Rietveld A W, Ferreira S T
Departamento de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Brazil.
Biochemistry. 1996 Jun 18;35(24):7743-51. doi: 10.1021/bi952118b.
Subunit dissociation and unfolding of dimeric rabbit muscle triose phosphate isomerase (TIM) induced by hydrostatic pressure were investigated. Changes in fluorescence emission of TIM (both intrinsic and of covalently attached probes) indicated that pressure ranging from 1 bar to 3.5 kbar promoted subunit dissociation and unfolding. Instrinsic fluorescence changes upon unfolding by pressure included a 27 nm red-shift of the emission, a decrease in fluorescence anisotropy from 0.14 to about 0.01, and a 1.5-fold increase in fluorescence quantum yield, similar to that observed in the presence of guanidine hydrochloride. Kinetics of pressure-induced fluorescence changes were slow (t 1/2 approximately 15 min) and little dependent on pressure. In order to selectively monitor subunit dissociation, fluorescence resonance energy transfer (FRET) measurements were carried out with TIM that was separately labeled with 5-((((2-iodoacetyl)-amino)ethyl)amino)naphthalene-1-sulfonic acid (1,5-IAEDANS) or fluorescein-5-isothiocyanate (FITC). FRET measurements indicated that subunit dissociation and unfolding took place concomitantly, both under equilibrium conditions and in kinetic experiments in which dissociation/unfolding was triggered by a sudden increase in pressure. Release of pressure caused monomer refolding and dimerization. Contrary to what would be expected for a process involving subunit dissociation, pressure effects on TIM were not dependent on protein concentration. Experiments involving a series of pressure jumps demonstrated persistent heterogeneity in sensitivity toward pressure in the ensemble of TIM dimers. This kind of deterministic behavior is similar to that exhibited by higher order protein aggregates and indicates that not all individual dimers are energetically identical in solution. The heterogeneity of native TIM revealed by sensitivity to pressure could not be detected by traditional means of protein separation, such as polyacrylamide gel electrophoresis (under both native and denaturing conditions) and size exclusion gel chromatography. This suggests that energetic heterogeneity originates from conformational heterogeneity of the protein. The possible biological relevance of the deterministic character of stability of TIM is discussed.
研究了静水压力诱导的二聚体兔肌肉磷酸丙糖异构酶（TIM）的亚基解离和去折叠。TIM（固有荧光和共价连接探针的荧光）发射荧光的变化表明，1巴至3.5千巴的压力促进了亚基解离和去折叠。压力诱导去折叠时固有荧光的变化包括发射峰红移27纳米、荧光各向异性从0.14降至约0.01以及荧光量子产率增加1.5倍，这与在盐酸胍存在下观察到的情况相似。压力诱导荧光变化的动力学较慢（半衰期约15分钟），且对压力的依赖性较小。为了选择性监测亚基解离，对分别用5 -（（（2 - 碘乙酰基）-氨基）乙基）氨基萘 - 1 - 磺酸（1,5 - IAEDANS）或异硫氰酸荧光素（FITC）标记的TIM进行了荧光共振能量转移（FRET）测量。FRET测量表明，在平衡条件下以及在压力突然升高触发解离/去折叠的动力学实验中，亚基解离和去折叠同时发生。压力释放导致单体重新折叠和二聚化。与涉及亚基解离的过程预期相反，压力对TIM的影响不依赖于蛋白质浓度。一系列压力跃升实验表明，TIM二聚体总体对压力的敏感性存在持续的异质性。这种确定性行为类似于高阶蛋白质聚集体表现出的行为，表明并非所有单个二聚体在溶液中的能量状态都相同。传统的蛋白质分离方法，如聚丙烯酰胺凝胶电泳（在天然和变性条件下）和尺寸排阻凝胶色谱法，无法检测到压力敏感性所揭示的天然TIM的异质性。这表明能量异质性源于蛋白质的构象异质性。讨论了TIM稳定性的确定性特征可能具有的生物学相关性。
