Kotake S, Hey P, Mirmira R G, Copeland R A
Department of Biochemistry and Molecular Biology, University of Chicago, Illinois 60637.
Arch Biochem Biophys. 1991 Feb 15;285(1):126-33. doi: 10.1016/0003-9861(91)90338-j.
The native conformation of bovine retinal arrestin has been characterized by a variety of spectroscopic methods. The purified protein gives rise to a near uv absorption band centered at 279 nm which results from the absorbance of its 14 tyrosine and one tryptophan residue. The extinction coefficient for this absorption band was determined to be 38.64 mM-1, cm-1 using the tyrosinate-tyrosine difference spectrum method; this extinction coefficient is ca. 17% lower than the previously reported value, and provides estimates of protein concentration which are in good agreement with estimates from the Bradford colorimetric assay. When native arrestin is purified to homogeneity, it displays a fluorescence spectrum which is dominated by tyrosine emission with no discernible contribution from tryptophan. Observation of the tyrosine-like fluorescence is dependent on the purity and structural integrity of the protein. Denaturation of arrestin by guanidine hydrochloride results in a diminution of tyrosine fluorescence and the concomitant appearance of a second fluorescence maximum at ca. 340 nm, presumably due to the single tryptophan residue. Thermal denaturation of arrestin leads to a conformation characterized by a broad fluorescence band centered at ca. 325 nm. Study of the arrestin fluorescence spectrum as a function of temperature indicates that the thermal denaturation is well modeled as a two-state transition with a transition midpoint of 60 degrees C. Temperature-dependent far uv circular dichroism studies indicate that changes in secondary structure occur coincident with the change in fluorescence. Studies of the temperature dependence of arrestin binding to light-adapted phosphorylated rhodopsin shows a strong correlation between the fluorescence spectral features of arrestin and its ability to bind rhodopsin. These data suggest that the relative intensities of tyrosine and tryptophan fluorescence are sensitive to the structural integrity of the native (i.e., rhodopsin binding) state of arrestin, and can thus serve as useful markers of conformational transitions of this protein. The lack of tryptophan fluorescence for native arrestin suggests an unusual environment for this residue. Possible mechanisms for this tryptophan fluorescence quenching are discussed.
牛视网膜抑制蛋白的天然构象已通过多种光谱方法进行了表征。纯化后的蛋白质在279nm处产生一个近紫外吸收带，这是由其14个酪氨酸和1个色氨酸残基的吸光度导致的。使用酪氨酸盐 - 酪氨酸差光谱法测定该吸收带的消光系数为38.64 mM-1·cm-1；该消光系数比先前报道的值低约17%，并且提供的蛋白质浓度估计值与Bradford比色法的估计值高度一致。当将天然抑制蛋白纯化至同质时，它会显示出一个荧光光谱，该光谱以酪氨酸发射为主，色氨酸没有明显贡献。酪氨酸样荧光的观察取决于蛋白质的纯度和结构完整性。盐酸胍使抑制蛋白变性会导致酪氨酸荧光减弱，并同时在约340nm处出现第二个荧光最大值，这可能是由于单个色氨酸残基引起的。抑制蛋白的热变性导致一种构象，其特征是在约325nm处有一个宽荧光带。对抑制蛋白荧光光谱随温度变化的研究表明，热变性可以很好地模拟为一个两态转变，转变中点为60℃。温度依赖性远紫外圆二色性研究表明，二级结构的变化与荧光变化同时发生。对抑制蛋白与光适应磷酸化视紫红质结合的温度依赖性研究表明，抑制蛋白的荧光光谱特征与其结合视紫红质的能力之间存在很强的相关性。这些数据表明，酪氨酸和色氨酸荧光的相对强度对抑制蛋白天然（即视紫红质结合）状态的结构完整性敏感，因此可以作为该蛋白质构象转变的有用标记。天然抑制蛋白缺乏色氨酸荧光表明该残基所处环境异常。讨论了这种色氨酸荧光猝灭的可能机制。
