Wells T A, Nakazawa M, Manabe K, Song P S
Department of Chemistry, University of Nebraska, Lincoln 68588-0304.
Biochemistry. 1994 Jan 25;33(3):708-12. doi: 10.1021/bi00169a012.
Dynamic quenching of the two lifetime component tryptophan fluorescence of Pisum phytochrome has revealed differential accessibility of certain residues. Both acrylamide and Tl+ ions showed preferential exposure of some tryptophans in Pfr-phytochrome. Greater kq's for Pfr are, however, in contrast with values for Avena phytochrome in which Pr-->Pfr conversion impedes Tl+ access. The Pr short lifetime component was more accessible to Cs+; however, the long component accessibility was approximately 2-fold higher in Pfr. 2-Hydroxy-5-nitrobenzyl bromide (HNB-Br) modification of native Pisum phytochrome was used to reduce the total number of fluorescent tryptophans. The absence of the fluorescence contributions of the three residues which reacted with HNB-Br in both photoisomers increased the Tl+ Ksv's for Pr and Pfr. The two additional HNB-Br modifications specific for Pfr resulted in a reversal of the Stern-Volmer plots relative to the unmodified protein. The regions around four of the 10 tryptophans may represent conformationally photoresponsive areas in Pisum phytochrome A. Furthermore, topographic changes associated with the phytochrome phototransformation are not confined to the 58-kDa chromphore domain, and they involve most if not all of the region from Trp-365 to Trp-787. We also provide evidence that the protein conformation in this region is not completely conserved between Pisum and Avena phytochromes.
豌豆光敏色素两个寿命组分色氨酸荧光的动态猝灭揭示了某些残基的不同可及性。丙烯酰胺和Tl⁺离子均显示出一些色氨酸在Pfr - 光敏色素中优先暴露。然而，Pfr的猝灭常数（kq）更大，这与燕麦光敏色素的值形成对比，在燕麦光敏色素中Pr→Pfr的转变会阻碍Tl⁺的进入。Pr的短寿命组分对Cs⁺更易接近；然而，在Pfr中长寿命组分的可及性大约高2倍。用2 - 羟基 - 5 - 硝基苄基溴（HNB - Br）修饰天然豌豆光敏色素以减少荧光色氨酸的总数。在两种光异构体中与HNB - Br反应的三个残基的荧光贡献缺失增加了Pr和Pfr的Tl⁺的猝灭常数（Ksv）。另外两个对Pfr特异的HNB - Br修饰导致相对于未修饰蛋白质的斯特恩 - 沃尔默曲线反转。10个色氨酸中4个周围的区域可能代表豌豆光敏色素A中构象光响应区域。此外，与光敏色素光转化相关的拓扑变化并不局限于58 kDa的发色团结构域，并且它们涉及从Trp - 365到Trp - 787的大部分（如果不是全部）区域。我们还提供证据表明该区域的蛋白质构象在豌豆和燕麦光敏色素之间并非完全保守。
