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首页> 外文期刊>Biochemistry >SIDE CHAIN PACKING OF THE N- AND C-TERMINAL HELICES PLAYS A CRITICAL ROLE IN THE KINETICS OF CYTOCHROME C FOLDING
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SIDE CHAIN PACKING OF THE N- AND C-TERMINAL HELICES PLAYS A CRITICAL ROLE IN THE KINETICS OF CYTOCHROME C FOLDING

机译：N末端和C末端的侧面链包装在细胞色素C折叠动力学中起着至关重要的作用

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The pairing of two alpha-helices at opposite ends of the chain is a highly conserved structural motif found throughout the cytochrome c family of proteins. It has previously been shown that association of the N- and C-terminal helices is a critical early event in the folding process of horse cytochrome c and is responsible for the formation of a partially folded intermediate (I-NC). In order to gain further insight into the structural and energetic basis of helix packing interactions and their role in folding, we prepared a series of horse cytochrome c variants in which Leu94, a critical residue at the helix contact site, was replaced by Ile, Val, or Ala. The Ile and Val substitutions resulted in minor changes in the stability of the native state, indicating that conservative mutations can be accommodated at the helix interface with only minor structural perturbations. In contrast, the L94A mutation resulted in a 3.5 kcal/mol decrease in unfolding free energy, suggesting that the smaller Ala side chain causes severe packing defects at the helix interface. The effect of these mutations on the kinetics of folding and unfolding as a function of denaturant concentration was studied by a systematic series of stopped-flow fluorescence measurements. The proteins with Leu, Ile, or Val at position 94 exhibit a major unresolved fluorescence change during the 1-ms dead time of the stopped-flow refolding measurements, while this effect is less pronounced in L94A, indicating that the rapid formation of a compact state (I-C) with largely quenched Trp59 fluorescence is favored by a large hydrophobic side chain at the helix-helix interface. Despite their small effects on overall stability, the L94I and L94V mutations result in a substantial reduction in the relative amplitude of the fastest observable folding phase (formation of I-NC) consistent with a strong decrease in the population of I-NC compared to the wild-type protein. This effect is amplified in the case of the destabilizing L94A variant, which exhibits slower folding kinetics and negligible accumulation of I-NC. Whereas the presence of a large hydrophobic side chain at position 94 is sufficient for the stabilization of I-C, the subsequent partially folded intermediate, I-NC, is stabilized by specific interactions that are responsible for the proper packing of the two alpha-helices.
机译：在链相反两端的两个α螺旋配对是在整个细胞色素c家族蛋白中发现的高度保守的结构基序。先前已经证明，N末端和C末端螺旋的缔合是马细胞色素c折叠过程中的关键早期事件，并负责部分折叠的中间体（I-NC）的形成。为了进一步了解螺旋堆积相互作用的结构和能量基础及其在折叠中的作用，我们制备了一系列马细胞色素c变体，其中在螺旋接触位点的关键残基Leu94被Ile，Val取代Ile和Val取代导致天然状态稳定性的微小变化，这表明保守的突变可以在螺旋界面处被容纳，而只有很小的结构扰动。相反，L94A突变导致展开自由能下降3.5 kcal / mol，这表明较小的Ala侧链会在螺旋界面处引起严重的堆积缺陷。通过一系列系统的停止流荧光测量，研究了这些突变对折叠和展开动力学的影响，该动力学是变性剂浓度的函数。在停止流重折叠测量的1毫秒死时间期间，在94位具有Leu，Ile或Val的蛋白质表现出主要的未分辨荧光变化，而在L94A中这种作用不太明显，表明紧密体的快速形成Trp59荧光在很大程度上被淬灭的I（IC）状态被螺旋-螺旋界面处的大疏水侧链所偏爱。尽管L94I和L94V突变对整体稳定性的影响很小，但导致可观察到的最快折叠相（I-NC形成）的相对幅度大大降低，与I-NC群体相比，其显着降低野生型蛋白。在不稳定的L94A变体的情况下放大了这种效果，该变体表现出较慢的折叠动力学和可忽略的I-NC积累。尽管在位置94处存在大的疏水侧链足以稳定I-C，但随后的部分折叠的中间体I-NC通过负责两个α-螺旋正确包装的特定相互作用而得以稳定。

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