Investigating the role of Substrate Binding in LeuT Transport With RosettaEPR

描述（由申请人提供）：尽管它们在自然界中普遍存在，并作为药物和治疗靶点，但许多膜蛋白仍然无法通过X射线晶体学和NMR确定结构。定点自旋标记与电子顺磁共振（SDSL-EPR）的组合正在成为一个越来越受欢迎的膜蛋白的结构表征的方法，由于它们可以被研究的相对容易。然而，SDSL-EPR并不直接产生高分辨率的结构。目前的建议描述了一种新的方法，ROSETTAEPR，以克服这一障碍。ROSETTAEPR将是一个工具包，其中EPR确定的距离和可及性限制将与基于蒙特卡罗的计算方法相结合，用于蛋白质的从头结构预测。在开发出基于知识的潜力，从EPR实验数据，它将基准上的蛋白质的已知结构，使用模拟和真实的EPR数据。此外，ROSETTAEPR和EPR实验距离和可及性数据将用于确定参与亮氨酸转运的LeuT apo、Na+和Na+/亮氨酸结合结构中间体。LeuT是神经递质钠同向转运体（NSS）蛋白家族的细菌同系物，该蛋白家族包括多巴胺、血清素和去甲肾上腺素转运体。虽然没有NSS转运蛋白的高分辨率结构，但LeuT的细胞外基质结合构象已通过X射线晶体学确定。然而，目前的结构是静态快照的LeuT运输周期，此外，他们被认为已被抓获的潜在抑制形式。因此，EPR光谱已被用来阐明蛋白质的动力学。有人发现，Na+结合导致增加蛋白质的灵活性，在细胞外环和水合的底物渗透途径，而随后的结合亮氨酸导致细胞外前庭关闭，并成为刚性。ROSETTAEPR将允许基于低分辨率EPR数据对这些中间体进行高分辨率结构解析。

DESCRIPTION (provided by applicant): Despite their prevalence in nature and as drug and therapeutic targets, many membrane proteins continue to evade structure determination by X-ray crystallography and NMR. The combination of site-directed spin labeling with electron paramagnetic resonance (SDSL-EPR) is becoming an increasingly popular method for the structural characterization of membrane proteins due to the relative ease with which they can be studied. However, SDSL-EPR does not yield high-resolution structures directly. The current proposal describes a new method, ROSETTAEPR, to overcome this obstacle. ROSETTAEPR will be a toolkit in which distance and accessibility restraints determined by EPR will be combined with Monte Carlo-based computational methods for the de novo structure prediction of proteins. After developing knowledge-based potentials derived from EPR experimental data, it will be benchmarked on proteins of known structure using both simulated and real EPR data. In addition, ROSETTAEPR and EPR experimental distance and accessibility data will be used to determine the LeuT apo, Na+, and Na+/leucine bound structural intermediates involved in leucine transport. LeuT is a bacterial homolog of the neurotransmitter sodium symporter (NSS) protein family, which includes the dopamine, serotonin, and norepinephrine transporters. While there are no high-resolution structures of the NSS transporters, extracellular-facing substrate-bound conformations of LeuT have been determined by X-ray crystallography. However, the current structures are static snapshots of the LeuT transport cycle; furthermore, they are believed to have been captured in a potentially inhibited form. Therefore, EPR spectroscopy has been employed to shed light on the dynamics of the protein. It was found that Na+ binding causes an increase in protein flexibility in the extracellular loops and hydration of the substrate permeation pathway, while subsequent binding of leucine causes the extracellular vestibule to close and become rigid. ROSETTAEPR will allow for the high-resolution structural elucidation of these intermediates based on low-resolution EPR data.