Photocontrolled Reversible Amyloid Fibril Formation of Parathyroid Hormone-Derived Peptides.

Peptide fibrillization is crucial in biological processes such as amyloid-related diseases and hormone storage, involving complex transitions between folded, unfolded, and aggregated states. We here employ light to induce reversible transitions between aggregated and nonaggregated states of a peptide, linked to the parathyroid hormone (PTH). The artificial light-switch 3-{[(4-aminomethyl)phenyl]diazenyl}benzoic acid (AMPB) is embedded into a segment of PTH, the peptide PTH, to control aggregation, revealing position-dependent effects.

Structural Impact of N-terminal Pyroglutamate in an Amyloid-β(3-42) Fibril Probed by Solid-State NMR Spectroscopy.

Extracellular amyloid-β (Aβ) plaques, primarily formed by Aβ(1-40) and Aβ(1-42) fibrils, are a hallmark of Alzheimer's disease. The Aβ peptide can undergo a high variety of different post-translational modifications including formation of a pyroglutamate (pGlu, pE) at N-terminal Glu3 or Glu11 of truncated Aβ(3-x) or Aβ(11-x), respectively. Here we studied structural similarities and differences between pEAβ(3-42) and LS-shaped Aβ(1-42) fibrils grown under identical conditions (pH 2) using solid-state NMR spectroscopy.

Amyloid fibril formation kinetics of low-pH denatured bovine PI3K-SH3 monitored by three different NMR techniques.

Misfolding of amyloidogenic proteins is a molecular hallmark of neurodegenerative diseases in humans. A detailed understanding of the underlying molecular mechanisms is mandatory for developing innovative therapeutic approaches. The bovine PI3K-SH3 domain has been a model system for aggregation and fibril formation.

Atomic Resolution Insights into pH Shift Induced Deprotonation Events in LS-Shaped Aβ(1-42) Amyloid Fibrils.

Alzheimer's disease is a neurodegenerative disorder associated with the deposition of misfolded aggregates of the amyloid-β protein (Aβ). Aβ(1-42) is one of the most aggregation-prone components in senile plaques of AD patients. We demonstrated that relatively homogeneous Aβ(1-42) fibrils with one predominant fold visible in solid-state NMR spectra can be obtained at acidic pH.

Conformational heterogeneity coupled with β-fibril formation of a scaffold protein involved in chronic mental illnesses.

Chronic mental illnesses (CMIs) pose a significant challenge to global health due to their complex and poorly understood etiologies and hence, absence of causal therapies. Research of the past two decades has revealed dysfunction of the disrupted in schizophrenia 1 (DISC1) protein as a predisposing factor involved in several psychiatric disorders. DISC1 is a multifaceted protein that serves myriads of functions in mammalian cells, for instance, influencing neuronal development and synapse maintenance.

A novel strategy for site selective spin-labeling to investigate bioactive entities by DNP and EPR spectroscopy.

A novel specific spin-labeling strategy for bioactive molecules is presented for eptifibatide (integrilin) an antiplatelet aggregation inhibitor, which derives from the venom of certain rattlesnakes. By specifically labeling the disulfide bridge this molecule becomes accessible for analytical techniques such as Electron Paramagnetic Resonance (EPR) and solid state Dynamic Nuclear Polarization (DNP). The necessary spin-label was synthesized and inserted into the disulfide bridge of eptifibatide via reductive followed by insertion by a double Michael addition under physiological conditions.

Amyloid-type Protein Aggregation and Prion-like Properties of Amyloids.

This review will focus on the process of amyloid-type protein aggregation. Amyloid fibrils are an important hallmark of protein misfolding diseases and therefore have been investigated for decades. Only recently, however, atomic or near-atomic resolution structures have been elucidated from various in vitro and ex vivo obtained fibrils.

Structural details of amyloid β oligomers in complex with human prion protein as revealed by solid-state MAS NMR spectroscopy.

Human PrP (huPrP) is a high-affinity receptor for oligomeric amyloid β (Aβ) protein aggregates. Binding of Aβ oligomers to membrane-anchored huPrP has been suggested to trigger neurotoxic cell signaling in Alzheimer's disease, while an N-terminal soluble fragment of huPrP can sequester Aβ oligomers and reduce their toxicity. Synthetic oligomeric Aβ species are known to be heterogeneous, dynamic, and transient, rendering their structural investigation particularly challenging.

Binding modes of thioflavin T and Congo red to the fibril structure of amyloid-β(1-42).

Binding modes for the amyloid-β(1-42) fibril fluorescent dyes thioflavin T and Congo red were predicted by molecular dynamics simulations and binding free energy calculations. Both probes bind on the fibril surface to primarily hydrophobic grooves, with their long axis oriented almost parallel to the fibril axis. The computed binding affinities are in agreement with experimental values.

Heteronuclear cross-relaxation effect modulated by the dynamics of N-functional groups in the solid state under N DP-MAS DNP.

In a typical magic-angle spinning (MAS) dynamic nuclear polarization (DNP) nuclear magnetic resonance (NMR) experiment, several mechanisms are simultaneously involved when transferring much larger polarization of electron spins to NMR active nuclei of interest. Recently, specific cross-relaxation enhancement by active motions under DNP (SCREAM-DNP) [Daube et al. JACS 2016] has been reported as one of these mechanisms.

Determining isoleucine side-chain rotamer-sampling in proteins from C chemical shift.

Chemical shifts are often the only nuclear magnetic resonance parameter that can be obtained for challenging macromolecular systems. Here we present a framework to derive the conformational sampling of isoleucine side chains from C chemical shifts and demonstrate that side-chain conformations in a low-populated folding intermediate can be determined.

Hyperpolarized MAS NMR of unfolded and misfolded proteins.

In this article we give an overview over the use of DNP-enhanced solid-state NMR spectroscopy for the investigation of unfolded, disordered and misfolded proteins. We first provide an overview over studies in which DNP spectroscopy has successfully been applied for the structural investigation of well-folded amyloid fibrils formed by short peptides as well as full-length proteins. Sample cooling to cryogenic temperatures often leads to severe line broadening of resonance signals and thus a loss in resolution.

Structural insights from lipid-bilayer nanodiscs link α-Synuclein membrane-binding modes to amyloid fibril formation.

The protein α-Synuclein (αS) is linked to Parkinson's disease through its abnormal aggregation, which is thought to involve cytosolic and membrane-bound forms of αS. Following previous studies using micelles and vesicles, we present a comprehensive study of αS interaction with phospholipid bilayer nanodiscs. Using a combination of NMR-spectroscopic, biophysical, and computational methods, we structurally and kinetically characterize αS interaction with different membrane discs in a quantitative and site-resolved way.

A d-enantiomeric peptide interferes with heteroassociation of amyloid-β oligomers and prion protein.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that affects millions of people worldwide. One AD hallmark is the aggregation of β-amyloid (Aβ) into soluble oligomers and insoluble fibrils. Several studies have reported that oligomers rather than fibrils are the most toxic species in AD progression.

Origin of metastable oligomers and their effects on amyloid fibril self-assembly.

Assembly of rigid amyloid fibrils with their characteristic cross-β sheet structure is a molecular signature of numerous neurodegenerative and non-neuropathic disorders. Frequently large populations of small globular amyloid oligomers (gOs) and curvilinear fibrils (CFs) precede the formation of late-stage rigid fibrils (RFs), and have been implicated in amyloid toxicity. Yet our understanding of the origin of these metastable oligomers, their role as on-pathway precursors or off-pathway competitors, and their effects on the self-assembly of amyloid fibrils remains incomplete.

DNP-Enhanced MAS NMR: A Tool to Snapshot Conformational Ensembles of α-Synuclein in Different States.

Intrinsically disordered proteins dynamically sample a wide conformational space and therefore do not adopt a stable and defined three-dimensional conformation. The structural heterogeneity is related to their proper functioning in physiological processes. Knowledge of the conformational ensemble is crucial for a complete comprehension of this kind of proteins.

Advancements of the sFIDA method for oligomer-based diagnostics of neurodegenerative diseases.

Early diagnosis of Alzheimer's disease (AD) is of great importance for the development of therapeutics and their application in the clinical environment. Amyloid β (Aβ) oligomers are crucial for the onset and progression of AD and represent a popular drug target, being presumably the most direct biomarker. Efforts to measure Aβ oligomers in body fluids are hampered by the low analyte concentration and presence of Aβ monomers.

Fibril structure of amyloid-β(1-42) by cryo-electron microscopy.

Amyloids are implicated in neurodegenerative diseases. Fibrillar aggregates of the amyloid-β protein (Aβ) are the main component of the senile plaques found in brains of Alzheimer's disease patients. We present the structure of an Aβ(1-42) fibril composed of two intertwined protofilaments determined by cryo-electron microscopy (cryo-EM) to 4.

Structural Characterization of Fibrils from Recombinant Human Islet Amyloid Polypeptide by Solid-State NMR: The Central FGAILS Segment Is Part of the β-Sheet Core.

Amyloid deposits formed from islet amyloid polypeptide (IAPP) are a hallmark of type 2 diabetes mellitus and are known to be cytotoxic to pancreatic β-cells. The molecular structure of the fibrillar form of IAPP is subject of intense research, and to date, different models exist. We present results of solid-state NMR experiments on fibrils of recombinantly expressed and uniformly 13C, 15N-labeled human IAPP in the non-amidated, free acid form.

Selective Protein Hyperpolarization in Cell Lysates Using Targeted Dynamic Nuclear Polarization.

Nuclear magnetic resonance (NMR) spectroscopy has the intrinsic capabilities to investigate proteins in native environments. In general, however, NMR relies on non-natural protein purity and concentration to increase the desired signal over the background. We here report on the efficient and specific hyperpolarization of low amounts of a target protein in a large isotope-labeled background by combining dynamic nuclear polarization (DNP) and the selectivity of protein interactions.

Progress towards structural understanding of infectious sheep PrP-amyloid.

The still elusive structural difference of non-infectious and infectious amyloid of the mammalian prion protein (PrP) is a major pending milestone in understanding protein-mediated infectivity in neurodegenerative diseases. Preparations of PrP-amyloid proven to be infectious have never been investigated with a high-resolution technique. All available models to date have been based on low-resolution data.

Liposome reconstitution and modulation of recombinant prenylated human Rac1 by GEFs, GDI1 and Pak1.

Small Rho GTPases are well known to regulate a variety of cellular processes by acting as molecular switches. The regulatory function of Rho GTPases is critically dependent on their posttranslational modification at the carboxyl terminus by isoprenylation and association with proper cellular membranes. Despite numerous studies, the mechanisms of recycling and functional integration of Rho GTPases at the biological membranes are largely unclear.

Expression, purification, and solid-state NMR characterization of the membrane binding heme protein nitrophorin 7 in two electronic spin states.

The nitrophorins (NPs) comprise a group of NO transporting ferriheme b proteins found in the saliva of the blood sucking insect Rhodnius prolixus . In contrast to other nitrophorins (NP1-4), the recently identified membrane binding isoform NP7 tends to form oligomers and precipitates at higher concentrations in solution. Hence, solid-state NMR (ssNMR) was employed as an alternative method to gain structural insights on the precipitated protein.