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 (ΔΔ G °) between the duplex and quadruplex indicates that an increase in loop length favors duplex formation and out competes the quadruplex

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

The 17-amino-acid N-terminal segment (httNT) that leads into the polyglutamine (polyQ) segment in the Huntington\u27s 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 httNT itself, form α-helix-rich oligomeric intermediates, only peptides with QN of eight or longer mature into amyloid-like aggregates, doing so by a slow increase in β-structure. Concentration-dependent circular dichroism and analytical ultracentrifugation suggest that the httNT 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 httNTQN peptides with N=8 or more undergo conversion into polyQ β-sheet aggregates. These final amyloid-like aggregates not only feature the expected high β-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. © 2011 Elsevier Ltd

On the Stability of the Soluble Amyloid Aggregates

Many amyloid proteins form metastable soluble aggregates (or protofibrils, or protein nanoparticles, with characteristic sizes from ∼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-β 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

Spontaneous formation of a protein corona prevents the loss of quantum dot fluorescence in physiological buffers

We report that proteins in physiological buffers spontaneously attach to polyethylene glycol coated fluorescent quantum dots (QDs) and strongly inhibit the gradual loss of QD fluorescence. This interaction does not strongly depend on the nature or solubility of the proteins tested (amyloid beta, barstar, cytochrome c, bovine serum albumin and lysozyme). Fluorescence correlation spectroscopy shows that each QD acquires a ‘corona’ of ∼100 protein molecules, which confers protection against ionic attacks in aqueous solutions. This effect would be important for any biological application, and also offers a potential in vitro assay for soluble proteins

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

Summary of FCS data showing molecular sizes under various conditions.

<p>Summary of FCS data showing molecular sizes under various conditions.</p

Structures and assembly mechanism of HTT exon1 polypeptides.

<p>A. Previously proposed mechanism of amyloid nucleation (HTT^NT, green; polyQ, orange; PRD, black). B. Sequence of HTT exon1. C. Sequences of peptides studied. C* = Cys residue modified with Alexa Fluor 555 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155747#sec009" target="_blank">Materials and Methods</a>).</p

Self-assembly in PBS of chemically synthesized HTT exon1 analogs.

<p>A. Raw FCS time-dependent fluorescence fluctuations of HTT^NTQ₂₃P₁₀C*K₂ (red) and HTT^NTQ₃₇P₁₀C*K₂ (black). B. Autocorrelation functions for the 10 mins FCS data shown in A, with the data points as filled squares and solid lines representing the fits (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155747#sec009" target="_blank">Materials and Methods</a>) and the same color scheme as A. Lines below the graph show the residuals between the data points and the fit curve. C. Concentration dependence of molecular size estimated from diffusion times for HTT^NTQ₃₇P₁₀C*K₂ (■) and HTT^NTQ₂₃P₁₀C*K₂ (). D. EM detail of different time points from the PBS incubation of a mixture of 2.0 μM HTT^NTQ₃₇P₁₀K₂. (More EM data is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155747#pone.0155747.s001" target="_blank">S1 Fig</a>.)</p