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

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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.