The First Step of Amyloidogenic Aggregation

The structural and dynamic characterization of the on-pathway intermediates involved in the mechanism of amyloid fibril formation is one of the major remaining biomedical challenges of our time. In addition to mature fibrils, various oligomeric structures are implicated in both the rate-limiting step of the nucleation process and the neuronal toxicity of amyloid deposition. Single-molecule fluorescence spectroscopy (SMFS) is an excellent tool for extracting most of the relevant information on these molecular systems, especially advanced multiparameter approaches, such as pulsed interleaved excitation (PIE). In our investigations of an amyloidogenic SH3 domain of α-spectrin, we have found dynamic oligomerization, even prior to incubation. Our single-molecule PIE experiments revealed that these species are small, mostly dimeric, and exhibit a loose and dynamic molecular organization. Furthermore, these experiments have allowed us to obtain quantitative information regarding the oligomer stability. These pre-amyloidogenic oligomers may potentially serve as the first target for fibrillization-prevention strategies

Bulk and Single-Molecule Fluorescence Studies of the Saturation of the DNA Double Helix Using YOYO‑3 Intercalator Dye

We report a thorough photophysical characterization of the interactions between double-stranded DNA (dsDNA) and the trimethine cyanine homodimer dye YOYO-3. The fluorescence emission of this dye is enhanced by intercalation within the DNA double helix. We have explored the saturation of the dsDNA by bound YOYO-3 at the single-molecule level by studying the single-pair Förster resonance energy transfer (FRET) from an energy donor, Alexa Fluor 488, tagged at the 5′ end of the double helix and the energy acceptor, YOYO-3, bound to the same DNA molecule. The spontaneous binding of YOYO-3 gives rise to an effective distribution of different FRET efficiencies and, therefore, donor–acceptor (D–A) distances. These distributions reveal the existence of multiple states of YOYO-3. Steady-state and time-resolved fluorescence and circular dichroism confirmed the presence of a DNA-bound aggregate of YOYO-3, conspicuous at high dye/base pair ratios. The spectral features of the aggregate suggest that it may have the structure of a parallel H-aggregate

The effect of a C-terminal tag in ubiquitin on SH3 binding.

<p><b>a</b>. Titration curve monitored by NMR chemical shift perturbations of T283 (black) and E303 (red) in CD2AP SH3-C. The upper curves correspond to titrations with C-terminal His-tagged ubiquitin, while the lower curves correspond to titrations with non-tagged ubiquitin. <b>b</b>. Comparison of the chemical shift deviations (Δδ) between the first and last titration point of titrating ¹⁵N-labelled CD2AP SH3-C with C-terminal His-tagged ubiquitin (green) and non-tagged ubiquitin (red).</p

Solution structures of the complexes between ubiquitin and the first and third SH3 domains of CD2AP.

<p>Stereo representation of the ensemble of 10 lowest combined target function structures of the CD2AP SH3-A (<b>a</b>) and SH3-C (<b>b</b>) domains in complex with ubiquitin. Ubiquitin is represented in blue and the SH3 domain in red. Structures were superimposed on the backbone atoms of residues 7-70 in ubiquitin.</p

SH3: Ubiquitin binding interfaces.

<p>Surface representations of the free forms of CD2AP SH3-A (<b>a</b>), ubiquitin (<b>b</b>) and CD2AP SH3-C (<b>c</b>), showing the surface-charge distribution. Electrostatic surface representation of the orientation of the CD2AP SH3-A and C complexes with ubiquitin (green and orange respectively), zooming in at the SH3 RT-loop (<b>d</b> & <b>g</b> respectively), SH3 n-Src loop (<b>e</b> & <b>h</b> respectively) and ubiquitin C-terminus (<b>f</b> & <b>i</b> respectively). Ubiquitin residues with chemical shift perturbation higher than the mean are coloured in magenta. Charged residues with high CSP are represented in coloured sticks (blue and red for positively and negatively charged) for clarity. The electrostatic surface representation was drawn with Pymol (<a href="http://www.pymol.org" target="_blank">www.pymol.org</a>) using a Poisson-Boltzmann electrostatics calculation [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073018#B40" target="_blank">40</a>].</p

Monitoring the binding between ubiquitin and the CD2AP SH3 domains and the CIN85 SH3-C domain by NMR chemical shift perturbations.

<p><b>a</b>. ¹H and ¹⁵N chemical shift changes upon titrating ubiquitin into a ¹⁵N-labelled CD2AP SH3-A solution. Region of ¹H-¹⁵N HSQC spectra recorded at increasing amounts of ubiquitin up to a final ratio of 1:1.6 (SH3: Ubi) (red to blue). <b>b</b>. ¹H and ¹⁵N chemical shift changes upon titrating ubiquitin into a ¹⁵N-labelled CD2AP SH3-C solution. Region of ¹H-¹⁵N HSQC spectra recorded at increasing amounts of ubiquitin up to a ratio of 1:2.4 (SH3: Ubi) (red to blue). <b>c</b>. ¹H and ¹⁵N chemical shift changes upon titrating CD2AP SH3-A into a ¹⁵N-labelled ubiquitin solution. Region of ¹H-¹⁵N HSQC spectra recorded at increasing amounts of CD2AP SH3-A up to a ratio of 1:1.4 (Ubi: SH3) (red to blue). <b>d</b>. ¹H and ¹⁵N chemical shift changes upon titrating CD2AP SH3-C into a ¹⁵N-labelled ubiquitin solution. Region of ¹H-¹⁵N HSQC spectra recorded at increasing amounts of CD2AP SH3-C up to a ratio of 1:2.5 (Ubi: SH3) (red to blue). <b>e</b> & <b>f</b>. Chemical shift deviations (Δδ) between the first and last titration point of the ubiquitin titration into ¹⁵N-labelled CD2AP SH3-A and SH3-C, respectively. <b>g</b>, <b>h</b>, <b>i</b> & <b>j</b>. Chemical shift deviations (Δδ) between the first and last titration point of titrating CD2AP SH3-A (G), SH3-C (H), SH3-B (I) and CIN85 SH3-C (J) into ¹⁵N-labelled ubiquitin corresponding to a ratio of 1:1.4, 1:2.5, 1:1.2 and 1:1.1 (Ubi: SH3) respectively. Blue solid lines indicate the mean chemical shift deviations.</p

Structural comparison of different SH3: Ubiquitin complexes.

<p>Cartoon representation of the lowest target function or lowest energy structure of different SH3: Ubiquitin complexes. <b>a</b>. CD2AP SH3-A:Ubiquitin complex. Ubiquitin is represented in blue and CD2AP SH3-A in green. <b>b</b>. CD2AP SH3-C:Ubiquitin complex. Ubiquitin is represented in blue and CD2AP SH3-C in red. <b>c</b>. Sla1 SH3-3 in complex with ubiquitin (PDB entry 2JT4; ubiquitin in blue, Sla1 SH3-3 in magenta). <b>d</b>. CIN85 SH3-C in complex with ubiquitin (PDB entry 2K6D; ubiquitin in blue, CIN85 SH3-C in cyan). Phenylalanine and tryptophan residues equivalent to F53 and W41 in CD2AP SH3-A are given in stick representation and coloured in orange in all SH3 domains for clarity. All complexes were superimposed on the backbone atoms of residues 7-70 of ubiquitin.</p