7 research outputs found
Repurposing triphenylmethane dyes to bind to trimers derived from Aß
Accepted author manuscriptSoluble oligomers of the β-amyloid peptide, Aβ, are associated with the progression of Alzheimer’s disease. Although many small molecules bind to these assemblies, the details of how these molecules interact with Aβ oligomers remain unknown. This paper reports that crystal violet, and other C3 symmetric triphenylmethane dyes, bind to C3 symmetric trimers derived from Aβ17–36. Binding changes the color of the dyes from purple to blue, and causes them to fluoresce red when irradiated with green light. Job plot and analytical ultracentrifugation experiments reveal that two trimers complex with one dye molecule. Studies with several triphenylmethane dyes reveal that three N,N-dialkylamino substituents are required for complexation. Several mutant trimers, in which Phe19, Phe20, and Ile31 were mutated to cyclohexylalanine, valine, and cyclohexylglycine, were prepared to probe the triphenylmethane dye binding site. Size exclusion chromatography, SDS-PAGE, and X-ray crystallographic studies demonstrate that these mutations do not impact the structure or assembly of the triangular trimer. Fluorescence spectroscopy and analytical ultracentrifugation experiments reveal that the dye packs against an aromatic surface formed by the Phe20 side chains and is clasped by the Ile31 side chains. Docking and molecular modeling provide a working model of the complex in which the triphenylmethane dye is sandwiched between two triangular trimers. Collectively, these findings demonstrate that the X-ray crystallographic structures of triangular trimers derived from Aβ can be used to guide the discovery of ligands that bind to soluble oligomers derived from Aβ.Ye
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Correction to “Repurposing Triphenylmethane Dyes To Bind to Trimers Derived from Aβ”
There was an error in listing the PDB ID numbers in the captionof Figure 7. The corrected caption should read as follows: (Figure Presented)
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Controlling the Oligomerization State of Aβ-Derived Peptides with Light
A key
challenge in studying the biological and biophysical properties
of amyloid-forming peptides is that they assemble to form heterogeneous
mixtures of soluble oligomers and insoluble fibrils. Photolabile protecting
groups have emerged as tools to control the properties of biomolecules
with light. Blocking intermolecular hydrogen bonds that stabilize
amyloid oligomers provides a general strategy to control the biological
and biophysical properties of amyloid-forming peptides. In this paper
we describe the design, synthesis, and characterization of macrocyclic
β-hairpin peptides that are derived from amyloidogenic peptides
and contain the <i>N</i>-2-nitrobenzyl photolabile protecting
group. Each peptide contains two heptapeptide segments from Aβ<sub>16–36</sub> or Aβ<sub>17–36</sub> constrained
into β-hairpins. The <i>N</i>-2-nitrobenzyl group
is appended to the amide backbone of Gly<sub>33</sub> to disrupt the
oligomerization of the peptides by disrupting intermolecular hydrogen
bonds. X-ray crystallography reveals that <i>N</i>-2-nitrobenzyl
groups can either block assembly into discrete oligomers or permit
formation of trimers, hexamers, and dodecamers. Photolysis of the <i>N</i>-2-nitrobenzyl groups with long-wave UV light unmasks the
amide backbone and alters the assembly and the biological properties
of the macrocyclic β-hairpin peptides. SDS–PAGE studies
show that removing the <i>N</i>-2-nitrobenzyl groups alters
the assembly of the peptides. MTT conversion and LDH release assays
show that decaging the peptides induces cytotoxicity. Circular dichroism
studies and dye leakage assays with liposomes reveal that decaging
modulates interactions of the peptides with lipid bilayers. Collectively,
these studies demonstrate that incorporating <i>N</i>-2-nitrobenzyl
groups into macrocyclic β-hairpin peptides provides a new strategy
to probe the structures and the biological properties of amyloid oligomers
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Asymmetric dimerization of adenosine deaminase acting on RNA facilitates substrate recognition.
Adenosine deaminases acting on RNA (ADARs) are enzymes that convert adenosine to inosine in duplex RNA, a modification that exhibits a multitude of effects on RNA structure and function. Recent studies have identified ADAR1 as a potential cancer therapeutic target. ADARs are also important in the development of directed RNA editing therapeutics. A comprehensive understanding of the molecular mechanism of the ADAR reaction will advance efforts to develop ADAR inhibitors and new tools for directed RNA editing. Here we report the X-ray crystal structure of a fragment of human ADAR2 comprising its deaminase domain and double stranded RNA binding domain 2 (dsRBD2) bound to an RNA duplex as an asymmetric homodimer. We identified a highly conserved ADAR dimerization interface and validated the importance of these sequence elements on dimer formation via gel mobility shift assays and size exclusion chromatography. We also show that mutation in the dimerization interface inhibits editing in an RNA substrate-dependent manner for both ADAR1 and ADAR2