Riccardo Cadalbert

De Novo 3D Structure Determination from Sub‐milligram Protein Samples by Solid‐State 100 kHz MAS NMR Spectroscopy

Solid-state NMR spectroscopy is an emerging tool for structural studies of crystalline, membrane-associated, sedimented, and fibrillar proteins. A major limitation for many studies is still the large amount of sample needed for the experiments, typically several isotopically labeled samples of 10-20 mg each. Here we show that a new NMR probe, pushing magic-angle sample rotation to frequencies around 100 kHz, makes it possible to narrow the proton resonance lines sufficiently to provide the necessary sensitivity and spectral resolution for efficient and sensitive proton detection. Using restraints from such spectra, a well-defined de novo structure of the model protein ubiquitin was obtained from two samples of roughly 500 μg protein each. This proof of principle opens new avenues for structural studies of proteins available in microgram, or tens of nanomoles, quantities that are, for example, typically achieved for eukaryotic membrane proteins by in-cell or cell-free expression.

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

Despite its central importance for understanding the molecular basis of Alzheimers 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.

Polymorphism in an Amyloid-Like Fibril-Forming Model Peptide†

The conversion of peptides or proteins from their soluble forms into amyloid fibrils is frequently associated with pathological conditions ranging from neurodegenerative disorders to systemic amyloidoses. Although amyloid fibrils and non-disease-associated amyloid-like fibrils can be formed by peptides and proteins that share no sequence identity, they display several common properties. One hallmark of amyloid and amyloid-like fibrils is their highly ordered organization into a laminated cross-b structure, in which the b strands run perpendicular to the long fibril axis. Another characteristic is that the same protein or peptide can form fibrils of different morphologies. It has been suggested that the structural and morphological variability of fibrils is likely to form the molecular basis for the phenomenon of strains, and may play a role in amyloid diseases. Although the basis of amyloid fibril polymorphism is not well understood, there is spectroscopic evidence that it is accompanied by specific changes in the conformation and packing of the individual polypeptide chains. It has been shown that fibril polymorphism can partially be controlled by variation of the growth conditions and that seeds from fibrils with a particular morphology can induce the sample to polymerize into fibrils of the same morphology. Elucidation of the factors that control the polymorphism of amyloid fibrils is therefore of major importance for understanding amyloid and prion diseases at the molecular level. Herein we address the molecular basis of polymorphism using the example of the de novo designed peptide ccb-p as a model system. Previous studies have shown that ccb-p (Ac-SIRELEARIRELELRIG-NH2) adopts a three-stranded a-helical coiled-coil structure in aqueous solution at low temperatures. However, the peptide forms amyloid-like fibrils spontaneously and irreversibly upon raising the temperature. When formed from a solution buffered at pH 7.3, the b strands within the fibrils were shown to assume a laminated cross-b conformation in which the extended b strands form antiparallel b sheets. The b strands were found to be shifted by three amino acid residues from an in-register arrangement (see Figure 1b,d). We denote this arrangement as “+ 3 out-of-register” (+ 3-or). It was suggested that, in addition to the clustering of hydrophobic residues, extensive salt-bridge formation between the charged side chains of Glu and Arg is a stabilizing factor for this arrangement. Therefore, the protonation of the Glu side chains at low pH was suspected to potentially change the register. As a result of its sensitivity to the inverse third power of the internuclear distance, solid-state NMR spectroscopy, and more specifically rotational echo double-resonance (REDOR) experiments, are a powerful tool to unambiguously determine the register of constituent b strands within an amyloid fibril. The distance between the carbonyl carbon atom and the amide nitrogen atom is close to 4.2 ? if two amino acid residues are hydrogen-bonded partners, and larger than 5.5 ? otherwise. If the samples investigated are selectively labeled with a single C and a single N atom and the distance measured is about 4.2 ?, the corresponding register is unambiguously established. To investigate the structure of ccb-p amyloid-like fibrils at the atomic level, differently labeled peptides were prepared. Of particular interest in the following are the results from two compounds: for compound I the N label was located on Ala7, and for compound II on Ile2. Both samples contained, in addition, a C label on the carbonyl of Leu14. Compound I will lead to a strong REDOR effect for a + 3-or antiparallel bsheet structure, known to form at pH 7.3, and sample II for a 2-or arrangement (see Figure 1), which will be shown to form at low pH. Figure 2 shows the REDOR dephasing on fibrils of compound I prepared from solution at different pH values. The dephasing increases with increasing pH in the range from 2.0 to 7.3, indicating an increase of the abundance of the + 3or fibril polymorph, which indeed is the dominant structure at neutral pH. Figure 3 shows the REDOR data obtained from samples of compound II. For samples prepared at low pH values, a strong REDOR effect is visible, attesting the existence of a 2-or structure. The solid lines in Figures 2 and 3 indicate the best fit of the data by a model in which the dephasing is described by a superposition of the + 3-or and the 2-or register dephasing curves. This approach is justified because compound I will [*] Dr. R. Verel, I. T. Tomka, C. Bertozzi, R. Cadalbert, Prof. B. H. Meier Physical Chemistry ETH Zurich, Wolfgang-Pauli-Strasse 10, 8093, Zurich (Switzerland) Fax: (+41)44-632-1621 E-mail: beme@nmr.phys.chem.ethz.ch Homepage: http://www.ssnmr.ethz.ch

Characterization of fibril dynamics on three timescales by solid-state NMR

A multi-timescale analysis of the backbone dynamics of HET-s (218–289) fibrils is described based on multiple site-specific R1 and R1ρ data sets and S2 measurements via REDOR for most backbone 15N and 13Cα nuclei. 15N and 13Cα data are fitted with motions at three timescales. Slow motion is found, indicating a global fibril motion. We further investigate the effect of 13C–13C transfer in measurement of 13Cα R1. Finally, we show that it is necessary to go beyond the Redfield approximation for slow motions in order to obtain accurate numerical values for R1ρ.

Enantiomeric and Diastereoisomeric (Mixed) L/ D-Octaarginine Derivatives – A Simple Way of Modulating the Properties of Cell-Penetrating Peptides†

Cell‐penetrating peptides (CPPs) are promising vehicles for delivery of drugs, antibiotics, proteins, nucleic acid derivatives, etc. into eukaryotic and prokaryotic target cells. To prevent premature degradation, CPPs consisting of D‐ or β‐amino acid residues have been used. We present simple models for the various modes of delivery of physiologically active cargoes by CPPs, depending on the nature of their conjugation (Fig. 1), and we describe the plasma stability of oligoarginines (OAs) 1–4, the most common unnatural CPPs. Fluorescein‐labeled L‐octaarginine 1 was found to have a half‐life (t1/2) of <0.5 min, the D‐enantiomer (2) of >7 d (Fig. 2). For possible medicinal applications, the former type of derivative would be too unstable, and the latter one undesirably persistent. Thus, seven of the 256 possible ‘mixed’ Flua‐L/D‐octaarginine amides, 4a–4g, were synthesized and shown to have half‐lives in heparine‐stabilized human plasma between 8 min and 5.5 h (Figs. 3 and 4). The cell penetration of the new OAs was investigated with ‘healthy’ and with apoptotic HEK cells (Figs. 5–8), and their interactions with phospholipid bilayers were studied, using anionic lipid vesicles (Figs. 9 and 10). There are surprisingly large differences in the rates of cell penetration and binding to vesicle walls between the various stereoisomeric octaarginine derivatives 1, 2, and 4a–4g (Figs. 5 and 7). – The role of D‐amino acids and D‐peptides in nature and in drug design is briefly discussed and referenced.

Microsecond Dynamics in Ubiquitin Probed by Solid-State 15N NMR Spectroscopy R1ρ Relaxation Experiments under Fast MAS (60–110 kHz)

15 N R1ρ relaxation experiments in solid-state NMR spectroscopy are sensitive to timescales and amplitudes of internal protein motions in the hundreds of nano- to microsecond time window, which is difficult to probe by solution-state NMR spectroscopy. By using 15 N R1ρ relaxation experiments, a simplified approach to detect low microsecond protein dynamics is described and residue-specific correlation times are determined from the ratio of 15 N R1ρ rate constants at different magic angle spinning frequencies. Microcrystalline ubiquitin exhibits small-amplitude dynamics on a timescale of about 1 μs across the entire protein, and larger amplitude motions, also on the 1 μs timescale, for several sites, including the β1 -β2 turn and the N terminus of the α helix. According to the analysis, the microsecond protein backbone dynamics are of lower amplitude than that concluded in previous solid-state NMR spectroscopy studies, but persist across the entire protein with a rather uniform timescale of 1 μs.

Partially-deuterated samples of HET-s(218–289) fibrils: assignment and deuterium isotope effect

Fast magic-angle spinning and partial sample deuteration allows direct detection of 1H in solid-state NMR, yielding significant gains in mass sensitivity. In order to further analyze the spectra, 1H detection requires assignment of the 1H resonances. In this work, resonance assignments of backbone HN and Hα are presented for HET-s(218–289) fibrils, based on the existing assignment of Cα, Cβ, C’, and N resonances. The samples used are partially deuterated for higher spectral resolution, and the shifts in resonance frequencies of Cα and Cβ due to the deuterium isotope effect are investigated. It is shown that the deuterium isotope effect can be estimated and used for assigning resonances of deuterated samples in solid-state NMR, based on known resonances of the protonated protein.

Solid-state NMR sequential assignments of the N-terminal domain of HpDnaB helicase

We present solid-state NMR assignments of the N-terminal domain of the DnaB helicase from Helicobacter pylori (153 residues) in its microcrystalline form. We use a sequential resonance assignment strategy based on three-dimensional NMR experiments. The resonance assignments obtained are compared with automated resonance assignments computed with the ssFLYA algorithm. An analysis of the 13C secondary chemical shifts determines the position of the secondary structure elements in this α-helical protein.

Solid-state NMR and EPR Spectroscopy of Mn2+-Substituted ATP-Fueled Protein Engines

Paramagnetic metal ions deliver structural information both in EPR and solid-state NMR experiments, offering a profitable synergetic approach to study bio-macromolecules. We demonstrate the spectral consequences of Mg2+ / Mn2+ substitution and the resulting information contents for two different ATP:Mg2+ -fueled protein engines, a DnaB helicase from Helicobacter pylori active in the bacterial replisome, and the ABC transporter BmrA, a bacterial efflux pump. We show that, while EPR spectra report on metal binding and provide information on the geometry of the metal centers in the proteins, paramagnetic relaxation enhancements identified in the NMR spectra can be used to localize residues at the binding site. Protein engines are ubiquitous and the methods described herein should be applicable in a broad context.

Identification of m‐Mentha‐3(8),6‐diene (Isosylveterpinolene) in Black Pepper Oil

m-Mentha-3(8),6-diene (s isosylveterpinolene; 5), which has not been found in nature before, was shown by GC/MS analysis and co-injection with a synthetic sample to be present to the extent of 0.12 – 0.17% in black and green pepper oil. The synthetic reference 5 was prepared by deconjugation of 3-methylcyclohex-2-en-1-one (1), subsequent alkylation with lithium 2-lithio-2-methylpropanoate, and decarboxylative dehydration of the resulting hydroxycarboxylic acid 3 employing dimethylformamide dimethyl acetal. On polar GC columns, 5 was eluted between p-cymene and terpinolene, and was characterized by an MS similar to that of terpinolene, but with the fragment m/z 121 (59, [M−Me]+) being less intense than that of the molecular ion at m/z 136 (67, M+). Since m-mentha-3(8),6-diene was also found to the extent of 0.24% in the headspace of ground black peppercorns, it can be considered to be of genuine natural origin, and thus is the first established example of a naturally occurring m-menthadiene.