Φ-Value analysis of a three-state protein folding pathway by NMR relaxation dispersion spectroscopy
Φ-Value analysis of a three-state protein folding pathway by NMR relaxation dispersion spectroscopy
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DOI：
10.1073/pnas.0705097104
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发表时间：
2007-10-02
影响因子：
11.1
通讯作者：
Kay, Lewis E.
中科院分区：
文献类型：
--
作者：
Neudecker, Philipp;Zarrine-Afsar, Arash;Kay, Lewis E.
蛋白质折叠的实验研究通常与双态折叠动力学相一致。然而，最近的NMR弛豫色散的几个快速折叠突变体的Fyn Src同源3（SH 3）域的研究已经确定，折叠通过低人口的途径中间，这不能检测到与停流实验进行。分散实验提供了精确的动力学和热力学参数，描述了折叠途径，沿着与详细的特定位点的结构表征的中间和未折叠状态从NMR化学位移提取。在这里，我们描述了NMR弛豫色散Phi值分析的A39 V/N53 P/V55 L Fyn SH 3域，其中合适的点突变的能量景观的影响进行了量化，提供额外的洞察折叠中间体的结构沿着与每残基的结构信息的两个限速过渡态，这是不可用的从以前的研究。除了描绘完整的三态折叠途径的优点之外，使用NMR弛豫色散而不是停流动力学来量化Phi值有助于其解释，因为所获得的化学位移监测可能由突变引入的折叠途径沿着的任何潜在结构变化，这是其分析中的一个重要问题。A39 V/N53 P/V55 L Fyn SH 3的几个点突变的Phi值分析确定β（3）-β（4）-发夹已经在第一过渡态形成，而在中间体中形成非天然相互作用的链β（1）直到最终过渡态之后才完全采用其天然构象。结果进一步支持的概念，途径上的中间体可以通过非天然接触稳定。
Experimental studies of protein folding frequently are consistent with two-state folding kinetics. However, recent NMR relaxation dispersion studies of several fast-folding mutants of the Fyn Src homology 3 (SH3) domain have established that folding proceeds through a low-populated on-pathway intermediate, which could not be detected with stopped-flow experiments. The dispersion experiments provide precise kinetic and thermodynamic parameters that describe the folding pathway, along with a detailed site-specific structural characterization of both the intermediate and unfolded states from the NMR chemical shifts that are extracted. Here we describe NMR relaxation dispersion Phi-value analysis of the A39V/N53P/V55L Fyn SH3 domain, where the effects of suitable point mutations on the energy landscape are quantified, providing additional insight into the structure of the folding intermediate along with per-residue structural information of both rate-limiting transition states that was not available from previous studies. In addition to the advantage of delineating the full three-state folding pathway, the use of NMR relaxation dispersion as opposed to stopped-flow kinetics to quantify Phi values facilitates their interpretation because the obtained chemical shifts monitor any potential structural changes along the folding pathway that might be introduced by mutation, a significant concern in their analysis. Phi-Value analysis of several point mutations of A39V/N53P/V55L Fyn SH3 establishes that the beta(3)-beta(4)-hairpin already is formed in the first transition state, whereas strand beta(1), which forms nonnative interactions in the intermediate, does not fully adopt its native conformation until after the final transition state. The results further support the notion that on-pathway intermediates can be stabilized by nonnative contacts.