In order to monitor conformational changes following photoactivation and phosphorylation of bovine rhodopsin, the two reactive sulfhydryl groups at Cys¹⁴⁰ and Cys³¹⁶ were specifically labeled with the monobromobimane (mBBr) fluorophore. Although alterations in conformation after light exposure of rhodopsin were not detected by fluorescence excitation scans (300-450 nm) of the mBBr-labeled protein, the fluorescence signal was reduced ∼ 90% in samples containing photoactivated phosphorhodopsin. Predominant labeling at either Cys¹⁴⁰ or Cys³¹⁶ in light-activated and phosphorylated rhodopsin merely generated a decrease of ∼ 38% and 28%, respectively, in the fluorescence excitation intensity. Thus, neither mBBr-modified Cys¹⁴⁰ nor mBBr-modified Cys³¹⁶ were involved single-handedly in the remarkable fall seen on the signal following phosphorylation of the protein; rather, the incorporation of phosphate groups on the mBBr-labeled light-activated rhodopsin appeared to affect its fluorescence signal in a cooperative or synergistic manner. These findings demonstrated that the phosphorylation of specific hydroxyl groups at the carboxyl terminal tail of rhodopsin causes definite conformational changes in the three-dimensional fold of the protein. Apparently, amino acid residues that are buried in the interior of the inactive protein become accessible following bleaching and phosphorylation of rhodopsin, quenching in turn the fluorescence excitation signal of mBBr-modified rhodopsin.

为了监测牛视紫红质的光活化和磷酸化后的构象变化，Cys ¹⁴⁰和Cys ^316上的两个反应性巯基分别用单溴二mane（mBBr）荧光团标记。尽管通过mBBr标记的蛋白的荧光激发扫描（300-450 nm）未检测到视紫红质暴露后构象的变化，但在含有光激活的视紫红质的样品中，荧光信号降低了约90％。在光激活的和磷酸化的视紫红质中，在Cys ¹⁴⁰或Cys ³¹⁶处的显着标记仅使荧光激发强度分别降低了约38％和28％。因此，两种mBBr修饰的Cys ¹⁴⁰mBBr修饰的Cys ³¹⁶也不参与蛋白质磷酸化后信号的明显下降。相反，在mBBr标记的光活化视紫红质上引入磷酸基团似乎以协同或协同方式影响其荧光信号。这些发现表明，视紫红质的羧基末端尾端的特定羟基的磷酸化引起了蛋白质三维折叠的确定构象变化。显然，在视紫红质的漂白和磷酸化作用之后，埋藏在无活性蛋白质内部的氨基酸残基变得可访问，进而淬灭了mBBr修饰的视紫红质的荧光激发信号。