Structure-Free Validation of Residual Dipolar Coupling and Paramagnetic Relaxation Enhancement Measurements of Disordered Proteins

Abstract

Residual dipolar couplings (RDCs) and paramagnetic relaxation enhancements (PREs) have emerged as valuable parameters for defining the structures and dynamics of disordered proteins by nuclear magnetic resonance (NMR) spectroscopy. Procedures for their measurement, however, may lead to conformational perturbations because of the presence of the alignment media necessary for recording RDCs, or of the paramagnetic groups that must be introduced for measuring PREs. We discuss here experimental methods for quantifying these effects by considering the case of the 40-residue isoform of the amyloid β peptide (Aβ40), which is associated with Alzheimer's disease. By conducting RDC measurements over a range of concentrations of certain alignment media, we show that perturbations arising from transient binding of Aβ40 can be characterized, allowing appropriate corrections to be made. In addition, by using NMR experiments sensitive to long-range interactions, we show that it is possible to identify relatively nonperturbing sites for attaching nitroxide radicals for PRE measurements. Thus, minimizing the conformational perturbations introduced by RDC and PRE measurements should facilitate their use for the rigorous determination of the conformational properties of disordered proteins.

Molecular determinants of the interaction of EGCG with ordered and disordered proteins

“…By incubating the proteins with relatively low ratios of EGCG relative to the protein molecules, however, the broadening of the protein resonances is not excessive and allows the acquisition of three‐dimensional NMR experiments to measure the 13 C resonances of the protein. We therefore measured the backbone resonances, including 13 Cα, 13 Cβ, 13 CO, 1 Hα, 1 HN, and 13 N of Aβ40 and α‐synuclein in the presence and absence of three molar excess of EGCG (Figure B, Supporting Information Figure S1A), and analyzed the chemical shifts of the resonances that were previously assigned to assess the effects of the interaction with EGCG on the conformational properties of the monomeric states of the two proteins. Backbone chemical shifts of 13 Cα or 13 Cβ in particular are extremely sensitive probes of the secondary structure content of disordered proteins, and their values can be used in methods such as δ2D to obtain accurate information on the nature of the residual structure and of the dynamical properties of the residues in the sequence …”

“…NMR experiments were carried out using a 700 MHz spectrometer at 5°C in 50 mM phosphate buffer at pH 7.4. Resonance assignments of the 1 H‐ 15 N‐HSQC spectrum of Aβ40 were derived from our previous studies and directly transferred easily to the spectra measured in the presence of EGCG. The strategy of assignment of the 3D experiments followed the protocol described above for α‐synuclein.…”

Structural Insight into IAPP‐Derived Amyloid Inhibitors and Their Mechanism of Action

“…[1b] Notably,A b40 in solution exhibits ac onformational preference for b-arch-like structures as well. [28] Thus, R3-GI and related ISMs may exert their inhibitory function Figure 7. Differentm echanistic pathways of ligand binding to amyloidogenic proteins.…”

“…Thedesign of the ISMs was based on the finding that amyloids are generally composed of a b-sheet-turn-b-sheet structural motif and that IAPP uses the same two binding regions for both its amyloid self-and its cross-amyloid hetero-assembly with Ab40/42. [1a, 2] ISMs were thus derived by linking the two hot segments IAPP (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18) and IAPP (22)(23)(24)(25)(26)(27)(28) in native or N-methylated form to each other via different linkers,mostly tripeptide sequences consisting of identical amino acids;notably,these two segments are highly homologous to segments of the amyloid core of Ab. [3] High Ab40/42 anti-amyloidogenic activity was found for seven out of the 16 studied ISMs with six of them containing bulky hydrophobic/aromatic residues (e.g.L LL, III, FFF) in the linker tripeptide and one of them, termed R3-GI, the RRR tripeptide.…”

NMR Characterization of Conformational Dynamics and Molecular Assemblies of Proteins

“…24,25) In aqueous solution, Aβ exists mostly as a monomeric unstructured polypeptide, although molecular dynamics (MD) simulations identified local non-random conformations in Aβ molecules. 26,27) A paramagnetism-assisted NMR technique has been developed for characterizing the dynamic conformational ensemble and the transient intra-molecular interactions of Aβ in solution. 28) This technique exploits paramagnetic probes such as spin labels attached to specific positions in Aβ molecules, which can induce paramagnetic relaxation enhancement (PRE) effects as NOE-independent sources of long-distance information.…”