCO.