Otzen D E, Fersht A R
MRC Unit for Protein Function and Design, Cambridge Centre for Protein Engineering, University Chemical Laboratory, U.K.
Biochemistry. 1998 Jun 2;37(22):8139-46. doi: 10.1021/bi980250g.
The 64-residue chymotrypsin inhibitor 2 (CI2) folds by a two-state nucleation-condensation mechanism, whereby secondary and tertiary structure coalesce concomitantly in the transition state around Ala 16 in the helical N-cap. Permutation of the SH3-domain of alpha-spectrin apparently shifts its folding nucleus to another region of the protein, suggesting that a protein's transition state may be altered by altering the protein's connectivity. We have characterized the structure of the transition state of a circular and a permuted version of CI2 by a protein engineering study encompassing 11 mutations. Circular CI2 was obtained by the introduction of cysteines at residues 3 and 63 and linking them by disulfide bond formation. Subsequent cyanogen-bromide cleavage of the scissile bond, Met 40-Glu 41, yielded permuted CI2. Circular and permuted CI2 also fold according to a two-state mechanism. Permutation does not affect the folding rate constant, but circularization increases it 7-fold. The transition states of circular and permuted CI2 are essentially unchanged from that of wild-type CI2. Importantly, the folding nucleus around Ala16 is retained. These results complement a previous observation that the transition state for association of two CI2 fragments (residues 1-40 and 41-64, generated by CNBr cleavage) is very similar to the folding transition state of intact CI2. The similarity of rate constants for folding of wild-type and permuted CI2, and their value relative to that for the association of fragments, allows us to estimate the gain in entropy of activation on having the separate fragments linked: 18.3 cal M-1 K-1; i.e. an effective molarity of 10(4) M. The contrast between the retention of the folding nucleus on permutation of CI2 and its change for the SH3-domain of alpha-spectrin probably arises because the latter was cleaved in its folding nucleus whereas cleavage at sites other than 40-41 in CI2 is very destabilizing. Whether or not a folding nucleus can be changed probably depends on the specific protein and its permissivity to permutation.
64个残基的胰凝乳蛋白酶抑制剂2（CI2）通过双态成核-凝聚机制进行折叠，在此过程中，二级和三级结构在螺旋N端帽中围绕Ala 16的过渡态同时合并。α-血影蛋白SH3结构域的重排显然将其折叠核心转移到了蛋白质的另一个区域，这表明蛋白质的过渡态可能会通过改变蛋白质的连接性而改变。我们通过一项包含11个突变的蛋白质工程研究，对环状和重排版本的CI2的过渡态结构进行了表征。通过在残基3和63处引入半胱氨酸并通过形成二硫键将它们连接起来，得到了环状CI2。随后对可裂解键Met 40-Glu 41进行溴化氰裂解，得到了重排CI2。环状和重排CI2也根据双态机制进行折叠。重排不影响折叠速率常数，但环化使其增加了7倍。环状和重排CI2的过渡态与野生型CI2的过渡态基本没有变化。重要的是，围绕Ala16的折叠核心得以保留。这些结果补充了之前的一项观察结果，即两个CI2片段（由CNBr裂解产生的残基1-40和41-64）缔合的过渡态与完整CI2的折叠过渡态非常相似。野生型和重排CI2折叠速率常数的相似性，以及它们相对于片段缔合的值，使我们能够估计将单独的片段连接起来时活化熵的增加：18.3 cal M-1 K-1；即有效摩尔浓度为10(4) M。CI2重排时折叠核心的保留与其α-血影蛋白SH3结构域的变化之间的差异，可能是因为后者在其折叠核心处被裂解，而CI2中40-41以外位点的裂解非常不稳定。折叠核心是否可以改变可能取决于特定的蛋白质及其对重排的容许性。
