Lilie H, Rudolph R, Buchner J
Institut für Biophysik & Physikalische Biochemie, Universität Regensburg, Germany.
J Mol Biol. 1995 Apr 21;248(1):190-201. doi: 10.1006/jmbi.1995.0211.
The folding pathways of multi-domain proteins are still poorly understood due to the complexity of the reaction involving domain folding, association and, in many cases, prolyl cis/trans isomerization. Here, we have established a kinetic model for the folding of the Fab fragment of the antibody MAK 33 with intact disulfide bonds. Folding of the hetero-dimeric protein from the completely denatured, oxidized state comprises the pairwise association of the two domains of each chain with those of the partner protein. Both the reactivation of the Fab fragment in which the two constituent polypeptide chains were covalently linked via a cystine bond (Fab) and that of a mutant lacking this covalent linkage (Fab/-cys) were monitored by ELISA. Folding of the Fab fragment is a slow process, which can be described by a single exponential term. The kinetic phase reflects a folding step after the association of the two chains. The same reaction was detected in the folding of Fab/-cys but an additional rate-limiting step is involved that is due to a unimolecular step in the folding of the isolated light chain. This implies that, during Fab reactivation, Fd associates with the light chain at the stage of an earlier folding intermediate, thus eliminating the additional slow folding step of the light chain observed with Fab/-cys. Both in Fab and Fab/-cys renaturation, the folding reaction after association is determined by prolyl isomerization. Therefore, at least four different association-competent folding intermediates have to be postulated according to the folding stage of light chain and the configuration of at least one prolyl-peptide bond. Using the different substrate specificities of cyclophilin and FK506 binding protein, we have obtained evidence that Pro159 within the Fd fragment may be responsible for the observed slow folding phase after association, although three other proline residues adopt a cis configuration in the native protein. Furthermore, the data suggest that in the case of the Fab fragment, association is a prerequisite for cis/trans isomerization of prolyl peptide bonds, implying that the quaternary but not the tertiary structure determines the cis-configuration of the prolyl residue in Fd involved in the rate-limiting folding reaction.
由于涉及结构域折叠、缔合以及在许多情况下的脯氨酰顺反异构化反应的复杂性，多结构域蛋白质的折叠途径仍未得到很好的理解。在此，我们建立了一个具有完整二硫键的抗体MAK 33的Fab片段折叠的动力学模型。从完全变性的氧化状态折叠异源二聚体蛋白质包括每条链的两个结构域与伙伴蛋白质的结构域成对缔合。通过酶联免疫吸附测定法监测了其中两条组成多肽链通过胱氨酸键共价连接的Fab片段（Fab）和缺乏这种共价连接的突变体（Fab/-cys）的再活化。Fab片段的折叠是一个缓慢的过程，可以用一个单指数项来描述。动力学阶段反映了两条链缔合后的一个折叠步骤。在Fab/-cys的折叠中检测到相同的反应，但涉及一个额外的限速步骤，这是由于分离的轻链折叠中的一个单分子步骤。这意味着，在Fab再活化过程中，Fd在较早的折叠中间体阶段与轻链缔合，从而消除了在Fab/-cys中观察到的轻链额外的缓慢折叠步骤。在Fab和Fab/-cys复性过程中，缔合后的折叠反应均由脯氨酰异构化决定。因此，根据轻链的折叠阶段和至少一个脯氨酰肽键的构型，必须假定至少四种不同的具有缔合能力的折叠中间体。利用亲环蛋白和FK506结合蛋白的不同底物特异性，我们获得了证据表明Fd片段内的Pro159可能是缔合后观察到的缓慢折叠阶段的原因，尽管在天然蛋白质中其他三个脯氨酸残基呈顺式构型。此外，数据表明，就Fab片段而言，缔合是脯氨酰肽键顺反异构化的先决条件，这意味着四级结构而非三级结构决定了参与限速折叠反应的Fd中脯氨酰残基的顺式构型。
