Institute of Chemistry, HALOmem, Charles Tanford Protein Center, Martin Luther University of Halle, Halle (Saale), Germany.
Institute of Chemistry, HALOmem, Charles Tanford Protein Center, Martin Luther University of Halle, Halle (Saale), Germany; Institute of Biochemistry and Biotechnology, HALOmem, Charles Tanford Protein Center, Martin Luther University of Halle, Halle (Saale), Germany.
Biophys J. 2021 Apr 20;120(8):1333-1342. doi: 10.1016/j.bpj.2021.02.011. Epub 2021 Feb 18.
Membrane insertion of protein domains is an important step in many membrane remodeling processes, for example, in vesicular transport. The membrane area taken up by the protein insertion influences the protein binding affinity as well as the mechanical stress induced in the membrane and thereby its curvature. To our knowledge, this is the first optical measurement of this quantity on a system in equilibrium with direct determination of the number of inserted protein and no further assumptions concerning the binding thermodynamics. Whereas macroscopic total area changes in lipid monolayers are typically measured on a Langmuir film balance, finding the number of inserted proteins without perturbing the system and quantitating any small area changes has posed a challenge. Here, we address both issues by performing two-color fluorescence correlation spectroscopy directly on the monolayer. With a fraction of the protein being fluorescently labeled, the number of inserted proteins is determined in situ without resorting to invasive techniques such as collecting the monolayer by aspiration. The second color channel is exploited to monitor a small fraction of labeled lipids to determine the total area increase. Here, we use this method to determine the insertion area per molecule of Sar1, a protein of the COPII complex, which is involved in transport vesicle formation. Sar1 has an N-terminal amphipathic helix, which is responsible for membrane binding and curvature generation. An insertion area of (3.4 ± 0.8) nm was obtained for Sar1 in monolayers from a lipid mixture typically used in COPII reconstitution experiments, in good agreement with the expected insertion area of the Sar1 amphipathic helix. By using the two-color approach, determining insertion areas relies only on local fluorescence measurements. No macroscopic area measurements are needed, giving the method the potential to also be applied to laterally heterogeneous monolayers and bilayers.
蛋白质结构域的膜插入是许多膜重塑过程中的重要步骤，例如囊泡运输。蛋白质插入所占据的膜面积会影响蛋白质的结合亲和力以及在膜中诱导的机械应力，从而影响膜的曲率。据我们所知，这是首次在与直接确定插入蛋白质数量且不进一步假设结合热力学的平衡系统中对该数量进行的光学测量。虽然在 Langmuir 膜天平上通常测量脂质单层的宏观总面积变化，但在不干扰系统的情况下找到插入蛋白质的数量并定量任何小的面积变化一直是一个挑战。在这里，我们通过直接在单层上进行双色荧光相关光谱学来解决这两个问题。通过对一部分蛋白质进行荧光标记，可以在不使用收集单层等侵入性技术的情况下原位确定插入蛋白质的数量。第二个颜色通道用于监测一小部分标记的脂质，以确定总面积增加。在这里，我们使用这种方法来确定参与运输囊泡形成的 COPII 复合物的 Sar1 蛋白的插入面积。Sar1 具有一个 N 端的两亲性螺旋，负责膜结合和曲率生成。从通常用于 COPII 重建实验的脂质混合物的单层中获得的 Sar1 的插入面积为（3.4 ± 0.8）nm，与 Sar1 两亲性螺旋的预期插入面积非常吻合。通过使用双色方法，确定插入面积仅依赖于局部荧光测量。不需要进行宏观面积测量，这使得该方法有可能也适用于横向不均匀的单层和双层。
