Ivan V. Sergeyev

Atomic Resolution Structure of Monomorphic Aβ42 Amyloid Fibrils

Amyloid-β (Aβ) is a 39-42 residue protein produced by the cleavage of the amyloid precursor protein (APP), which subsequently aggregates to form cross-β amyloid fibrils that are a hallmark of Alzheimers disease (AD). The most prominent forms of Aβ are Aβ1-40 and Aβ1-42, which differ by two amino acids (I and A) at the C-terminus. However, Aβ42 is more neurotoxic and essential to the etiology of AD. Here, we present an atomic resolution structure of a monomorphic form of AβM01-42 amyloid fibrils derived from over 500 (13)C-(13)C, (13)C-(15)N distance and backbone angle structural constraints obtained from high field magic angle spinning NMR spectra. The structure (PDB ID: 5KK3 ) shows that the fibril core consists of a dimer of Aβ42 molecules, each containing four β-strands in a S-shaped amyloid fold, and arranged in a manner that generates two hydrophobic cores that are capped at the end of the chain by a salt bridge. The outer surface of the monomers presents hydrophilic side chains to the solvent. The interface between the monomers of the dimer shows clear contacts between M35 of one molecule and L17 and Q15 of the second. Intermolecular (13)C-(15)N constraints demonstrate that the amyloid fibrils are parallel in register. The RMSD of the backbone structure (Q15-A42) is 0.71 ± 0.12 Å and of all heavy atoms is 1.07 ± 0.08 Å. The structure provides a point of departure for the design of drugs that bind to the fibril surface and therefore interfere with secondary nucleation and for other therapeutic approaches to mitigate Aβ42 aggregation.

Solvation and aggregation of n,n'-dialkylimidazolium ionic liquids: a multinuclear NMR spectroscopy and molecular dynamics simulation study.

The solvation and aggregation of the ionic liquid (IL) 1-n-butyl-3-methylimidazolium chloride ([C4mim]Cl) in water and dimethylsulfoxide (DMSO) were examined by analysis of (1)H and (35/37)Cl chemical shift perturbations and molecular dynamics (MD) simulations. Evidence of aggregation of the IL n-butyl chains in aqueous environments at IL concentrations of 75-80 wt% was observed both in the NMR experiments and in the MD simulations. The studies also show that [C4mim]Cl behaves as a typical electrolyte in water, with both ions completely solvated at low concentrations. On the other hand, the data reveal that the interactions between the [C4mim](+) and Cl(-) ions strengthen as the DMSO content of the solutions increases, and IL-rich clusters persist in this solvent even at concentrations below 10 wt%. These results provide an experimentally supported atomistic explanation of the effects that these two solvents have on some of the macroscopic properties of [C4mim]Cl. The implications that these findings could have on the design of IL-based solvent systems are briefly discussed.

Efficient assignment and NMR analysis of an intact virus using sequential side-chain correlations and DNP sensitization

Significance This work presents a technique for dynamic nuclear polarization (DNP)-enhanced magic-angle spinning (MAS) solid-state NMR studies of complex proteins and biological assemblies. The sequential side-chain correlation approach streamlines the site-specific assignment of NMR peaks in multidimensional spectra, a critical step in determining structural information such as distances. When combined with DNP enhancement, fast MAS, and nonuniform sampling, this technique allows for faster data acquisition than previously possible. Applied to the intact Pf1 bacteriophage, sequential side-chain correlation spectra have enabled a virtually complete assignment using DNP data alone. These assignments shed insight into the chemical shift and linewidth changes associated with cryogenic temperatures. Our data point to hydration as a key variable influencing these parameters. An experimental strategy has been developed to increase the efficiency of dynamic nuclear polarization (DNP) in solid-state NMR studies. The method makes assignments simpler, faster, and more reliable via sequential correlations of both side-chain and Cα resonances. The approach is particularly suited to complex biomolecules and systems with significant chemical-shift degeneracy. It was designed to overcome the spectral congestion and line broadening that occur due to sample freezing at the cryogenic temperatures required for DNP. Nonuniform sampling (NUS) is incorporated to achieve time-efficient collection of multidimensional data. Additionally, fast (25 kHz) magic-angle spinning (MAS) provides optimal sensitivity and resolution. Data collected in <1 wk produced a virtually complete de novo assignment of the coat protein of Pf1 virus. The peak positions and linewidths for samples near 100 K are perturbed relative to those near 273 K. These temperature-induced perturbations are strongly correlated with hydration surfaces.

Cholesterol-binding site of the influenza M2 protein in lipid bilayers from solid-state NMR

Significance Cholesterol is important for membrane protein function, but cholesterol-binding structures of membrane proteins are difficult to determine by X-ray crystallography and electron microscopy due to the small size and dynamic nature of cholesterol. We have developed a solid-state NMR approach to determine the cholesterol-binding structure of membrane proteins in lipid bilayers. Applied to the influenza M2 protein, the measured interatomic distances and cholesterol orientational angles indicate that cholesterol binds M2 in a substoichiometric fashion, flanking methyl-rich transmembrane (TM) residues near an amphipathic helix, without requiring a cholesterol recognition sequence motif, and this substoichiometric binding uniquely correlates with membrane curvature generation. These results give unprecedented insights into how cholesterol clusters M2 to the neck of the budding virus to mediate membrane scission. The influenza M2 protein not only forms a proton channel but also mediates membrane scission in a cholesterol-dependent manner to cause virus budding and release. The atomic interaction of cholesterol with M2, as with most eukaryotic membrane proteins, has long been elusive. We have now determined the cholesterol-binding site of the M2 protein in phospholipid bilayers using solid-state NMR spectroscopy. Chain-fluorinated cholesterol was used to measure cholesterol proximity to M2 while sterol-deuterated cholesterol was used to measure bound-cholesterol orientation in lipid bilayers. Carbon–fluorine distance measurements show that at a cholesterol concentration of 17 mol%, two cholesterol molecules bind each M2 tetramer. Cholesterol binds the C-terminal transmembrane (TM) residues, near an amphipathic helix, without requiring a cholesterol recognition sequence motif. Deuterium NMR spectra indicate that bound cholesterol is approximately parallel to the bilayer normal, with the rough face of the sterol rings apposed to methyl-rich TM residues. The distance- and orientation-restrained cholesterol-binding site structure shows that cholesterol is stabilized by hydrophobic interactions with the TM helix and polar and aromatic interactions with neighboring amphipathic helices. At the 1:2 binding stoichiometry, lipid 31P spectra show an isotropic peak indicative of high membrane curvature. This M2–cholesterol complex structure, together with previously observed M2 localization at phase boundaries, suggests that cholesterol mediates M2 clustering to the neck of the budding virus to cause the necessary curvature for membrane scission. The solid-state NMR approach developed here is generally applicable for elucidating the structural basis of cholesterol’s effects on membrane protein function.

Dynamic Nuclear Polarization Signal Enhancement with High-Affinity Biradical Tags

Dynamic nuclear polarization is an emerging technique for sensitizing solid-state NMR experiments by transferring polarization from electrons to nuclei. Stable biradicals, the polarization source for the cross effect mechanism, are typically codissolved at millimolar concentrations with proteins of interest. Here we describe the high-affinity biradical tag TMP-T, created by covalently linking trimethoprim, a nanomolar affinity ligand of dihydrofolate reductase (DHFR), to the biradical polarizing agent TOTAPOL. With TMP-T bound to DHFR, large enhancements of the protein spectrum are observed, comparable to when TOTAPOL is codissolved with the protein. In contrast to TOTAPOL, the tight binding TMP-T can be added stoichiometrically at radical concentrations orders of magnitude lower than in previously described preparations. Benefits of the reduced radical concentration include reduced spectral bleaching, reduced chemical perturbation of the sample, and the ability to selectively enhance signals for the protein of interest.

NMR Signal Quenching from Bound Biradical Affinity Reagents in DNP Samples

We characterize the effect of specifically bound biradicals on the NMR spectra of dihydrofolate reductase from E. coli. Dynamic nuclear polarization methods enhance the signal-to-noise of solid state NMR experiments by transferring polarization from unpaired electrons of biradicals to nuclei. There has been recent interest in colocalizing the paramagnetic polarizing agents with the analyte of interest through covalent or noncovalent specific interactions. This experimental approach broadens the scope of dynamic nuclear polarization methods by offering the possibility of selective signal enhancements and the potential to work in a broad range of environments. Paramagnetic compounds can have other effects on the NMR spectroscopy of nearby nuclei, including broadening of nuclear resonances due to the proximity of the paramagnetic agent. Understanding the distance dependence of these interactions is important for the success of the technique. Here we explore paramagnetic signal quenching due to a bound biradical, specifically a biradical-derivatized trimethoprim ligand of E. coli dihydrofolate reductase. Biradical-derivatized trimethoprim has nanomolar affinity for its target, and affords strong and selective signal enhancements in dynamic nuclear polarization experiments. In this work, we show that, although the trimethoprim fragment is well ordered, the biradical (TOTAPOL) moiety is disordered when bound to the protein. The distance dependence in bleaching of NMR signal intensity allows us to detect numerous NMR signals in the protein. We present the possibility that static disorder and electron spin diffusion play roles in this observation, among other contributions. The fact that the majority of signals are observed strengthens the case for the use of high affinity or covalent radicals in dynamic nuclear polarization solid state NMR enhancement.

Strategies for Efficient Sample Preparation for Dynamic Nuclear Polarization Solid-State NMR of Biological Macromolecules

Solid-state NMR (SSNMR) is a powerful tool for the elucidation of structure and dynamics in biological macromolecules. Over the years, SSNMR spectroscopists have developed an array of techniques enabling the measurement of internuclear correlations, distances, and torsional angles; these have been applied to the study of a number of biological systems that are difficult to study by X-ray crystallography and solution NMR, including key biological targets such as membrane proteins and amyloid fibrils. Applications of SSNMR to other topic areas, including materials science, pharmaceuticals, and small molecules, have also flourished in recent years. These studies, however, have always been hampered by the low inherent sensitivity of SSNMR, requiring large amounts of both sample and time for data collection. By taking advantage of unpaired electrons doped into a sample as a ready source of additional nuclear polarization, dynamic nuclear polarization (DNP) has brought about large improvements in SSNMR sensitivity. These, in turn, have enabled structural studies of previously inaccessible targets, such as large protein complexes, nucleic acids, viral capsids, and membrane proteins in vivo. Herein, we focus on sample preparation strategies and considerations for scientists interested in applying DNP to challenging systems.