Fluorescence techniques applied to root-state detection in protein folding

蛋白质的一维氨基酸序列决定了其三维天然结构、折叠机制和生物活性。关于多肽链如何折叠的一种假设是，它开始于由环节点闭合的长环的形成，由沿1D序列间隔20到100个残基的短残基片段对形成。这一假设已经在大肠杆菌腺苷酸激酶上得到了广泛的验证，通过稳态和时间分辨Förster-resonance能量转移光谱实验结合停流双混合。虽然这些实验揭示了哪些回路形成得早，哪些形成得晚，但它们无法回答以下问题：什么是环的独立稳定性，只涉及环内残基而不涉及非环残基的辅助接触的稳定性？2. 对于早期环路形成至关重要的环路节点的独立稳定性是什么？3. 如果环路节点在早期重折叠过程中只被少量填充，我们如何测量它的亚稳态？4. 环节点形成的分子驱动力和机制是什么？我们认为这些问题对于序列到结构预测的最终目标至关重要。我们的目标是通过应用一种基于独特光谱探针的方法来解决这四个问题，该方法将二氮杂环-[2.2.2]-辛-2-烯或DBO结合到多肽中，其中多肽模型要么是单个环节点，要么是整个环腺苷酸激酶。当DBO与色氨酸、酪氨酸或其他合适的伙伴探针联合使用时，目前提供了最高距离分辨率的FRET方法，称为短距离FRET或sdFRET，并且需要这种分辨率来检测环路节点的原生相似性。DBO的使用同时提供了一种接触猝灭方法，称为碰撞诱导荧光猝灭或CIFQ。这些方法及其组合使我们能够确定亚稳态淋巴结的边缘种群。CIFQ和压力跳变测量使我们能够确定在水中充分填充或在添加稳定助剂后变得充分填充的环的折叠速率：这构成了探索环节点形成的分子机制所需的基础。

The one-dimensional amino acid sequence of a protein determines its three-dimensional native structure, its folding mechanism, and biological activity. One hypothesis on how folding of the polypeptide chain proceeds is that it starts with the formation of long loops closed by loop nodes, by pairs of short residue segments separated by twenty to hundred residues along the 1D sequence. This hypothesis has been extensively tested on E.coli adenylate kinase by steady-state and time-resolved Förster-resonance energy transfer spectroscopic experiments coupled to stopped-flow double mixing. While these experiments revealed which loops are formed early and which only later, they could not answer the following questions: 1. What is the independent stability of a loop, the stability that only involves intra-loop residues and no assisting contacts with non-loop residues? 2. What is the independent stability of the loop node, crucial for early loop formation? 3. How can we measure the metastability of a loop node if it becomes only marginally populated during early refolding? 4. What is the molecular driving force and mechanism of loop-node formation? We consider these questions critical for the ultimate goal of sequence-to-structure prediction.We aim to address the four questions by applying methods based on a unique spectroscopic probe, on diazabicyclo-[2.2.2]-oct-2-ene or DBO, incorporated into polypeptides, where the polypeptides model either a single loop node or a whole loop of adenylate kinase. DBO, when used in combination with tryptophan, tyrosine or other suitable partner probes, currently provides the FRET method of highest distance resolution, named short-distance FRET or sdFRET, and this resolution is required to detect the native-likeness of a loop node. The use of DBO simultaneously affords a contact-quenching method, named collision-induced fluorescence quenching or CIFQ. These methods and their combination allow us to determine even marginal populations of metastable nodes. CIFQ and pressure jump measurements allow us to determine the folding rates of loops that are sufficiently populated in water or become sufficiently populated upon addition of stabilizing co-agents: This constitutes the required basis to probe the molecular mechanism of loop-node formation.