Fluorescence techniques applied to root-state detection in protein folding

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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.