STUDIES OF PROTEIN FOLDING

在过去的几年里，我们分配了四个核心领域， 细胞色素c和它们的折叠顺序。 前人研究 也表明细胞色素c的折叠可能是 由核心域-域交互驱动。 自1988年以来 其他几组研究人员已经发现， 不同蛋白质的折叠中间体， 与我们的核心领域概念保持一致。 因此，在本发明中， 核心域-域交互可以是通用的 驱动球状蛋白质的折叠。 知道是否 核心域-域交互是否不同于 我们已经确定了已知类型的非共价相互作用 热力学和动力学效应的差异 在Leu 32和Leu 33之间用正缬氨酸（Nva）取代 马细胞色素c三片段复合物的Leu 35 前几年。 该络合物由血红素组成 残基1至5的片段，（1-25）H，和两个脱辅基片段 （28-38）和（39-104）。 它类似于天然的细胞色素c 除了残基39 - 55是柔性的。 我们有 现在使用复杂的模型分析和解释数据。 研究结果首次表明， 在标准吉布斯能量变化中，ddGs，与 片段（28-38）与 铁或亚铁络合物定性地与 活化吉布斯能的差异，dGa，相关 片段（39-104）的直接解离。 这些 ddG和dGa也与 的695 nm波段的热稳定性的差异， 铁络合物，已知指示Fe-S的条带 邦德 这些相关性被解释为 这表明一些额外的非共价键 相互作用可以由堆积的原子团产生 在疏水核中以稳定基态。 这些相互作用可以具有使得 它们产生的稳定能量会受到扰动 Leu 32 Nva取代比Leu 35 Nva多得多。 这些相互作用的扰动是由 Leu 32的γ-甲基基团的去除将传播 通过核心本身，并影响铁的稳定性- S键和（39-104）的结合强度。 这些 属性与核心域一致 互动 此外，分析，结合 其他证据，似乎表明，这些传播 核心中的非共价相互作用可能会有所不同 来自于货车范德华相互作用，疏水能， 氢键和电荷-电荷相互作用。 映射 核心侧链参与这些繁殖的非共价 酵母iso-2-细胞色素c相互作用的研究 定向诱变正在进行中。 去约会八个变种人 已经或正在生成。 精确地识别 细胞色素c核磁共振研究的核心领域也在 使用二维NOESY、COSY和TOCSY的进展 光谱与结构生物学 第节，LBC，NHLBI。

In the previous years we have assigned four core domains of cytochrome c and their folding order. The previous studies have also suggested that the folding of cytochrome c may be driven by the core domain-domain interaction. Since 1988 several groups of other investigators have identified folding intermediates of different proteins which appear to be consistent with our concept of the core domains. Thus, the core domain-domain interaction may be general in driving the folding of globular proteins. To know whether or not the core domain-domain interaction differs from the known types of non-covalent interactions we have determined the differences in their thermodynamic and kinetic effects of substitution with norvaline (Nva) between Leu32 and Leu35 of the horse cytochrome c three-fragment complex in the previous years. The complex consists of a heme fragment of residues 1 to 5, (1-25)H, and two apofragments (28-38) and (39-104). It resembles native cytochrome c except for residues 39 to 55 which are flexible. We have now analyzed and interpreted the data using complex models. The results suggest for the first time that the difference in the standard Gibbs energy change, ddGs, associated with the equilibrium constant of fragment (28-38) with the ferric or ferrous complex qualitatively correlates with the difference in the activation Gibbs energy, dGa, associated with the direct dissociation of fragment (39-104). These ddGs and dGa also qualitatively correlate with the difference in the heat stability of the 695 nm band of the ferric complex, a band known to be indicative of the Fe-S bond. These correlations have been interpreted as suggesting that some propagative extra non-covalent interactions may be generated by the packed atomic groups in the hydrophobic core to stabilize the ground state. These interactions may have characteristics such that the stabilizing energy generated by them would be perturbed much more by the Leu32 Nva substitution than the Leu35 Nva. The perturbation of these interactions imposed by the removal of the gamma-methyl group of Leu32 would propagate itself through the core and affect the stability of the Fe- S bond and the binding strength of (39-104). These properties are consistent with the core domain-domain interaction. Furthermore, the analysis, combined with other evidence, appears to suggest that these propagative non-covalent interactions in the core are likely to differ from van der Waals interactions, hydrophobic energy, hydrogen bonds and charge-charge interactions. To map the core side chains involved in these propagative non-covalent interactions studies of yeast iso-2-cytochrome c using site directed mutagenesis is in progress. To date eight mutants have been or are being generated. To precisely identify the core domains of cytochrome c NMR studies are also in progress using two-dimensional NOESY, COSY, and TOCSY spectra in collaboration with the structural Biology Section, LBC, NHLBI.