The route from the folded to the amyloid state: exploring the potential energy surface of a drug-like miniprotein

The amyloid formation of the folded segment of a variant of Exenatide (a marketed drug for Type-2 Diabetes Mellitus ) was studied by ECD and NMR. We found that the optimum temperature for E5 protein amyloidosis coincides with body temperature and requires well below physiological salt concentration. Decomposition of the ECD spectra and its barycentric representation on the folded-unfolded-amyloid potential energy surface allowed us to monitor the full range of molecular transformation of amyloidogenesis. We identified points of no return ( e.g. T =37°C, pH =4.1, c E5 =250µM, c NaCl =50mM, t >4-6 h) which will inevitably gravitate into the amyloid-state. The strong B-type FUV-ECD spectra and an unexpectedly strong NUV-ECD signal (Θ ~275-285nm ) indicate that the amyloid phase of E5 is built from monomers of quasi -elongated backbone structure ( φ ~-145°, ψ ~+145°) with strong interstrand Tyr↔Trp interaction. Misfolded intermediers and the buildup of "toxic" early-stage oligomers leading to self-association were identified and monitored as function of time. Results indicate that the amyloid transition is triggered by subtle misfolding of the α-helix exposing aromatic and hydrophobic side chains that may provide the first centers for an intermolecular reorganization. These initial clusters provide the spatial closeness and sufficient time for a transition to the β-structured amyloid nucleus thus the process follows a nucleated growth mechanism

The Importance of Mg2 + -Free State in Nucleotide Exchange of Oncogenic K-Ras Mutants

For efficient targeting of oncogenic K-Ras interaction sites, a mechanistic picture of the Ras-cycle is necessary. Herein, we used NMR relaxation techniques and molecular dynamics simulations to decipher the role of slow dynamics in wild-type and three oncogenic P-loop mutants of K-Ras. Our measurements reveal a dominant two-state conformational exchange on the ms timescale in both GDP- and GTP-bound K-Ras. The identified low-populated higher energy state in GDP-loaded K-Ras has a conformation reminiscent of a nucleotide-bound/Mg2+ -free state characterized by shortened β2/β3-strands and a partially released switch-I region preparing K-Ras for the interaction with the incoming nucleotide exchange factor and subsequent reactivation. By providing insight into mutation-specific differences in K-Ras structural dynamics, our systematic analysis improves our understanding of prolonged K-Ras signaling and may aid the development of allosteric inhibitors targeting nucleotide exchange in K-Ras