Venkitakrishnan Rani Parvathy, Benard Outhiriaradjou, Max Marianna, Markley John L, Assadi-Porter Fariba M
Department of Biochemistry, University of Wisconsin-Madison, Madison, WI, USA.
Methods Mol Biol. 2012;914:47-63. doi: 10.1007/978-1-62703-023-6_4.
Detection of weak ligand binding to membrane-spanning proteins, such as receptor proteins at low physiological concentrations, poses serious experimental challenges. Saturation transfer difference nuclear magnetic resonance (STD-NMR) spectroscopy offers an excellent way to surmount these problems. As the name suggests, magnetization transferred from the receptor to its bound ligand is measured by directly observing NMR signals from the ligand itself. Low-power irradiation is applied to a (1)H NMR spectral region containing protein signals but no ligand signals. This irradiation spreads quickly throughout the membrane protein by the process of spin diffusion and saturates all protein (1)H NMR signals. (1)H NMR signals from a ligand bound transiently to the membrane protein become saturated and, upon dissociation, serve to decrease the intensity of the (1)H NMR signals measured from the pool of free ligand. The experiment is repeated with the irradiation pulse placed outside the spectral region of protein and ligand, a condition that does not lead to saturation transfer to the ligand. The two resulting spectra are subtracted to yield the difference spectrum. As an illustration of the methodology, we review here STD-NMR experiments designed to investigate binding of ligands to the human sweet taste receptor, a member of the large family of G-protein-coupled receptors. Sweetener molecules bind to the sweet receptor with low affinity but high specificity and lead to a variety of physiological responses.
检测弱配体与跨膜蛋白（如处于低生理浓度的受体蛋白）的结合，存在严峻的实验挑战。饱和转移差核磁共振（STD-NMR）光谱法提供了一种解决这些问题的绝佳方法。顾名思义，通过直接观测配体自身的NMR信号来测量从受体转移至其结合配体的磁化强度。对包含蛋白质信号但无配体信号的(1)H NMR光谱区域施加低功率辐射。这种辐射通过自旋扩散过程迅速在整个膜蛋白中传播，并使所有蛋白质(1)H NMR信号饱和。短暂结合到膜蛋白上的配体的(1)H NMR信号会饱和，解离时，会降低从游离配体池测量的(1)H NMR信号强度。将辐射脉冲置于蛋白质和配体光谱区域之外重复该实验，此条件不会导致向配体的饱和转移。将得到的两个光谱相减以产生差异光谱。作为该方法的一个示例，我们在此回顾旨在研究配体与人类甜味受体（G蛋白偶联受体大家族的一员）结合的STD-NMR实验。甜味剂分子以低亲和力但高特异性结合甜味受体，并引发多种生理反应。
