Moon C Preston, Fleming Karen G
T.C. Jenkins Department of Biophysics, Johns Hopkins University, Baltimore, Maryland, USA.
Methods Enzymol. 2011;492:189-211. doi: 10.1016/B978-0-12-381268-1.00018-5.
Accurate measurements of the thermodynamic stability of folded membrane proteins require methods for monitoring their conformation that are free of experimental artifacts. For tryptophan fluorescence emission experiments with membrane proteins folded into liposomes, there are two significant sources of artifacts: the first is light scattering by the liposomes; the second is the nonlinear relationship of some tryptophan spectral parameters with changes in protein conformation. Both of these sources of error can interfere with the method of determining the reversible equilibrium thermodynamic stability of proteins using titrations of chemical denaturants. Here, we present methods to manage light scattering by liposomes for tryptophan emission experiments and to properly monitor tryptophan spectra as a function of protein conformation. Our methods are tailored to the titrations of membrane proteins using common chemical denaturants. One of our recommendations is to collect and analyze the right-angle light scattering peak that occurs around the excitation wavelength in a fluorescence experiment. Another recommendation is to use only those tryptophan spectral parameters that are linearly proportional to the protein conformational population. We show that other commonly used spectral parameters lead to errors in protein stability measurements.
准确测量折叠膜蛋白的热力学稳定性需要能够监测其构象且不受实验假象影响的方法。对于将膜蛋白折叠到脂质体中的色氨酸荧光发射实验，存在两个重要的假象来源：第一个是脂质体的光散射；第二个是一些色氨酸光谱参数与蛋白质构象变化之间的非线性关系。这两个误差来源都会干扰使用化学变性剂滴定来确定蛋白质可逆平衡热力学稳定性的方法。在此，我们提出了一些方法来处理脂质体在色氨酸发射实验中的光散射，并根据蛋白质构象正确监测色氨酸光谱。我们的方法是针对使用常见化学变性剂对膜蛋白进行滴定而量身定制的。我们的一项建议是在荧光实验中收集并分析激发波长附近出现的直角光散射峰。另一项建议是仅使用那些与蛋白质构象群体呈线性比例关系的色氨酸光谱参数。我们表明，其他常用的光谱参数会导致蛋白质稳定性测量出现误差。
