Choi J K, Kim I S, Kwon T I, Parker W, Song P S
Department of Chemistry, University of Nebraska, Lincoln 68588-0304.
Biochemistry. 1990 Jul 24;29(29):6883-91. doi: 10.1021/bi00481a018.
We have studied the effects of pH, ionic strength, and hydrophobic fluorescence probes, 8-anilinonaphthalene-1-sulfonate (ANS) and bis-ANS, on the structure of intact (124-kDa) Avena phytochrome. The Pfr form of phytochrome forms oligomers in solution to a greater extent than the Pr form. Hydrophobic forces play a major role in the oligomerization of phytochrome, as suggested by fluorescence and monomerization by bis-ANS. However, electrostatic charges also take part in the phytochrome oligomerization. The partial proteolytic digestion patterns for the Pr and Pfr species are different, but binding of bis-ANS to the phytochrome abolishes this difference and yields an identical proteolytic peptide mapping for both spectral forms of phytochrome. This appears to result from bis-ANS binding at the carboxy-terminal domain, which induces monomerization of phytochrome oligomers. A second bis-ANS binding at an amino-terminal site blocks cleavage sites of trypsin and alpha-chymotrypsin. Bis-ANS especially blocks access of the proteases to the amino-terminal cleavage site that produces an early proteolytic product (114/118 kDa) on SDS gels. The bis-ANS binding does not, however, affect the proteolytic cleavage site that occurs in the hinge region between the two structural domains of phytochrome, the chromophore domain and the C-terminal non-chromophore domain. A chromophore binding site in the Pfr form is apparently exposed for preferential binding of bis-ANS, causing cyclization of the chromophore and bleaching of its absorbance at 730 nm. These observations have been discussed in terms of a photoreversible topographic change of the chromophore/apoprotein during the phototransformation of phytochrome.
我们研究了pH值、离子强度以及疏水荧光探针8-苯胺基萘-1-磺酸盐（ANS）和双ANS对完整的（124 kDa）燕麦光敏色素结构的影响。光敏色素的Pfr形式在溶液中比Pr形式更易形成寡聚体。如荧光和双ANS诱导的单体化所示，疏水力在光敏色素的寡聚化过程中起主要作用。然而，静电荷也参与了光敏色素的寡聚化。Pr和Pfr形式的部分蛋白酶解消化模式不同，但双ANS与光敏色素的结合消除了这种差异，并为两种光谱形式的光敏色素产生了相同的蛋白酶解肽图谱。这似乎是由于双ANS在羧基末端结构域结合，从而诱导光敏色素寡聚体的单体化。双ANS在氨基末端位点的第二次结合会阻断胰蛋白酶和α-胰凝乳蛋白酶的切割位点。双ANS尤其会阻断蛋白酶接近氨基末端切割位点，该位点在SDS凝胶上产生早期蛋白酶解产物（114/118 kDa）。然而，双ANS的结合并不影响在光敏色素的两个结构域（发色团结构域和C末端非发色团结构域）之间的铰链区发生的蛋白酶解切割位点。Pfr形式中的发色团结合位点显然暴露，有利于双ANS的优先结合，导致发色团环化并使其在730 nm处的吸光度漂白。已根据光敏色素光转化过程中发色团/脱辅基蛋白的光可逆地形变化对这些观察结果进行了讨论。
