Gorovits B M, Horowitz P M
Department of Biochemistry, University of Texas Health Science Center at San Antonio 78284, USA.
J Biol Chem. 1995 Jun 2;270(22):13057-62. doi: 10.1074/jbc.270.22.13057.
Steady-state fluorescence polarization was used to examine the chaperonin cpn60 that was covalently labeled with pyrene. Two compounds, 1-pyrenesulfonyl chloride or N-(1-pyrene)maleimide, were used to incorporate up to 8 mol of pyrene per mol of cpn60 14-mer. The fluorescence lifetime of the cpn60-pyrenesulfonyl chloride conjugate exhibited a double exponential decay: 5.36 ns, with a fractional contribution to the intensity of 7%, and 48.77 ns, with a fractional contribution to the intensity of 93%. These yield a second-order average lifetime of 45.58 ns at 20 degrees C. Analysis of the fluorescence polarization data for the pyrene probe by the Perrin-Weber treatment revealed the existence of two components that account for the depolarization. The fast component accounted for 24% of the depolarization at 20 degrees C. The rotational relaxation time for the cpn60 14-mer derived from the low viscosity part of the Perrin-Weber plot which accentuates the slow motion gave rho h = 1113 +/- 55 ns. When this value of rho h is compared with the rho h calculated based on the Stokes radius of cpn60 from ultracentrifugation, rho Stokes, it leads to rho h/rho Stokes = 0.4 which is considerably smaller than the value expected (rho h/rho Stokes = 1) or actually found in the cpn60-rhodanese complex (rho h/rho Stokes = 0.93). These considerations and the observed presence of the fast motion suggest that cpn60 is not a rigid protein. Analysis of the polarization data as a function of temperature, which is weighted more toward the fast motion, showed that the rotational relaxation time assessed by temperature variation is greatly increased (from 552.5 to 2591 ns) for the complex of cpn60 with partially folded rhodanese (34-kDa monomeric protein). No change in rho h was observed upon formation of the cpn60.ATP complex (rho h = 556.9 ns). These data indicate that there is local motion in the cpn60 14-mer molecule that can be frozen by formation of a binary complex with partially folded proteins. This conclusion is in keeping with results showing that the structure of cpn60 is generally stabilized when it forms complexes with passenger proteins (Mendoza, J. A., and Horowitz, P. M. (1994) J. Biol. Chem. 269, 25963-25965).
稳态荧光偏振被用于检测与芘共价标记的伴侣蛋白cpn60。两种化合物，1-芘磺酰氯或N-(1-芘基)马来酰亚胺，被用于每摩尔cpn60 14聚体中掺入多达8摩尔的芘。cpn60-芘磺酰氯共轭物的荧光寿命呈现双指数衰减：5.36纳秒，对强度的贡献率为7%，以及48.77纳秒，对强度的贡献率为93%。在20摄氏度时，这些产生了45.58纳秒的二阶平均寿命。通过佩林-韦伯处理对芘探针的荧光偏振数据进行分析，揭示了存在两个导致去极化的组分。快速组分在20摄氏度时占去极化的24%。从佩林-韦伯图的低粘度部分得出的cpn60 14聚体的旋转弛豫时间，该部分突出了慢速运动，得到ρh = 1113 ± 55纳秒。当将这个ρh值与基于超速离心法得到的cpn60的斯托克斯半径计算出的ρStokes进行比较时，得到ρh/ρStokes = 0.4，这比预期值（ρh/ρStokes = 1）或在cpn60-硫氰酸酶复合物中实际发现的值（ρh/ρStokes = 0.93）要小得多。这些考虑因素以及观察到的快速运动的存在表明cpn60不是一种刚性蛋白质。对作为温度函数的偏振数据进行分析，该分析更多地侧重于快速运动，结果表明，对于cpn60与部分折叠的硫氰酸酶（34 kDa单体蛋白）的复合物，通过温度变化评估的旋转弛豫时间大幅增加（从552.5纳秒增加到2591纳秒）。在形成cpn60·ATP复合物时未观察到ρh的变化（ρh = 556.9纳秒）。这些数据表明，cpn60 14聚体分子中存在局部运动，该运动可通过与部分折叠的蛋白质形成二元复合物而被冻结。这一结论与结果相符，即当cpn60与乘客蛋白形成复合物时，其结构通常会得到稳定（门多萨，J. A.，和霍洛维茨，P. M.（1994年）《生物化学杂志》269，25963 - 25965）。
