Mass Determination of Entire Amyloid Fibrils by Using Mass Spectrometry

Amyloid fibrils are self‐assembled protein structures with important roles in biology (either pathogenic or physiological), and are attracting increasing interest in nanotechnology. However, because of their high aspect ratio and the presence of some polymorphism, that is, the possibility to adopt various structures, their characterization is challenging and basic information such as their mass is unknown. Here we show that charge‐detection mass spectrometry, recently developed for large self‐assembled systems such as viruses, provides such information in a straightforward manner.     

Unique Identification of Supramolecular Structures in Amyloid Fibrils by Solid‐State NMR Spectroscopy

The fibril structure formed by the amyloidogenic fragment SNNFGAILSS of the human islet amyloid polypeptide (hIAPP) is determined with 0.52 Å resolution. Symmetry information contained in the easily obtainable resonance assignments from solid‐state NMR spectra (see picture), along with long‐range constraints, can be applied to uniquely identify the supramolecular organization of fibrils.

     

Molecular Structure of Amyloid Fibrils Formed by Residues 127 to 147 of the Human Prion Protein

Amyloid fibrils are filamentous and insoluble forms of peptides or proteins. Proline has long been considered to be incompatible with the cross‐β structural motif of amyloid fibrils. On the basis of solid‐state NMR spectroscopy data, we present a structural model of an in‐register parallel β sheet for the amyloid fibrils formed from a human prion protein fragment, huPrP_127–47. We have developed a simple solid‐state NMR spectroscopy technique to identify solvent‐protected backbone amide protons in a H/D exchange experiment without disaggregating the amyloid fibrils, from which we find that proline residue P₁₃₇ does not disrupt the β‐sheet structure from G₁₂₇ to G₁₄₂. We suggest that the resultant kink at P₁₃₇ generates a twist between adjacent peptide strands to maintain hydrogen bonding in the β‐sheet regions flanking the P₁₃₇ residue. Although proline can be well integrated into the cross‐β structure of amyloid fibrils, the kink formed at the position of the proline residue will considerably weaken the hydrogen bonding between the neighboring strands, especially when the mutation site is near the central region of a β sheet.     

A Non‐damaging Method to Analyze the Configuration and Dynamics of Nitrotyrosines in Proteins

Often, deregulation of protein activity and turnover by tyrosine nitration drives cells toward pathogenesis. Hence, understanding how the nitration of a protein affects both its function and stability is of outstanding interest. Nowadays, most of the in vitro analyses of nitrated proteins rely on chemical treatment of native proteins with an excess of a chemical reagent. One such reagent, peroxynitrite, stands out for its biological relevance. However, given the excess of the nitrating reagent, the resulting in vitro modification could differ from the physiological nitration. Here, we determine unequivocally the configuration of distinct nitrated‐tyrosine rings in single‐tyrosine mutants of cytochrome c. We aimed to confirm the nitration position by a non‐destructive method. Thus, we have resorted to ¹H‐¹⁵N heteronuclear single quantum coherence(HSQC) spectra to identify the ³J(N? H) correlation between a ¹⁵N‐tagged nitro group and the adjacent aromatic proton. Once the chemical shift of this proton was determined, we compared the ¹H‐¹³C HSQC spectra of untreated and nitrated samples. All tyrosines were nitrated at ε positions, in agreement to previous analysis by indirect techniques. Notably, the various nitrotyrosine residues show a different dynamic behaviour that is consistent with molecular dynamics computations.     

A Detailed Analysis of the Morphology of Fibrils of Selectively Mutated Amyloid β (1–40)

A small library of rationally designed amyloid β [Aβ(1–40)] peptide variants is generated, and the morphology of their fibrils is studied. In these molecules, the structurally important hydrophobic contact between phenylalanine 19 (F19) and leucine 34 (L34) is systematically mutated to introduce defined physical forces to act as specific internal constraints on amyloid formation. This Aβ(1–40) peptide library is used to study the fibril morphology of these variants by employing a comprehensive set of biophysical techniques including solution and solid‐state NMR spectroscopy, AFM, fluorescence correlation spectroscopy, and XRD. Overall, the findings demonstrate that the introduction of significant local physical perturbations of a crucial early folding contact of Aβ(1–40) only results in minor alterations of the fibrillar morphology. The thermodynamically stable structure of mature Aβ fibrils proves to be relatively robust against the introduction of significantly altered molecular interaction patterns due to point mutations. This underlines that amyloid fibril formation is a highly generic process in protein misfolding that results in the formation of the thermodynamically most stable cross‐β structure.     

NMR Spectroscopic Assignment of Backbone and Side‐Chain Protons in Fully Protonated Proteins: Microcrystals,Sedimented Assemblies,and Amyloid Fibrils

We demonstrate sensitive detection of alpha protons of fully protonated proteins by solid‐state NMR spectroscopy with 100–111 kHz magic‐angle spinning (MAS). The excellent resolution in the Cα‐Hα plane is demonstrated for 5 proteins, including microcrystals, a sedimented complex, a capsid and amyloid fibrils. A set of 3D spectra based on a Cα–Hα detection block was developed and applied for the sequence‐specific backbone and aliphatic side‐chain resonance assignment using only 500 μg of sample. These developments accelerate structural studies of biomolecular assemblies available in submilligram quantities without the need of protein deuteration.     

Probing Polyoxometalate–Protein Interactions Using Molecular Dynamics Simulations

The molecular interactions between the Ce^IV‐substituted Keggin anion [PW₁₁O₃₉Ce(OH₂)₄]^3? ( CeK ) and hen egg‐white lysozyme (HEWL) were investigated by molecular dynamics simulations. The analysis of CeK was compared with the Ce^IV‐substituted Keggin dimer [(PW₁₁O₃₉)₂Ce]^10? ( CeK₂ ) and the Zr^IV‐substituted Lindqvist anion [W₅O₁₈Zr(OH₂)(OH)]^3? ( ZrL ) to understand how POM features such as shape, size, charge, or type of incorporated metal ion influence the POM???protein interactions. Simulations revealed two regions of the protein in which the CeK anion interacts strongly: cationic sites formed by Arg21 and by Arg45 and Arg68. The POMs chiefly interact with the side chains of the positively charged (arginines, lysines) and the polar uncharged residues (tyrosines, serines, aspargines) via electrostatic attraction and hydrogen bonding with the oxygen atoms of the POM framework. The CeK anion shows higher protein affinity than the CeK₂ and ZrL anions, because it is less hydrophilic and it has the right size and shape for establishing interactions with several residues simultaneously. The larger, more negatively charged CeK₂ anion has a high solvent‐accessible surface, which is sub‐optimal for the interaction, while the smaller ZrL anion is highly hydrophilic and cannot efficiently interact with several residues simultaneously.     

Probing the Interactions of Porphyrins with Macromolecules Using NMR Spectroscopy Techniques

Porphyrinic compounds are widespread in nature and play key roles in biological processes such as oxygen transport in blood, enzymatic redox reactions or photosynthesis. In addition, both naturally derived as well as synthetic porphyrinic compounds are extensively explored for biomedical and technical applications such as photodynamic therapy (PDT) or photovoltaic systems, respectively. Their unique electronic structures and photophysical properties make this class of compounds so interesting for the multiple functions encountered. It is therefore not surprising that optical methods are typically the prevalent analytical tool applied in characterization and processes involving porphyrinic compounds. However, a wealth of complementary information can be obtained from NMR spectroscopic techniques. Based on the advantage of providing structural and dynamic information with atomic resolution simultaneously, NMR spectroscopy is a powerful method for studying molecular interactions between porphyrinic compounds and macromolecules. Such interactions are of special interest in medical applications of porphyrinic photosensitizers that are mostly combined with macromolecular carrier systems. The macromolecular surrounding typically stabilizes the encapsulated drug and may also modify its physical properties. Moreover, the interaction with macromolecular physiological components needs to be explored to understand and control mechanisms of action and therapeutic efficacy. This review focuses on such non-covalent interactions of porphyrinic drugs with synthetic polymers as well as with biomolecules such as phospholipids or proteins. A brief introduction into various NMR spectroscopic techniques is given including chemical shift perturbation methods, NOE enhancement spectroscopy, relaxation time measurements and diffusion-ordered spectroscopy. How these NMR tools are used to address porphyrin–macromolecule interactions with respect to their function in biomedical applications is the central point of the current review.     

Atomic‐Resolution Three‐Dimensional Structure of Amyloid β Fibrils Bearing the Osaka Mutation

Despite its central importance for understanding the molecular basis of Alzheimer's disease (AD), high‐resolution structural information on amyloid β‐peptide (Aβ) fibrils, which are intimately linked with AD, is scarce. We report an atomic‐resolution fibril structure of the Aβ1‐40 peptide with the Osaka mutation (E22Δ), associated with early‐onset AD. The structure, which differs substantially from all previously proposed models, is based on a large number of unambiguous intra‐ and intermolecular solid‐state NMR distance restraints.     

Probing the Limits of Selectivity in a Recognition‐Mediated Reaction Network Embedded within a Dynamic Covalent Library

Two recognition‐mediated reaction processes operating through reactive binary complexes drive resolution of a 24‐component dynamic covalent library, assembled from individual aldehydes and nucleophiles. The effectiveness of the library resolution and selective amplification of one recognition‐enabled species over another is limited by the difference in the rates of the recognition‐mediated reactive processes and the strength of the recognition processes employed in the dynamic system.     

Novel zwitterionic reverse micelles for encapsulation of proteins in low-viscosity media

Large proteins remain inaccessible to structural NMR studies because of their unfavorable relaxation properties. Their solubilization in the aqueous core of reverse micelles, in a low-viscosity medium, represents a promising approach, provided that their native tertiary structure is maintained. However, the use of classical ionic surfactants may lead to protein unfolding, due to strong electrostatic interactions between the polar head groups and the protein charges. To design reverse micelles in which these interactions are weakened, a new zwitterionic surfactant molecule was synthesized and studied by high-resolution NMR spectroscopy, for which cytochrome C and 15N-labeled ubiquitin were used as guest candidates. At different ionization states, both proteins are encapsulated in the absence of salts or other additives, in a folded conformation similar to the native one.     

Conformational Dynamics in the Core of Human Y145Stop Prion Protein Amyloid Probed by Relaxation Dispersion NMR

Microsecond to millisecond timescale backbone dynamics of the amyloid core residues in Y145Stop human prion protein (PrP) fibrils were investigated by using ¹⁵N rotating frame (R_1ρ) relaxation dispersion solid-state nuclear magnetic resonance spectroscopy over a wide range of spin-lock fields. Numerical simulations enabled the experimental relaxation dispersion profiles for most of the fibril core residues to be modelled by using a two-state exchange process with a common exchange rate of 1000 s⁻¹, corresponding to protein backbone motion on the timescale of 1 ms, and an excited-state population of 2 %. We also found that the relaxation dispersion profiles for several amino acids positioned near the edges of the most structured regions of the amyloid core were better modelled by assuming somewhat higher excited-state populations (∼5–15 %) and faster exchange rate constants, corresponding to protein backbone motions on the timescale of ∼100–300 μs. The slow backbone dynamics of the core residues were evaluated in the context of the structural model of human Y145Stop PrP amyloid.