Fratti Rutilio, Calderin Jorge D, Starr Matthew L
Dept of Biochemistry & Center for Biophysics and Quantitative Biology, University of Illinois at Urbana-Champaign, Urbana, IL, USA.
Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL, USA.
Methods Mol Biol. 2025;2887:119-132. doi: 10.1007/978-1-0716-4314-3_8.
Vacuole fusion is driven by SNARE proteins that require activation-or priming-by the AAA+ protein Sec18 (NSF) before they can bring membranes together and trigger the merger of two bilayers into a continuous membrane. Sec18 resides on vacuoles prior to engaging inactive cis-SNARE complexes through its interaction with the regulatory lipid phosphatidic acid (PA). Binding PA causes Sec18 to undergo large conformational changes that keeps it bound to the membrane, thus precluding its interactions with SNAREs. Such conformational changes can be measured by various biochemical and biophysical assays. The conversion of PA to diacylglycerol by the PA phosphatase Pah1 releases Sec18 from the membrane-bound pool and promotes its transfer to SNARE complexes allowing priming to occur. Here we describe four spectroscopy-based methods to distinguish conformational changes from alterations in secondary structure during PA binding. These methods only require purified protein and short chain soluble lipids, making the methods rapid and affordable ways to screen the effects of specific protein-lipid interactions. The assays described in this chapter include 1-anilino-8-naphthalenesulfonate (ANS) spectroscopy and intrinsic tryptophan fluorescence to detect exposure of hydrophobic regions; differential scanning fluorometry to measure changes in protein stability across a temperature gradient; and circular dichroism to examine changes in secondary structure.
液泡融合由SNARE蛋白驱动，这些蛋白在将膜聚集在一起并触发两个双层膜合并成一个连续膜之前，需要由AAA +蛋白Sec18（NSF）进行激活或引发。在通过与调节性脂质磷脂酸（PA）相互作用与无活性的顺式SNARE复合物结合之前，Sec18存在于液泡上。结合PA会导致Sec18发生大的构象变化，使其与膜结合，从而排除其与SNARE的相互作用。这种构象变化可以通过各种生化和生物物理测定来测量。PA磷酸酶Pah1将PA转化为二酰基甘油，使Sec18从膜结合池中释放出来，并促进其转移到SNARE复合物中，从而引发反应。在这里，我们描述了四种基于光谱的方法，以区分PA结合过程中构象变化与二级结构改变。这些方法只需要纯化的蛋白质和短链可溶性脂质，使这些方法成为筛选特定蛋白质-脂质相互作用效果的快速且经济实惠的方法。本章所述的测定方法包括1-苯胺基-8-萘磺酸盐（ANS）光谱法和内在色氨酸荧光法以检测疏水区域的暴露；差示扫描荧光法以测量蛋白质稳定性在温度梯度上的变化；以及圆二色性以检查二级结构的变化。
