Exploring local and non-local interactions for protein stability by structural motif engineering
Exploring local and non-local interactions for protein stability by structural motif engineering
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DOI：
10.1006/jmbi.1999.3385
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发表时间：
2000-02-11
影响因子：
5.6
通讯作者：
Steipe, B
中科院分区：
文献类型：
--
作者：
Niggemann, M;Steipe, B
为了探索局部和非局部相互作用对蛋白质热力学稳定性的相对贡献，我们设计了一种基于基序工程和序列改组相结合的实验方法。免疫球蛋白V-L结构域中的候选链段被鉴定，其构象被提出为由非局部相互作用主导。然后通过引入基序共有序列，构建不同构象的局部相互作用结构基序作为替换。我们发现，我们构建的所有九个替代系统地减少折叠协同。通过比较这种不稳定的效果与这些图案的三个改组序列的折叠过渡，我们估计本地，本地相互作用的折叠自由能的贡献。我们的结果表明，局部和非局部相互作用对稳定性的贡献大致相等，但局部相互作用通过增加对变性的抵抗力来稳定，而非局部相互作用则增加折叠协同性。在这些主-客体实验中通过序列改组的稳定性的系统性损失表明所设计的相互作用确实存在于天然状态中，因此共有序列工程可能是结构设计中的有用工具，但非局部相互作用必须考虑全局稳定性工程。统计方法是工程蛋白质结构和稳定性的有力工具，但仅基于局部序列倾向的分析并不能充分代表蛋白质结构中序列和背景的平衡。(C)北京大学出版社.
In order to probe the relative contribution of local and non-local interactions to the thermodynamic stability of proteins, we have devised an experimental approach based on a combination of motif engineering and sequence shuffling. Candidate chain segments in an immunoglobulin V-L domain were identified whose conformation is proposed to be dominated by non-local interactions. Locally interacting structural motifs of a different conformation were then constructed as replacements, by introducing motif consensus sequences. We find that all nine replacements we constructed systematically reduce the folding cooperativity. By comparing this destabilising effect with the folding transitions of shuffled sequences for three of these motifs, we estimate the contribution of local, native interactions to the free energy of folding. Our results suggest that local and non-local interactions contribute to stability by an approximately equal amount, but that local interactions stabilise by increasing the resistance to denaturation while non-local interactions increase folding cooperativity. The systematic loss of stability by sequence shuffling in these host-guest experiments suggests that the designed interactions indeed are present in the native state, thus consensus sequence engineering may be a useful tool in structure design, but non-local interactions must be taken into account for global stability engineering. Statistical approaches are powerful tools for engineering protein structure and stability, but an analysis based on local sequence propensities alone does not adequately represent the balance of sequence and context in protein structures. (C) 2000 Academic Press.