THE PRINCIPLES THAT GOVERN PROTEIN FOLDING--INTERACTION BETWEEN CLOSED LOOPS

这一部分的研究导致了这样的假设：在 蛋白质存在一些新的离域相互作用，这种相互作用是由 通过折叠后形成的接触基团的闭合环 产生额外的能量来稳定结构。如上所述 在另一份报告中，A.Fisher和H.Taniuchi找到了四个 假设细胞色素中的闭合环c.在这四个中，闭合 环路1位于亚铁血红素上方，在碎片中排在第一位 络合，闭合环2位于血红素的左侧 假设对残基28到38进行排序，并且闭合环3位于 右侧稳定蛋氨酸80-S-铁键。上一次 研究表明，闭合环2与闭合环2相互作用 回路3.分析这个闭合回路的热力学2-闭合 环路3相互作用我们使用三段络合物铁氧体(1- 25)H.(28-38).(39-104)和片段交换技术。这个 先前的研究表明，在过量的情况下 片段(28-38)可以直接测量 片段(39-104)的解离，即不经过两个 片段复合体(125)H.(39-104)。在这个原则的基础上 我们计划测量取代亮氨酸32和 亮氨酸35与降冰片碱(一次一个)对结合力的影响 片段(39-104)。因此，我们准备了亚铁血红素--和 阿夫林和放射性标记(39-104)测定解离 复铁率(1-25)H.(39-104)作为温度的函数， 导致活化吉布斯能，在25度时为18.25千卡/摩尔 C，活化热为52.8，在5.6千卡/摩尔范围内； 熵，在20EU内为116。这与之前的数据相结合 提示血红素的减少加强了 闭合环路1或闭合环路3或两者。使用此信息并 之前解离常数的数据，测量的 碎片(39104)与络合物铁(1-)的解离速率 25)H.(28-38)(39-104)作为游离态浓度的函数 片段(28-38)应该允许我们确定 直接解离。相同的过程将用于 在32位或35位含有取代的络合物。

The studies in this Section have led to the hypothesis that in proteins exists some new delocalized interaction that is mediated by a closed loop of contacting groups formed after folding and generates extra energy to stabilize the structure. As described in another report A. Fisher and H. Taniuchi have located four hypothetical closed loops in cytochrome c. Of these four, closed loop 1 is located above the heme and forms first in fragment complexation, closed loop 2 located at the left side of the heme is assumed to order residues 28 to 38 and closed loop 3 located at the right side to stabilize the Met 80-S-Fe bond. The previous studies have indicated that closed loop 2 interacts with closed loop 3. To analyze thermodynamics of this closed loop 2-closed loop 3 interaction we use the three fragment complex ferro(1- 25)H.(28-38).(39-104) and a fragment exchange technique. The previous studies have shown that in the presence of excess of fragment (28-38) it is possible to measure the rate of direct dissociation of fragment (39-104) i.e. without going through two fragment complex (125)H.(39-104). On the basis of this principle we plan to measure the effect of substitution of leucine 32 and leucine 35 with norvaline (one at a time) on the binding force of fragment (39-104). Thus, we have prepared the heme- and apofragments and radiolabelled (39-104) determined the dissociation rate of complex ferro(1-25)H.(39-104) as a function of temperature, resulting in activation Gibbs energy, 18.25 kcal/mol at 25 degrees C; activation enthalpy, 52.8 within 5.6 kcal/mol; and activation entropy, 116 within 20eu. This combined with the previous data suggest that reduction of heme strengthens the interaction of closed loop 1 or closed loop 3 or both. Using this information and the previous data of dissociation constants, measurements of the dissociation rate of fragment (39104) from complex ferro-(1- 25)H.(28-38).(39-104) as a function of concentration of free fragment (28-38) should allow us to determine the rate constant of the direct dissociation. The same procedure will be used for the complex containing substitution at position 32 or 35.