A Targetable Self-association Surface of the Huntingtin exon1 Helical Tetramer Required for Assembly of Amyloid Pre-nucleation Oligomers.

Polyglutamine (polyQ) sequences undergo repeat-length dependent formation of disease-associated, amyloid-like cross-ß core structures with kinetics and aggregate morphologies often influenced by the flanking sequences. In Huntington's disease (HD), the httNT segment on the polyQ's N-terminal flank enhances aggregation rates by changing amyloid nucleation from a classical homogeneous mechanism to a two-step process requiring an ɑ-helix-rich oligomeric intermediate. A folded, helix-rich httNT tetrameric structure suggested to be this critical intermediate was recently reported. Here we employ single alanine replacements along the httNT sequence to assess this proposed structure and refine the mechanistic model. We find that Ala replacement of hydrophobic residues within simple httNT peptides greatly suppresses helicity, supporting the tetramer model. These same helix-disruptive replacements in the httNT segment of an exon-1 analog greatly reduce aggregation kinetics, suggesting that an ɑ-helix rich multimer - either the tetramer or a larger multimer - plays an on-pathway role in nucleation. Surprisingly, several other Ala replacements actually enhance helicity and/or amyloid aggregation. The spatial localization of these residues on the tetramer surface suggests a self-association interface responsible for formation of the octomers and higher-order multimers most likely required for polyQ amyloid nucleation. Multimer docking of the tetramer, using the protein-protein docking algorithm ClusPro, predicts this symmetric surface to be a viable tetramer dimerization interface. Intriguingly, octomer formation brings the emerging polyQ chains into closer proximity at this tetramer-tetramer interface. Further supporting the potential importance of tetramer super-assembly, computational docking with a known exon-1 aggregation inhibitor predicts ligand contacts with residues at this interface.

Hsp70 Inhibits Aggregation of IAPP by Binding to the Heterogeneous Prenucleation Oligomers.

Molecular chaperone Hsp70 plays important roles in the pathology of amyloid diseases by inhibiting aberrant aggregation of proteins. However, the biophysical mechanism of the interaction of Hsp70 with the intrinsically disordered proteins (IDPs) is unclear. Here, we report that Hsp70 inhibits aggregation of islet amyloid polypeptide (IAPP) at substoichiometric concentrations under diverse solution conditions, including in the absence of ATP. The inhibitory effect is strongest if Hsp70 is added in the beginning of aggregation but progressively less if added later, indicating a role for Hsp70 in preventing nucleation of IAPP. However, ensemble measurement of the binding affinity suggests poor interactions between Hsp70 and IAPP. Therefore, we hypothesize that the interaction must involve a rare species (e.g., the oligomeric intermediates of IAPP). Size exclusion chromatography and field flow fractionation are then used to fractionate the constituent species. Multiangle light scattering and fluorescence correlation spectroscopy measurements indicate that the dominant fraction in size exclusion chromatography contains a few nanomolar Hsp70-IAPP complexes amid several µmoles of free Hsp70. Using single-particle two-color coincidence detection measurements, we detected a minor fraction that exhibits fluorescence bursts arising from heterogeneous oligomeric complexes of IAPP and Hsp70. Taken together, our results indicate that Hsp70 interacts poorly with the monomers but strongly with oligomers of IAPP. This is likely a generic feature of the interactions of Hsp70 chaperones with the amyloidogenic IDPs. Whereas high-affinity interactions with the oligomers prevent aberrant aggregation, poor interaction with the monomers averts interference with the physiological functions of the IDPs.

High-resolution structure of a partially folded insulin aggregation intermediate.

Insulin has long been served as a model for protein aggregation, both due to the importance of aggregation in the manufacture of insulin and because the structural biology of insulin has been extensively characterized. Despite intensive study, details about the initial triggers for aggregation have remained elusive at the molecular level. We show here that at acidic pH, the aggregation of insulin is likely initiated by a partially folded monomeric intermediate. High-resolution structures of the partially folded intermediate show that it is coarsely similar to the initial monomeric structure but differs in subtle details-the A chain helices on the receptor interface are more disordered and the B chain helix is displaced from the C-terminal A chain helix when compared to the stable monomer. The result of these movements is the creation of a hydrophobic cavity in the center of the protein that may serve as nucleation site for oligomer formation. Knowledge of this transition may aid in the engineering of insulin variants that retain the favorable pharamacokinetic properties of monomeric insulin but are more resistant to aggregation.

Design, Synthesis, and Self-Assembly Studies of a Suite of Monodisperse, Facially Amphiphilic, Protein-Dendron Conjugates.

The custom design of protein-dendron amphiphilic macromolecules is at the forefront of macromolecular engineering. Macromolecules with this architecture are very interesting because of their ability to self-assemble into various biomimetic nanoscopic structures. However, to date, there are no reports on this concept due to technical challenges associated with the chemical synthesis. Towards that end, herein, a new chemical methodology for the modular synthesis of a suite of monodisperse, facially amphiphilic, protein-dendron bioconjugates is reported. Benzyl ether dendrons of different generations (G1-G4) are coupled to monodisperse cetyl ethylene glycol to form macromolecular amphiphilic activity-based probes (AABPs) with a single protein reactive functionality. Micelle-assisted protein labeling technology is utilized for site-specific conjugation of macromolecular AABPs to globular proteins to make monodisperse, facially amphiphilic, protein-dendron bioconjugates. These biohybrid conjugates have the ability to self-assemble into supramolecular protein nanoassemblies. Self-assembly is primarily mediated by strong hydrophobic interactions of the benzyl ether dendron domain. The size, surface charge, and oligomeric state of protein nanoassemblies could be systematically tuned by choosing an appropriate dendron or protein of interest. This chemical method discloses a new way to custom-make monodisperse, facially amphiphilic, protein-dendron bioconjugates.

Building, Characterization, and Applications of Cuvette-FCS in Denaturant-Induced Expansion of Globular and Disordered Proteins.

Fluorescence correlation spectroscopy (FCS) is a single-molecule sensitive technique with widespread applications in biophysics. However, conventional microscope-based FCS setups have limitations in performing certain experiments such as those requiring agitations such as stirring or heating, and those involving measurements in solvents with the mismatch of refractive indices. We have recently developed an FCS setup that is suitable for performing measurements inside regular cuvettes. The cuvette-FCS is suitable for performing single-molecule measurements in experiments that are regularly performed in spectrofluorometers but are generally avoided in microscope-based FCS. Here we describe building and characterization of the performance of the cuvette-FCS setup in detail. Finally, we have used a natively folded protein and an intrinsically disordered protein to demonstrate and describe how cuvette-FCS can be applied conveniently to measure urea-dependent expansion of hydrodynamic size of proteins.

A Fluorescence Correlation Spectrometer for Measurements in Cuvettes.

We have developed a fluorescence correlation spectroscopy (FCS) setup for performing single-molecule measurements on samples inside regular cuvettes. The cuvette FCS uses a horizontally mounted extra-long working distance, 0.7 NA, air objective with a working distance of >1.8 mm instead of a high NA water or oil immersion objective. The performance of the cuvette FCS is found to be highly sensitive to the quality and alignment of the cuvette. The radial resolution and effective observation volume obtained using the optimized setup are ∼340 nm and 1.8 fL, respectively. The highest molecular brightness and the signal/noise ratio in the autocorrelation data achieved using an aqueous solution of rhodamine B are greater than 44 kHz and 110, respectively. Here, we demonstrate two major advantages of cuvette FCS. For example, the cuvette FCS can be used for measurements over a wide range of temperatures that is beyond the range permitted in the microscope-based FCS. Furthermore, cuvette FCS can be coupled to automatic titrators to study urea-dependent unfolding of proteins with unprecedented accuracy. The ease of use and compatibility with various accessories will enable applications of cuvette FCS in the experiments that are regularly performed in spectrofluorometers but are generally avoided in microscope-based FCS.

Quantitative Characterization of Metastability and Heterogeneity of Amyloid Aggregates.

Amyloids are heterogeneous assemblies of extremely stable fibrillar aggregates of proteins. Although biological activities of the amyloids are dependent on its conformation, quantitative evaluation of heterogeneity of amyloids has been difficult. Here we use disaggregation of the amyloids of tetramethylrhodamine-labeled Aß (TMR-Aß) to characterize its stability and heterogeneity. Disaggregation of TMR-Aß amyloids, monitored by fluorescence recovery of TMR, was negligible in native buffer even at low nanomolar concentrations but the kinetics increased exponentially with addition of denaturants such as urea or GdnCl. However, dissolution of TMR-Aß amyloids is different from what is expected in the case of thermodynamic solubility. For example, the fraction of soluble amyloids is found to be independent of total concentration of the peptide at all concentrations of the denaturants. Additionally, soluble fraction is dependent on growth conditions such as temperature, pH, and aging of the amyloids. Furthermore, amyloids undissolved in a certain concentration of the denaturant do not show any further dissolution after dilution in the same solvent; instead, these require higher concentrations of the denaturant. Taken together, our results indicate that amyloids are a heterogeneous ensemble of metastable states. Furthermore, dissolution of each structurally homogeneous member requires a unique threshold concentration of denaturant. Fraction of soluble amyloids as a function of concentration of denaturants is found to be sigmoidal. The sigmoidal curve becomes progressively steeper with progressive seeding of the amyloids, although the midpoint remains unchanged. Therefore, heterogeneity of the amyloids is a major determinant of the steepness of the sigmoidal curve. The sigmoidal curve can be fit assuming a normal distribution for the population of the amyloids of various kinetic stabilities. We propose that the mean and the standard deviation of the normal distribution provide quantitative estimates of mean kinetic stability and heterogeneity, respectively, of the amyloids in a certain preparation.

Insulin-eukaryotic model membrane interaction: Mechanistic insight of insulin fibrillation and membrane disruption.

Injection of exogenous insulin in the subcutaneous mass has been a proven therapy for type II diabetes. However, chronic administration of insulin often develops local amyloidosis at the injection site, pathologically known as "Insulin Ball". This reduces the insulin bioavailability and exacerbates the disease pathology. Thus, the molecular interaction between insulin and the recipient's membrane surface plays a co-operative role in accelerating the amyloidosis. This interaction, however, is different from the molecular interaction of insulin with the native membranous environment of the pancreatic ß-cells. The differential membrane mediated interaction that directly affects the aggregation kinetics of insulin remains elusive yet intriguing to understand the mechanism of pathological development. In this study we have characterized the interactions of insulin at different states with model eukaryotic membranes using high and low-resolution spectroscopic techniques in combination with microscopic investigation. Our results show that insulin amyloid intermediates are capable of interacting with model membranes with variable functional affinity towards the different compositions. Fluorescence correlation spectroscopy confirms the aggregation states of insulin in presence of the eukaryotic model membranes while solid-state NMR spectroscopy in conjugation with differential scanning calorimetry elucidates the molecular interaction of insulin intermediates with the lipid head groups along with the acyl chains. Additionally, dye leakage assays support the eukaryotic model membrane disruption by insulin intermediates, similar to hIAPP and Aß40, as previously reported. Thus, the present study establishes the distinct mode of interactions of insulin amyloid with pancreatic ß-cell and general mammalian cell mimicking membranes.

Curcumin Dictates Divergent Fates for the Central Salt Bridges in Amyloid-ß40 and Amyloid-ß42.

There are three specific regions in the Amyloid beta (Aß) peptide sequence where variations cause enhanced toxicity in Alzheimer's disease: the N-terminus, the central salt bridge, and the C-terminus. Here, we investigate if there is a close conformational connection between these three regions, which may suggest a concerted mechanism of toxicity. We measure the effects of Zn2+ and curcumin on Aß40, and compare these with their previously reported effects on Aß42. Aß42 and Aß40 differ only near the C-terminus, where curcumin interacts, while Zn2+ interacts near the N-terminus. Therefore, this comparison should help us differentiate the effect of modulating the C- and the N-termini. We find that curcumin allows fibril-like structures containing the salt bridge to emerge in the mature Aß40 aggregates, but not in Aß42. In contrast, we find no difference in the effects of Zn+2 on Aß40 and Aß42. In the presence of Zn+2, both of these fail to form proper fibrils, and the salt bridge remains disrupted. These results indicate that modulations of the Aß termini can determine the fate of a salt bridge far away in the sequence, and this has significant consequences for Aß toxicity. We also infer that small molecules can alter oligomer-induced toxicity by modulating the aggregation pathway, without substantially changing the final product of aggregation.

Folding Landscape of Mutant Huntingtin Exon1: Diffusible Multimers, Oligomers and Fibrils, and No Detectable Monomer.

Expansion of the polyglutamine (polyQ) track of the Huntingtin (HTT) protein above 36 is associated with a sharply enhanced risk of Huntington's disease (HD). Although there is general agreement that HTT toxicity resides primarily in N-terminal fragments such as the HTT exon1 protein, there is no consensus on the nature of the physical states of HTT exon1 that are induced by polyQ expansion, nor on which of these states might be responsible for toxicity. One hypothesis is that polyQ expansion induces an alternative, toxic conformation in the HTT exon1 monomer. Alternative hypotheses posit that the toxic species is one of several possible aggregated states. Defining the nature of the toxic species is particularly challenging because of facile interconversion between physical states as well as challenges to identifying these states, especially in vivo. Here we describe the use of fluorescence correlation spectroscopy (FCS) to characterize the detailed time and repeat length dependent self-association of HTT exon1-like fragments both with chemically synthesized peptides in vitro and with cell-produced proteins in extracts and in living cells. We find that, in vitro, mutant HTT exon1 peptides engage in polyQ repeat length dependent dimer and tetramer formation, followed by time dependent formation of diffusible spherical and fibrillar oligomers and finally by larger, sedimentable amyloid fibrils. For expanded polyQ HTT exon1 expressed in PC12 cells, monomers are absent, with tetramers being the smallest molecular form detected, followed in the incubation time course by small, diffusible aggregates at 6-9 hours and larger, sedimentable aggregates that begin to build up at 12 hrs. In these cell cultures, significant nuclear DNA damage appears by 6 hours, followed at later times by caspase 3 induction, mitochondrial dysfunction, and cell death. Our data thus defines limits on the sizes and concentrations of different physical states of HTT exon1 along the reaction profile in the context of emerging cellular distress. The data provide some new candidates for the toxic species and some new reservations about more well-established candidates. Compared to other known markers of HTT toxicity, nuclear DNA damage appears to be a relatively early pathological event.

Fluorescence Correlation Spectroscopy: A Tool to Study Protein Oligomerization and Aggregation In Vitro and In Vivo.

Fluorescence correlation spectroscopy (FCS) is a highly sensitive analytical technique used to measure dynamic molecular parameters, such as diffusion time (from which particle size can be calculated), conformation, and concentration of fluorescent molecules. It has been particularly powerful in characterizing size distributions in molecular associations (e.g., dimer/multimer formation) both in well-behaved thermodynamically equilibrated systems in vitro as well as in more complex environments in vivo. Protein aggregation reactions like amyloid formation, in contrast, are complex, often involving a series of uniquely structured aggregation intermediates appearing at different time scales. Nonetheless, FCS can be used in appropriate cases to characterize the early stages of some aggregation reactions. Here are described step-by-step protocols and experimental procedures for the study of molecular complex formation in aggregation systems as observed in simple buffer systems, cell extracts, and living cells. The methods described are illustrated with examples from studies of the self-assembly of huntingtin fragments, but in principle can be adapted for any aggregating system.

Aggregation behavior of chemically synthesized, full-length huntingtin exon1.

Repeat length disease thresholds vary among the 10 expanded polyglutamine (polyQ) repeat diseases, from about 20 to about 50 glutamine residues. The unique amino acid sequences flanking the polyQ segment are thought to contribute to these repeat length thresholds. The specific portions of the flanking sequences that modulate polyQ properties are not always clear, however. This ambiguity may be important in Huntington's disease (HD), for example, where in vitro studies of aggregation mechanisms have led to distinctly different mechanistic models. Most in vitro studies of the aggregation of the huntingtin (HTT) exon1 fragment implicated in the HD mechanism have been conducted on inexact molecules that are imprecise either on the N-terminus (recombinantly produced peptides) or on the C-terminus (chemically synthesized peptides). In this paper, we investigate the aggregation properties of chemically synthesized HTT exon1 peptides that are full-length and complete, containing both normal and expanded polyQ repeat lengths, and compare the results directly to previously investigated molecules containing truncated C-termini. The results on the full-length peptides are consistent with a two-step aggregation mechanism originally developed based on studies of the C-terminally truncated analogues. Thus, we observe relatively rapid formation of spherical oligomers containing from 100 to 600 HTT exon1 molecules and intermediate formation of short protofibril-like structures containing from 500 to 2600 molecules. In contrast to this relatively rapid assembly, mature HTT exon1 amyloid requires about one month to dissociate in vitro, which is similar to the time required for neuronal HTT exon1 aggregates to disappear in vivo after HTT production is discontinued.

Serine phosphorylation suppresses huntingtin amyloid accumulation by altering protein aggregation properties.

Aggregation of expanded polyglutamine repeat-containing fragments of the huntingtin (htt) protein may play a key role in Huntington's disease. Consistent with this hypothesis, two Ser-to-Asp mutations in the 17-amino-acid N-terminal htt(NT) segment abrogate both visible brain aggregates and disease symptoms in a full-length Q(97) htt mouse model while compromising aggregation kinetics and aggregate morphology in an htt fragment in vitro [Gu et al. (2009). Serines 13 and 16 are critical determinants of full-length human mutant huntingtin induced disease pathogenesis in HD mice. Neuron64, 828-840]. The htt(NT) segment has been shown to play a critical role in facilitating nucleation of amyloid formation in htt N-terminal exon1 fragments. We show here how these Ser-to-Asp mutations dramatically affect aggregation kinetics and aggregate structural integrity. First, these negatively charged Ser replacements impair the assembly of the α-helical oligomers that play a critical role in htt amyloid nucleation, thus providing an explanation for reduced amyloid formation rates. Second, these sequence modifications alter aggregate morphology, decrease aggregate stability, and enhance the steric accessibility of the htt(NT) segment within the aggregates. Together, these changes make the sequence-modified peptides kinetically and thermodynamically less likely to aggregate and more susceptible, if they do, to posttranslational modifications and degradation. These effects also show how phosphorylation of a protein might achieve cellular effects via direct impacts on the protein's aggregation properties. In fact, preliminary studies on exon1-like molecules containing phosphoryl-Ser residues at positions 13 and 16 show that they reduce aggregation rates and generate atypical aggregate morphologies similar to the effects of the Ser-to-Asp mutants.

Slow amyloid nucleation via α-helix-rich oligomeric intermediates in short polyglutamine-containing huntingtin fragments.

The 17-amino-acid N-terminal segment (htt(NT)) that leads into the polyglutamine (polyQ) segment in the Huntington's disease protein huntingtin (htt) dramatically increases aggregation rates and changes the aggregation mechanism, compared to a simple polyQ peptide of similar length. With polyQ segments near or above the pathological repeat length threshold of about 37, aggregation of htt N-terminal fragments is so rapid that it is difficult to tease out mechanistic details. We describe here the use of very short polyQ repeat lengths in htt N-terminal fragments to slow this disease-associated aggregation. Although all of these peptides, in addition to htt(NT) itself, form α-helix-rich oligomeric intermediates, only peptides with Q(N) of eight or longer mature into amyloid-like aggregates, doing so by a slow increase in ß-structure. Concentration-dependent circular dichroism and analytical ultracentrifugation suggest that the htt(NT) sequence, with or without added glutamine residues, exists in solution as an equilibrium between disordered monomer and α-helical tetramer. Higher order, α-helix rich oligomers appear to be built up via these tetramers. However, only htt(NT)Q(N) peptides with N=8 or more undergo conversion into polyQ ß-sheet aggregates. These final amyloid-like aggregates not only feature the expected high ß-sheet content but also retain an element of solvent-exposed α-helix. The α-helix-rich oligomeric intermediates appear to be both on- and off-pathway, with some oligomers serving as the pool from within which nuclei emerge, while those that fail to undergo amyloid nucleation serve as a reservoir for release of monomers to support fibril elongation. Based on a regular pattern of multimers observed in analytical ultracentrifugation, and a concentration dependence of α-helix formation in CD spectroscopy, it is likely that these oligomers assemble via a four-helix assembly unit. PolyQ expansion in these peptides appears to enhance the rates of both oligomer formation and nucleation from within the oligomer population, by structural mechanisms that remain unclear.

Nature of the amyloid-beta monomer and the monomer-oligomer equilibrium.

The monomer to oligomer transition initiates the aggregation and pathogenic transformation of Alzheimer amyloid-ß (Aß) peptide. However, the monomeric state of this aggregation-prone peptide has remained beyond the reach of most experimental techniques, and a quantitative understanding of this transition is yet to emerge. Here, we employ single-molecule level fluorescence tools to characterize the monomeric state and the monomer-oligomer transition at physiological concentrations in buffers mimicking the cerebrospinal fluid (CSF). Our measurements show that the monomer has a hydrodynamic radius of 0.9 ± 0.1 nm, which confirms the prediction made by some of the in silico studies. Surprisingly, at equilibrium, both Aß(40) and Aß(42) remain predominantly monomeric up to 3 µm, above which it forms large aggregates. This concentration is much higher than the estimated concentrations in the CSF of either normal or diseased brains. If Aß oligomers are present in the CSF and are the key agents in Alzheimer pathology, as is generally believed, then these must be released in the CSF as preformed entities. Although the oligomers are thermodynamically unstable, we find that a large kinetic barrier, which is mostly entropic in origin, strongly impedes their dissociation. Thermodynamic principles therefore allow the development of a pharmacological agent that can catalytically convert metastable oligomers into nontoxic monomers.

Critical nucleus size for disease-related polyglutamine aggregation is repeat-length dependent.

Because polyglutamine (polyQ) aggregate formation has been implicated as playing an important role in expanded CAG repeat diseases, it is important to understand the biophysics underlying the initiation of aggregation. Previously, we showed that relatively long polyQ peptides aggregate by nucleated growth polymerization and a monomeric critical nucleus. We show here that over a short range of repeat lengths, from Q(23) to Q(26), the size of the critical nucleus for aggregation increases from monomeric to dimeric to tetrameric. This variation in nucleus size suggests a common duplex antiparallel ß-sheet framework for the nucleus, and it further supports the feasibility of an organized monomeric aggregation nucleus for longer polyQ repeat peptides. The data also suggest that a change in the size of aggregation nuclei may have a role in the pathogenicity of polyQ expansion in this series of familial neurodegenerative diseases.

On the stability of the soluble amyloid aggregates.

Many amyloid proteins form metastable soluble aggregates (or protofibrils, or protein nanoparticles, with characteristic sizes from approximately 10 to a few hundred nm). These can coexist with protein monomers and amyloid precipitates. These soluble aggregates are key determinants of the toxicity of these proteins. It is therefore imperative to understand the physical basis underlying their stability. Simple nucleation theory, typically applied to explain the kinetics of amyloid precipitation, fails to predict such intermediate stable states. We examine stable nanoparticles formed by the Alzheimer's amyloid-beta peptide (40 and 42 residues), and by the protein barstar. These molecules have different hydrophobicities, and therefore have different short-range attractive interactions between the molecules. We also vary the pH and the ionic strength of the solution to tune the long-range electrostatic repulsion between them. In all the cases, we find that increased long-range repulsion results in smaller stable nanoparticles, whereas increased hydrophobicity produces the opposite result. Our results agree with a charged-colloid type of model for these particles, which asserts that growth-arrested colloid particles can result from a competition between short-range attraction and long-range repulsion. The nanoparticle size varies superlinearly with the ionic strength, possibly indicating a transition from an isotropic to a linear mode of growth. Our results provide a framework for understanding the stability and growth of toxic amyloid nanoparticles, and provide cues for designing effective destabilizing agents.

Protein aggregation probed by two-photon fluorescence correlation spectroscopy of native tryptophan.

Fluorescence correlation spectroscopy (FCS) has proven to be a powerful tool for the study of a range of biophysical problems including protein aggregation. However, the requirement of fluorescent labeling has been a major drawback of this approach. Here we show that the intrinsic tryptophan fluorescence, excited via a two-photon mechanism, can be effectively used to study the aggregation of tryptophan containing proteins by FCS. This method can also yield the tryptophan fluorescence lifetime in parallel, which provides a complementary parameter to understand the aggregation process. We demonstrate that the formation of soluble aggregates of barstar at pH 3.5 shows clear signatures both in the two-photon tryptophan FCS data and in the tryptophan lifetime analysis. The ability to probe the soluble aggregates of unmodified proteins is significant, given the major role played by this species in amyloid toxicity.

Effect of loop length variation on quadruplex-Watson Crick duplex competition.

The effect of loop length on quadruplex stability has been studied when the G-rich strand is present along with its complementary C-rich strand, thereby resulting in competition between quadruplex and duplex structures. Using model sequences with loop lengths varying from T to T5, we carried out extensive FRET to discover the influence of loop length on the quadruplex-Watson Crick duplex competition. The binding data show an increase in the binding affinity of quadruplexes towards their complementary strands upon increasing the loop length. Our kinetic data reveal that unfolding of the quadruplex in presence of a complementary strand involves a contribution from a predominant slow and a small population of fast opening conformer. The contribution from the fast opening conformer increases upon increasing the loop length leading to faster duplex formation. FCS data show an increase in the interconversion between the quadruplex conformers in presence of the complementary strand, which shifts the equilibrium towards the fast opening conformer with an increase in loop length. The relative free-energy difference (Delta DeltaG(o)) between the duplex and quadruplex indicates that an increase in loop length favors duplex formation and out competes the quadruplex.