17 research outputs found
Characterization of the Raf Kinase Inhibitory Protein (RKIP) Binding Pocket: NMR-Based Screening Identifies Small-Molecule Ligands
Raf kinase inhibitory protein (RKIP), also known as phoshaptidylethanolamine binding protein (PEBP), has been shown to inhibit Raf and thereby negatively regulate growth factor signaling by the Raf/MAP kinase pathway. RKIP has also been shown to suppress metastasis. We have previously demonstrated that RKIP/Raf interaction is regulated by two mechanisms: phosphorylation of RKIP at Ser-153, and occupation of RKIP's conserved ligand binding domain with a phospholipid (2-dihexanoyl-sn-glycero-3-phosphoethanolamine; DHPE). In addition to phospholipids, other ligands have been reported to bind this domain; however their binding properties remain uncharacterized.In this study, we used high-resolution heteronuclear NMR spectroscopy to screen a chemical library and assay a number of potential RKIP ligands for binding to the protein. Surprisingly, many compounds previously postulated as RKIP ligands showed no detectable binding in near-physiological solution conditions even at millimolar concentrations. In contrast, we found three novel ligands for RKIP that specifically bind to the RKIP pocket. Interestingly, unlike the phospholipid, DHPE, these newly identified ligands did not affect RKIP binding to Raf-1 or RKIP phosphorylation. One out of the three ligands displayed off target biological effects, impairing EGF-induced MAPK and metabolic activity.This work defines the binding properties of RKIP ligands under near physiological conditions, establishing RKIP's affinity for hydrophobic ligands and the importance of bulky aliphatic chains for inhibiting its function. The common structural elements of these compounds defines a minimal requirement for RKIP binding and thus they can be used as lead compounds for future design of RKIP ligands with therapeutic potential
Geometry and Efficacy of Trp-Trp, Trp-Tyr and Tyr-Tyr Aromatic Interaction in Cross-Strand Positions of a Designed β-Hairpin
The Influence of Monomer Structures on the Liquid Crystalline Order of Aramide Polymers: An NMR Analysis
The Influence of Monomer Structures on the Liquid Crystalline Order of Aramide Polymers: An NMR Analysis
Enhanced sensitivity to local dynamics in peptides by use of temperature-jump IR-spectroscopy and isotope labeling
Site-specific isotopic labeling of molecules is a widely used approach in IR-spectroscopy to resolve local contributions to vibrational modes. The induced frequency shift of the corresponding IR band depends on the substituted masses, but also on hydrogen bonding and on vibrational coupling. The impact of these different factors was analyzed with a designed three-stranded β-sheet peptide and by use of selected 13C isotope substitutions at multiple positions in the peptide backbone. Single strand labels give rise to isotopically shifted bands at different frequencies depending on the specific sites, demonstrating sensitivity to the local environment. Cross-strand double and triple labeled peptides exhibited two resolved bands, which could be uniquely assigned to specific residues, whose equilibrium IR indicated only weak local-mode coupling. Temperature-jump IR-laser spectroscopy was applied to monitor structural dynamics and revealed an impressive enhancement of the isotope sensitivity to both local positions and coupling between them as compared to equilibrium FTIR. Site-specific relaxation rates were altered upon introduction of additional cross-strand isotopes. Likewise, the rates for the global β-sheet dynamics were affected in a manner dependent on the distinct relaxation behavior of the labeled oscillator. The study demonstrates that isotope labels do not just provide local structural probes, but they rather sense the dynamic complexity of the molecular environment.publishe
E22G Pathogenic Mutation of β‑Amyloid (Aβ) Enhances Misfolding of Aβ40 by Unexpected Prion-like Cross Talk between Aβ42 and Aβ40
Cross-seeding of
misfolded amyloid proteins is postulated to induce
cross-species infection of prion diseases. In sporadic Alzheimer’s
disease (AD), misfolding of 42-residue β-amyloid (Aβ)
is widely considered to trigger amyloid plaque deposition. Despite
increasing evidence that misfolded Aβ mimics prions, interactions
of misfolded 42-residue Aβ42 with more abundant 40-residue Aβ40
in AD are elusive. This study presents <i>in vitro</i> evidence
that a heterozygous E22G pathogenic (“Arctic”) mutation
of Aβ40 can enhance misfolding of Aβ via cross-seeding
from wild-type (WT) Aβ42 fibril. Thioflavin T (ThT) fluorescence
analysis suggested that misfolding of E22G Aβ40 was enhanced
by adding 5% (w/w) WT Aβ42 fibril as “seed”, whereas
WT Aβ40 was unaffected by Aβ42 fibril seed. <sup>13</sup>C SSNMR analysis revealed that such cross-seeding prompted formation
of E22G Aβ40 fibril that structurally mimics the seed Aβ42
fibril, suggesting unexpected cross talk of Aβ isoforms that
potentially promotes early onset of AD. The SSNMR approach is likely
applicable to elucidate structural details of heterogeneous amyloid
fibrils produced in cross-seeding for amyloids linked to neurodegenerative
diseases
Isotopically Site-Selected Dynamics of a Three-Stranded -Sheet Peptide Detected with Temperature-Jump IR-Spectroscopy
Infrared detected temperature jump (T-jump) spectroscopy and site-specific isotopic labeling were applied to study a model three-stranded beta-sheet peptide with the goal of individually probing the dynamics of strand and turn structural elements. This peptide had two DPro-Gly (pG) turn sequences to stabilize the two component hairpins, which were labeled with 13C=O on each of the Gly residues to resolve them spectroscopically. Labeling the second turn on the amide preceding the DPro (XxxDPro amide) provided an alternate turn label as a control. Placing 13C=O labels on specific in-strand residues gave shifted modes that overlap the XxxDPro amide I’ modes. Their impact could be separated from the turn dynamics by a novel difference-transient analysis approach. FTIR spectra were modeled with DFTcomputations which showed the local, isotope-selected vibrations were effectively uncoupled from the other amide I modes. Our T-jump dynamics results, combined with NMR structures and equilibrium spectral measurements, showed the first turn to be most stable and best formed with the slowest dynamics, while the second turn and first strand (N-terminus) had similar dynamics, and the third strand (C-terminus) had the fastest dynamics and was the least structured. The relative dynamics of the strands, XxxDPro amides and 13C-labeled Gly residues on the turns also qualitatively corresponded to molecular dynamics (MD) simulations of turn and strand fluctuations. MD trajectories indicated the turns to be bistable, with the first turn being Type I’ and the second turn flipping from I’ to II’. The differences in relaxation times for each turn and the separate strands revealed that the folding process of this turnstabilized beta-sheet structure proceeds in a multi-step process
Role of Aromatic Cross-Links in Structure and Dynamics of Model Three-Stranded β‑Sheet Peptides
A series
of closely related peptide sequences that form triple-strand
structures was designed with a variation of cross-strand aromatic
interactions and spectroscopically studied as models for β-sheet
formation and stabilities. Structures of the three-strand models were
determined with NMR methods and temperature-dependent equilibrium
studies performed using circular dichroism and Fourier transform infrared
spectroscopies. Our equilibrium data show that the presence of a direct
cross-strand aromatic contact in an otherwise folded peptide does
not automatically result in an increased thermal stability and can
even distort the structure. The effect on the conformational dynamics
was studied with infrared-detected temperature-jump relaxation methods
and revealed a high sensitivity to the presence and the location of
the aromatic cross-links. Aromatic contacts in the three-stranded
peptides slow down the dynamics in a site-specific manner, and the
impact seems to be related to the distance from the turn. With a Xxx-<sup>D</sup>Pro linkage as a probe with some sensitivity for the turn,
small differences were revealed in the relative relaxation of the
sheet strands and turn regions. In addition, we analyzed the component
hairpins, which showed less uniform dynamics as compared to the parent
three-stranded β-sheet peptides