Cavagnero S, Zhou Z H, Adams M W, Chan S I
Arthur Amos Noyes Laboratories of Chemical Physics, California Institute of Technology, Pasadena 91125, USA.
Biochemistry. 1998 Mar 10;37(10):3377-85. doi: 10.1021/bi9721804.
As part of our studies on the structural and dynamic properties of hyperthermostable proteins, we have investigated the unfolding pathways of the small iron-sulfur protein rubredoxin from Pyrococcus furiosus (RdPf) at pH 2. Unfolding has been initiated by temperature jump, triggered by manual mixing of a concentrated protein solution into a thermally preequilibrated buffer. The process has been followed in real time by absorption, tryptophan fluorescence emission, and far-UV circular dichroism. Unlike the case of the mesophilic rubredoxin from Clostridium pasteurianum (RdCp), RdPf displays a complex unfolding kinetics, pointing to the formation of at least three intermediates. All of the steps, including the one involving metal ion release, are extremely slow. However, hydrophobic core relaxation--not Fe3+ loss--is rate-determining for RdPf unfolding. This clearly rules out the fact that Fe3+ is solely responsible for the kinetic stability of RdPf. Results have been discussed in terms of sequential vs parallel pathways, and the possible role of irreversible phenomena has been taken into consideration. Aggregation does not appear to play a significant role in the observed kinetic complexities. According to a proposed sequential mechanism, partial release of secondary structure elements precedes iron loss, which is then followed by further loss of beta-sheet content and, finally, by hydrophobic relaxation. Although the main features of the RdPf unfolding mechanism remain substantially unchanged over the experimentally accessible temperature range, final hydrophobic relaxation gets faster, relative to the other events, as the temperature is decreased. A qualitative assessment of the unfolding activation parameters suggests that this arises from the very low activation energies (Ea) that characterize this step.
作为我们对超嗜热蛋白的结构和动力学性质研究的一部分，我们研究了来自激烈火球菌（Pyrococcus furiosus）的小铁硫蛋白嗜热红氧还蛋白（RdPf）在pH 2时的去折叠途径。通过将浓缩蛋白溶液手动混合到热预平衡缓冲液中引发温度跃升来启动去折叠过程。该过程通过吸收、色氨酸荧光发射和远紫外圆二色性进行实时监测。与来自巴氏梭菌（Clostridium pasteurianum）的嗜温红氧还蛋白（RdCp）不同，RdPf呈现出复杂的去折叠动力学，表明至少形成了三种中间体。所有步骤，包括涉及金属离子释放的步骤，都极其缓慢。然而，对于RdPf去折叠来说，疏水核心松弛而非Fe3+的丢失是速率决定因素。这明确排除了Fe3+单独负责RdPf动力学稳定性的事实。已根据连续途径与平行途径对结果进行了讨论，并考虑了不可逆现象的可能作用。聚集似乎在观察到的动力学复杂性中不起重要作用。根据提出的连续机制，二级结构元件的部分释放先于铁的丢失，随后是β-折叠含量的进一步减少，最后是疏水松弛。尽管在实验可及的温度范围内，RdPf去折叠机制的主要特征基本保持不变，但随着温度降低，相对于其他事件，最终的疏水松弛变得更快。对去折叠活化参数的定性评估表明，这是由于该步骤具有非常低的活化能（Ea）所致。
