Studying Dynamics and Energetics of Membrane Protein Oligomerization using Single Molecule Fluorescence

细胞壁或细胞膜由双层脂膜和蛋白质组成，蛋白质是控制电信号和底物进出细胞的功能大分子。膜蛋白也感知外部刺激，并触发内部的信号级联反应。直到最近，脂膜本身被认为主要是一种被动的基质，将细胞内部与外部分开。然而，最近，越来越清楚的是，脂膜的组成影响蛋白质的功能。这也与我们的营养有关；众所周知，胆固醇和omega-3等化合物可以进入脂膜，改变其性质并调节蛋白质功能。相反，嵌入在脂膜中的蛋白质也会改变其相态行为和形态。*许多蛋白质齐聚成更大的复合体或簇。我们最近发现，膜蛋白的聚集，质子门控的KCSA通道，与膜的变形有关。在这个研究项目中，我们研究了蛋白质齐聚和膜变形的能量学及其对蛋白质功能的影响。我们将利用单分子荧光直接观察蛋白质齐聚的动力学，我们可以跟踪蛋白质在人造膜中的运动，并确定聚集过程的能量景观。我们将确定膜曲率对蛋白质功能和寡聚化的影响，并表征哪些种类的膜蛋白经历了脂质介导的聚集。

The cell wall, or cell membrane, is composed of a double-layered lipid film and proteins, functional macromolecules that control electrical signals and substrate transport in and out of the cell. Membrane proteins also sense external stimuli and trigger signaling cascades in the inside. Until recently, the lipid membrane itself was considered mainly a passive matrix that separates the cell interior from the exterior. However, recently, it became increasingly clear that the composition of the lipid membrane influences the functioning of the proteins. This also relates to our nutrition; compounds such as cholesterol and omega-3 are known to enter the lipid film, alter its properties and modulate protein function. Conversely, also the proteins embedded in the lipid membrane alter its phase behaviour and form. ***Many proteins oligomerize to larger complexes or clusters. We found recently that clustering of a membrane protein, the proton-gated KcsA channels, is related to deformations of the membrane. In this research program, we investigate the energetics of protein oligomerization and membrane deformations and their implication on protein function. We will directly observe the dynamics of protein oligomerization using single molecule fluorescence, where we can follow the movement of the proteins in an artificial membrane and determine the energy landscape of the clustering process. We will determine the effect of membrane curvature on protein function and oligomerization and characterize which classes of membrane proteins undergo lipid-mediated clustering.