Kemple M D, Yuan P, Nollet K E, Fuchs J A, Silva N, Prendergast F G
Department of Physics, Indiana University-Purdue University Indianapolis 46202-3273.
Biophys J. 1994 Jun;66(6):2111-26. doi: 10.1016/S0006-3495(94)81006-9.
The rotational motion of tryptophan side chains in oxidized and reduced wild-type (WT) Escherichia coli thioredoxin and in two single-tryptophan variants of E. coli thioredoxin was studied in solution in the temperature range 20-50 degrees C from 13C-NMR relaxation rate measurements at 75.4 and 125.7 MHz and at 20 degrees C from steady-state and time-resolved trp fluorescence anisotropy measurements. Tryptophan enriched with 13C at the delta 1 and epsilon 3 sites of the indole ring was incorporated into WT thioredoxin and into two single-trp mutants, W31F and W28F, in which trp-28 or trp-31 of WT thioredoxin was replaced, respectively, with phenylalanine. The NMR relaxation data were interpreted using the Lipari and Szabo "model-free" approach (G. Lipari and A. Szabo. 1982. J. Amer. Chem. Soc. 104:4546-4559) with trp steady-state anisotropy data included for the variants at 20 degrees C. Values for the correlation time for the overall rotational motion (tau m) from NMR of oxidized and reduced WT thioredoxin at 35 degrees C agree well with those given by Stone et al. (Stone, M. J., K. Chandrasekhar, A. Holmgren, P. E. Wright, and H. J. Dyson. 1993. Biochemistry. 32:426-435) from 15N NMR relaxation rates, and the dependence of tau m on viscosity and temperature was in accord with the Stokes-Einstein relationship. Order parameters (S2) near 1 were obtained for the trp side chains in the WT proteins even at 50 degrees C. A slight increase in the amplitude of motion (decrease in S2) of trp-31, which is near the protein surface, but not of trp-28, which is partially buried in the protein matrix, was observed in reduced relative to oxidized WT thioredoxin. For trp-28 in W31F, order parameters near 1 (S2 > or = 0.8) at 20 degrees C were found, whereas trp-31 in W28F yielded the smallest order parameters (S2 approximately 0.6) of any of the cases. Analysis of time-resolved anisotropy decays in W28F and W31F yielded S2 values in good agreement with NMR, but gave tau m values about 60% smaller. Generally, values of tau e, the effective correlation time for the internal motion, were < or = 60 ps from NMR, whereas somewhat longer times were obtained from fluorescence. The ability of NMR and fluorescence techniques to detect subnanosecond motions in proteins reliably is examined.
通过在75.4和125.7 MHz下进行的¹³C - NMR弛豫速率测量，以及在20℃下进行的稳态和时间分辨的色氨酸荧光各向异性测量，研究了氧化型和还原型野生型（WT）大肠杆菌硫氧还蛋白以及大肠杆菌硫氧还蛋白的两个单色氨酸变体在20 - 50℃温度范围内溶液中的色氨酸侧链旋转运动。在吲哚环的δ1和ε3位点富含¹³C的色氨酸被掺入野生型硫氧还蛋白以及两个单色氨酸突变体W31F和W28F中，其中野生型硫氧还蛋白的色氨酸 - 28或色氨酸 - 31分别被苯丙氨酸取代。使用Lipari和Szabo的“无模型”方法（G. Lipari和A. Szabo. 1982. J. Amer. Chem. Soc. 104:4546 - 4559）解释NMR弛豫数据，并纳入了20℃下变体的色氨酸稳态各向异性数据。在35℃下，氧化型和还原型野生型硫氧还蛋白NMR的整体旋转运动相关时间（τm）值与Stone等人（Stone, M. J., K. Chandrasekhar, A. Holmgren, P. E. Wright, and H. J. Dyson. 1993. Biochemistry. 32:426 - 435）从¹⁵N NMR弛豫速率得到的值非常吻合，并且τm对粘度和温度的依赖性符合斯托克斯 - 爱因斯坦关系。即使在50℃时，野生型蛋白质中色氨酸侧链的序参数（S²）也接近1。相对于氧化型野生型硫氧还蛋白，在还原型中观察到靠近蛋白质表面的色氨酸 - 31的运动幅度略有增加（S²减小），而部分埋在蛋白质基质中的色氨酸 - 28则没有。对于W31F中的色氨酸 - 28，在20℃时发现序参数接近1（S²≥0.8），而W28F中的色氨酸 - 31产生了所有情况中最小的序参数（S²约为(0.6)）。对W28F和W31F中时间分辨各向异性衰减的分析得出的S²值与NMR结果高度吻合，但τm值小约60%。一般来说，从NMR得到的内部运动有效相关时间τe的值≤60 ps，而从荧光得到的时间则稍长。研究了NMR和荧光技术可靠检测蛋白质中亚纳秒级运动的能力。
