Development and application of EPR-spectroscopic methods for the analysis of structure and dynamics of integral membrane proteins using the Na+/proline transporter PutP as a model

EPR 光谱方法的开发和应用，以 Na /脯氨酸转运蛋白 PutP 作为模型分析整合膜蛋白的结构和动力学

• 批准号: 5452540
• 负责人: Professor Dr. Heinrich Jung
• 金额: --
• 依托单位: Arbeitsgruppe Makromolekülstruktur
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2005
• 资助国家: 德国
• 起止时间: 2004-12-31 至 2015-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/5452540?language=en
• 关键词: Development; application; EPR; spectroscopic; methods

Understanding protein function at the molecular level requires information not only on protein structure and but also on its dynamics. Due to their hydrophobic nature, this information is particularly difficult to obtain for integral membrane proteins such as secondary transport proteins. This research proposal aims at the advancement of electron paramagnetic resonance (EPR) spectroscopic methods to probe structure and dynamics of membrane proteins in their native environment utilising the Na+/proline transporter PutP as a model. After modelling static structure based on pulsed EPR distance data, our research activities shall focus on conformational alterations required to translocate coupling ion and substrate across the membrane. Thereby, cw and pulse EPR mobility, accessibility, and distance data as well as spin label-phospholipid headgroup distances (31P ENDOR) shall be used to characterise individual conformational states under equilibrium conditions, and to follow the time course of conformational changes. An approach that translates resulting correlated distance changes in the nanometer range into a coarse-grained model of structural changes of PutP will be developed. Therein, data on spin label mobility and accessibility shall be used to control and refine the model. Based on known similarities at the tertiary structure level, information on PutP dynamics is expected to be transferable to transporters of various families.

在分子水平上理解蛋白质的功能不仅需要蛋白质结构的信息，还需要其动力学的信息。由于它们的疏水性质，对于整合膜蛋白如二级转运蛋白，该信息特别难以获得。本研究建议的目的是在电子顺磁共振（EPR）光谱方法的进步，以探测膜蛋白的结构和动力学在其原生环境中利用Na+/脯氨酸转运蛋白PutP作为模型。在基于脉冲EPR距离数据建模静态结构后，我们的研究活动将集中在跨膜转运耦合离子和底物所需的构象改变上。因此，cw和脉冲EPR迁移率、可及性和距离数据以及自旋标记-磷脂头基距离（31 P ENDOR）应用于确定平衡条件下的单个构象状态，并跟踪构象变化的时间过程。将开发一种方法，将产生的相关的距离变化在纳米范围内的PutP的结构变化的粗粒度模型。其中，关于自旋标记迁移率和可及性的数据应用于控制和改进模型。基于已知的相似性在三级结构水平上，PutP动力学的信息预计将被转移到运输的各个家庭。

项目成果

Extracellular loop 4 of the proline transporter PutP controls the periplasmic entrance to ligand binding sites.

脯氨酸转运蛋白 PutP 的细胞外环 4 控制配体结合位点的周质入口

• DOI: 10.1016/j.str.2014.03.011
• 发表时间: 2014
• 期刊: Structure
• 影响因子: 5.7
• 作者: Dunkel;Hilger;Lipiszko;Polyhach;Jeschke;Bracher;Steinhoff
• 通讯作者: Steinhoff