THERMAL UNFOLDING PATHWAY FOR THE THERMOSTABLE P22 TAILSPIKE ENDORHAMNOSIDASE
THERMAL UNFOLDING PATHWAY FOR THE THERMOSTABLE P22 TAILSPIKE ENDORHAMNOSIDASE
复制标题
DOI：
10.1021/bi00239a026
复制
发表时间：
1991-06-25
期刊：
影响因子：
2.9
通讯作者：
KING, J
中科院分区：
文献类型：
--
作者：
CHEN, BL;KING, J
蛋白质稳定性在生物学或工业上相关的条件常常不同于研究可逆变性的条件。噬菌体 P22 的三聚体尾刺内含鼠李糖苷酶是一种病毒结构蛋白，在各种环境条件下对热、蛋白酶和去污剂表现出高度稳定性。其细胞内折叠途径包括单体和三聚体折叠中间体，并已成为详细遗传分析的主题。为了了解尾峰热稳定性的基础，我们研究了热和洗涤剂展开的动力学。在尾刺热解折叠过程中，亚稳态解折叠中间体会积聚，在寒冷或存在 SDS 的情况下可能会被捕获。该物种仍然是三聚体，但失去了与病毒衣壳结合的能力，并且与天然三聚体不同，它部分容易受到蛋白酶消化。其N端区域包含约110个残基，是未折叠的，而多肽链的中央区域和C端仍处于折叠状态。因此，热变性的起始步骤是 N 末端的展开，但中间体的熔化代表变性过程中的第二个动力学障碍。这种两步展开在高温下异常缓慢。例如，在 65°C 的 2% SDS 中，从天然链到未折叠中间体的转变的解折叠速率常数为 1. 1 X 10(-3) s-1，从中间体到未折叠链的转变为 4.0 x 10(-5) s-1。顺序解折叠途径解释了表观 T(m) 对温度敏感折叠突变的不敏感性 [Sturtevant, J. M., Yu, M.-H., Haase-Pettingell, C., & King, J. (1989) J. Biol.化学。 264、10693-106981，位于该链的中心区域。在较低温度下发生的正向折叠途径中尚未检测到亚稳态展开中间体。高温热解折叠途径的早期阶段并不是低温折叠途径后期的逆转。
The conditions in which protein stability is biologically or industrially relevant frequently differ from those in which reversible denaturation is studied. The trimeric tailspike endorhamnosidase of phage P22 is a viral structural protein which exhibits high stability to heat, proteases, and detergents under a range of environmental conditions. Its intracellular folding pathway includes monomeric and trimeric folding intermediates and has been the subject of detailed genetic analysis. To understand the basis of tailspike thermostability, we have examined the kinetics of thermal and detergent unfolding. During thermal unfolding of the tailspike, a metastable unfolding intermediate accumulates which can be trapped in the cold or in the presence of SDS. This species is still trimeric, but has lost the ability to bind to virus capsids and, unlike the native trimer, is partially susceptible to protease digestion. Its N-terminal regions, containing about 110 residues, are unfolded whereas the central regions and the C-termini of the polypeptide chains are still in the folded state. Thus, the initiation step in thermal denaturation is the unfolding of the N-termini, but melting of the intermediate represents a second kinetic barrier in the denaturation process. This two-step unfolding is unusually slow at elevated temperature; for instance, in 2% SDS at 65-degrees-C, the unfolding rate constant is 1. 1 X 10(-3) s-1 for the transition from the native to the unfolding intermediate and 4.0 x 10(-5) s-1 for the transition from the intermediate to the unfolded chains. The sequential unfolding pathway explains the insensitivity of the apparent T(m) to the presence of temperature-sensitive folding mutations [Sturtevant, J. M., Yu, M.-H., Haase-Pettingell, C., & King, J. (1989) J. Biol. Chem. 264, 10693-106981 which are located in the central region of the chain. The metastable unfolding intermediate has not been detected in the forward folding pathway occurring at lower temperatures. The early stage of the high-temperature thermal unfolding pathway is not the reverse of the late stage of the low-temperature folding pathway.