Fluorescence techniques applied to root-state detection in protein folding

蛋白质的一维氨基酸序列决定了它的三维天然结构、折叠机制和生物活性。关于多肽链如何折叠的一个假设是，它始于由环节点闭合的长环的形成，由沿着1D序列被20到100个残基隔开的成对的短残基片段组成。这一假说已经通过稳态和时间分辨的Förster共振能量转移光谱实验与停流双混合相结合，在E.Coli腺苷酸激酶上得到了广泛的验证。虽然这些实验揭示了哪些环形成得早，哪些形成得晚，但它们不能回答以下问题：1.环的独立稳定性是什么，只涉及环内残基而不与非环残基辅助接触的稳定性是什么？2.环节点的独立稳定性是什么，对早期环形成至关重要？3.如果环节点在早期折叠过程中只有少量填充，我们如何衡量它的亚稳性？4.环节点形成的分子推动力和机制是什么？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基于独特的光谱探针的方法来解决这四个问题，这些方法基于引入多肽的重氮二环-[2.2.2]-OCT-2-烯或DBO，其中多肽模拟腺苷酸激酶的单环节点或整个环。当与色氨酸、酪氨酸或其他合适的伙伴探针结合使用时，dbo目前提供了距离分辨率最高的FRET方法，称为短距离FRET或sdFRET，这种分辨率是检测环节点的原生相似性所必需的。同时使用DBO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甚至可以确定亚稳态结节的边缘种群。CIFQ和压力跳跃测量使我们能够确定在水中充分填充或在添加稳定剂后变得足够填充的环路的折叠率：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.