Chen B, King J
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Biochemistry. 1991 Jun 25;30(25):6260-9. doi: 10.1021/bi00239a026.
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 Tm to the presence of temperature-sensitive folding mutations [Sturtevant, J. M., Yu, M.-H., Haase-Pettingell, C., & King, J. (1989) J. Biol. Chem. 264, 10693-10698] 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.
蛋白质稳定性在生物学或工业上具有相关性的条件，常常与研究可逆变性的条件有所不同。噬菌体P22的三聚体尾刺内鼠李糖苷酶是一种病毒结构蛋白，在一系列环境条件下，它对热、蛋白酶和去污剂都表现出高度稳定性。其细胞内折叠途径包括单体和三聚体折叠中间体，并且一直是详细遗传分析的对象。为了理解尾刺热稳定性的基础，我们研究了热变性和去污剂变性的动力学。在尾刺的热变性过程中，一种亚稳态的变性中间体积累起来，它可以在低温或SDS存在的情况下被捕获。这个物种仍然是三聚体，但已经失去了与病毒衣壳结合的能力，并且与天然三聚体不同，它对蛋白酶消化部分敏感。其N端区域包含约110个残基，处于未折叠状态，而多肽链的中心区域和C端仍处于折叠状态。因此，热变性的起始步骤是N端的展开，但中间体的解链代表了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℃的2% SDS中，从天然状态转变为展开中间体的展开速率常数为1.1×10⁻³ s⁻¹，从中间体转变为展开链的速率常数为4.0×10⁻⁵ s⁻¹。这种顺序展开途径解释了表观熔点对位于链中心区域的温度敏感折叠突变的不敏感性[斯特蒂文特，J.M.，于，M.-H.，哈斯-佩廷格尔，C.，& 金，J.（1989年）《生物化学杂志》264，10693 - 10698]。在较低温度下发生的正向折叠途径中未检测到亚稳态的变性中间体。高温热变性途径的早期阶段不是低温折叠途径后期阶段的逆过程。
