Kimura Tetsunari, Lee Jennifer C, Gray Harry B, Winkler Jay R
Beckman Institute, California Institute of Technology, Pasadena, CA 91125-7400, USA.
Proc Natl Acad Sci U S A. 2007 Jan 2;104(1):117-22. doi: 10.1073/pnas.0609413103. Epub 2006 Dec 19.
The evolution of tryptophan-to-heme (W/heme) distance distributions extracted from analysis of fluorescence energy transfer kinetics during the refolding of Rhodopseudomonas palustris cytochrome c' reveals dramatic differences between two variants [W32 (Q1A/F32W/W72F) and W72 (Q1A)]. Both W32/heme and W72/heme distance distributions measured at the earliest time point attainable with a continuous-flow mixer (150 mus) confirm that the polypeptide ensemble is not uniformly collapsed and that native structure is not formed. Time-resolved fluorescence spectra indicate that W32 is sequestered from the aqueous solution during the first 700 mus of folding, whereas W72 remains exposed to solvent. The first moment of the W32/heme distance distribution evolves to its native value faster than that of W72, suggesting that the approach of W32 to the heme precedes that of W72.
从沼泽红假单胞菌细胞色素c'重折叠过程中荧光能量转移动力学分析得出的色氨酸-血红素(W/血红素)距离分布的演变,揭示了两个变体[W32(Q1A/F32W/W72F)和W72(Q1A)]之间的显著差异。在连续流动混合器可达到的最早时间点(150微秒)测量的W32/血红素和W72/血红素距离分布均证实,多肽总体并非均匀折叠,且未形成天然结构。时间分辨荧光光谱表明,在折叠的前700微秒内,W32与水溶液隔离,而W72仍暴露于溶剂中。W32/血红素距离分布的一阶矩比W72更快地演变为其天然值,这表明W32比W72更早接近血红素。