Identification of a mechanical rheostat in the hydrophobic core of protein L.
Identification of a mechanical rheostat in the hydrophobic core of protein L.
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DOI：
10.1016/j.jmb.2009.08.015
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发表时间：
2009-10-16
影响因子：
5.6
通讯作者：
Brockwell, David J.
中科院分区：
文献类型：
--
作者：
Sadler, David P.;Petrik, Eva;Taniguchi, Yukinori;Pullen, James R.;Kawakami, Masaru;Radford, Sheena E.;Brockwell, David J.
蛋白质及其复合物承受或响应机械刺激的能力对于细胞维持其结构组织、传递外部信号以及促进展开和重塑至关重要。使用原子力显微镜的力谱允许单个蛋白质分子在所施加的延伸下的行为被研究，并且它们的机械强度被量化。蛋白L是一种简单的模型蛋白，显示出中等的机械强度，并被认为通过两个机械亚结构域的剪切而展开。在这里，我们调查的重要性，侧链包装的蛋白L的机械强度，通过测量一系列的蛋白L变体含有单一的保守的疏水体积缺失突变体的机械强度。在所表征的五种生物学不稳定的变体中，仅发现靠近两个机械亚结构域之间的界面的一个残基（I60 V）在机械性质上与野生型不同（Δ FI 60 V-WT =-36 pN at 447 nm s-1，Δ xuI 60 V-WT = 0.2 nm）。解折叠数据的Φ值分析揭示了高度天然的过渡态。为了测试机械界面上疏水接触的数量是否会影响蛋白L的机械强度，我们测量了另外两种变体的机械性能。蛋白质L L10 F增加了核心堆积，但不增强界面接触，在447 nm s− 1下增加了13 ± 11 pN的机械强度。相比之下，增加核心和交叉界面接触的蛋白质L I60 F在447 nm s− 1下的机械强度增加了72 ± 13 pN。这些数据表明了一种方法，通过这种方法，自然界可以从有限数量的拓扑结构中进化出不同的机械响应，并展示了一种通用但简单的方法，通过这种方法可以合理地改变蛋白质的机械强度。
The ability of proteins and their complexes to withstand or respond to mechanical stimuli is vital for cells to maintain their structural organisation, to relay external signals and to facilitate unfolding and remodelling. Force spectroscopy using the atomic force microscope allows the behaviour of single protein molecules under an applied extension to be investigated and their mechanical strength to be quantified. protein L, a simple model protein, displays moderate mechanical strength and is thought to unfold by the shearing of two mechanical sub-domains. Here, we investigate the importance of side-chain packing for the mechanical strength of protein L by measuring the mechanical strength of a series of protein L variants containing single conservative hydrophobic volume deletion mutants. Of the five thermodynamically destabilised variants characterised, only one residue (I60V) close to the interface between two mechanical sub-domains was found to differ in mechanical properties to wild type (ΔFI60V–WT = − 36 pN at 447 nm s− 1, ΔxuI60V–WT = 0.2 nm). Φ-value analysis of the unfolding data revealed a highly native transition state. To test whether the number of hydrophobic contacts across the mechanical interface does affect the mechanical strength of protein L, we measured the mechanical properties of two further variants. protein L L10F, which increases core packing but does not enhance interfacial contacts, increased mechanical strength by 13 ± 11 pN at 447 nm s− 1. By contrast, protein L I60F, which increases both core and cross-interface contacts, increased mechanical strength by 72 ± 13 pN at 447 nm s− 1. These data suggest a method by which nature can evolve a varied mechanical response from a limited number of topologies and demonstrate a generic but facile method by which the mechanical strength of proteins can be rationally modified.
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影响因子：
5.6
作者：
Ferguson, N;Capaldi, AP;Radford, SE
通讯作者：
Radford, SE
影响因子：
4.8
作者：
Graille, M;Harrison, S;Stura, EA
通讯作者：
Stura, EA
DOI：
10.1073/pnas.192351899
发表时间：
2002-09-17
影响因子：
11.1
作者：
Best, RB;Fowler, SB;Clarke, J
通讯作者：
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影响因子：
3.4
作者：
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通讯作者：
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影响因子：
64.8
作者：
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通讯作者：
Evans, E