Eder J, Rheinnecker M, Fersht A R
MRC Unit for Protein Function and Design, University Chemical Laboratory, Cambridge, U.K.
J Mol Biol. 1993 Sep 20;233(2):293-304. doi: 10.1006/jmbi.1993.1507.
Subtilisin BPN' is an extracellular serine protease from Bacillus amyloliquefaciens that requires an N-terminal 77 amino acid pro-sequence for correct folding of the catalytic domain. We have expressed an inactive, stable pro-subtilisin variant in Escherichia coli and show that it has structural properties similar to native subtilisin in terms of its near- and far-UV circular dichroism spectra, its compactness, and its capacity to bind calcium ions stoichiometrically. Unlike subtilisin, the pro-subtilisin variant unfolds reversibly with guanidinium chloride, and unfolding occurs via a folding intermediate. This intermediate is similar to the metastable intermediate state recently found for folding of subtilisin in the absence of the pro-sequence. The intermediate state has native-like secondary but little tertiary structure, and has a compactness between that of the native and unfolded state. Pro-subtilisin folds from the intermediate to the folded state in a single co-operative transition mediated by the pro-sequence. The isolated pro-sequence does not appear from its circular dichroism and 1H-NMR spectrum to have enough intrinsic stabilizing interactions to fold autonomously. However, the difference circular dichroism spectra of the pro-subtilisin variant and native subtilisin suggest that it is folded in the context of the pro-subtilisin molecule. The inability of the pro-subtilisin variant to bind a polypeptide inhibitor supports further the hypothesis that the pro-sequence interacts with subtilisin in the region where the active site is exposed. Our results suggest that the interactions provided by the pro-sequence are important only late on the folding pathway of pro-subtilisin and stabilize the transition state for folding. Kinetic analysis of the refolding reaction in the presence and absence of the pro-sequence reveal this stabilization to be in excess of 7.5 kcal/mol; folding is accelerated more than five orders of magnitude.
枯草杆菌蛋白酶BPN'是一种来自解淀粉芽孢杆菌的细胞外丝氨酸蛋白酶，其催化结构域的正确折叠需要一个N端77个氨基酸的前序列。我们在大肠杆菌中表达了一种无活性的、稳定的前枯草杆菌蛋白酶变体，并表明其在近紫外和远紫外圆二色光谱、紧凑性以及化学计量结合钙离子的能力方面具有与天然枯草杆菌蛋白酶相似的结构特性。与枯草杆菌蛋白酶不同，前枯草杆菌蛋白酶变体在氯化胍作用下可逆展开，且展开过程通过一个折叠中间体进行。这个中间体类似于最近在无前序列情况下枯草杆菌蛋白酶折叠过程中发现的亚稳态中间态。该中间态具有类似天然的二级结构但几乎没有三级结构，其紧凑性介于天然态和未折叠态之间。前枯草杆菌蛋白酶在由前序列介导的单一协同转变中从中间体折叠为折叠态。从其圆二色光谱和1H-NMR谱来看，分离出的前序列似乎没有足够的内在稳定相互作用来自主折叠。然而，前枯草杆菌蛋白酶变体和天然枯草杆菌蛋白酶的差示圆二色光谱表明它在前枯草杆菌蛋白酶分子的环境中是折叠的。前枯草杆菌蛋白酶变体无法结合多肽抑制剂进一步支持了前序列在活性位点暴露区域与枯草杆菌蛋白酶相互作用的假说。我们的结果表明，前序列提供的相互作用仅在枯草杆菌蛋白酶前体的折叠途径后期才重要，并稳定折叠的过渡态。对有无前序列时复性反应的动力学分析表明这种稳定作用超过7.5千卡/摩尔；折叠加速了五个多数量级。
