Imino Hydrogen Positions in Nucleic Acids from Density Functional Theory Validated by NMR Residual Dipolar Couplings

Hydrogen atom positions of nucleotide bases in RNA structures solved by X-ray crystallography are commonly derived from heavy-atom coordinates by assuming idealized geometries. In particular, N1–H1 vectors in G and N3–H3 vectors in U are commonly positioned to coincide with the bisectors of their respective heavy-atom angles. We demonstrate that quantum-mechanical optimization of the hydrogen positions relative to their heavy-atom frames considerably improves the fit of experimental residual dipolar couplings to structural coordinates. The calculations indicate that deviations of the imino N–H vectors in RNA U and G bases result from H-bonding within the base pair and are dominated by the attractive interaction between the H atom and the electron density surrounding the H-bond-acceptor atom. DFT optimization of H atom positions is impractical in structural biology studies. We therefore have developed an empirical relation that predicts imino N–H vector orientations from the heavy-atom coordinates of the base pair. This relation agrees very closely with the DFT results, permitting its routine application in structural studies

Impact of N-Terminal Acetylation of α-Synuclein on Its Random Coil and Lipid Binding Properties

N-Terminal acetylation of α-synuclein (aS), a protein implicated in the etiology of Parkinson’s disease, is common in mammals. The impact of this modification on the protein’s structure and dynamics in free solution and on its membrane binding properties has been evaluated by high-resolution nuclear magnetic resonance and circular dichroism (CD) spectroscopy. While no tetrameric form of acetylated aS could be isolated, N-terminal acetylation resulted in chemical shift perturbations of the first 12 residues of the protein that progressively decreased with the distance from the N-terminus. The directions of the chemical shift changes and small changes in backbone ³<i>J</i>_HH couplings are consistent with an increase in the α-helicity of the first six residues of aS, although a high degree of dynamic conformational disorder remains and the helical structure is sampled <20% of the time. Chemical shift and ³<i>J</i>_HH data for the intact protein are virtually indistinguishable from those recorded for the corresponding N-terminally acetylated and nonacetylated 15-residue synthetic peptides. An increase in α-helicity at the N-terminus of aS is supported by CD data on the acetylated peptide and by weak medium-range nuclear Overhauser effect contacts indicative of α-helical character. The remainder of the protein has chemical shift values that are very close to random coil values and indistinguishable between the two forms of the protein. No significant differences in the fibrillation kinetics were observed between acetylated and nonacetylated aS. However, the lipid binding properties of aS are strongly impacted by acetylation and exhibit distinct behavior for the first 12 residues, indicative of an initiation role for the N-terminal residues in an “initiation–elongation” process of binding to the membrane

Nuclear Magnetic Resonance Observation of α‑Synuclein Membrane Interaction by Monitoring the Acetylation Reactivity of Its Lysine Side Chains

The interaction between α-synuclein (αS) protein and lipid membranes is key to its role in synaptic vesicle homeostasis and plays a role in initiating fibril formation, which is implicated in Parkinson’s disease. The natural state of αS inside the cell is generally believed to be intrinsically disordered, but chemical cross-linking experiments provided evidence of a tetrameric arrangement, which was reported to be rich in α-helical secondary structure based on circular dichroism (CD). Cross-linking relies on chemical modification of the protein’s Lys C^ε amino groups, commonly by glutaraldehyde, or by disuccinimidyl glutarate (DSG), with the latter agent preferred for cellular assays. We used ultra-high-resolution homonuclear decoupled nuclear magnetic resonance experiments to probe the reactivity of the 15 αS Lys residues toward <i>N</i>-succinimidyl acetate, effectively half the DSG cross-linker, which results in acetylation of Lys. The intensities of both side chain and backbone amide signals of acetylated Lys residues provide direct information about the reactivity, showing a difference of a factor of 2.5 between the most reactive (K6) and the least reactive (K102) residue. The presence of phospholipid vesicles decreases reactivity of most Lys residues by up to an order of magnitude at high lipid:protein stoichiometries (500:1), but only weakly at low ratios. The decrease in Lys reactivity is found to be impacted by lipid composition, even for vesicles that yield similar αS CD signatures. Our data provide new insight into the αS–bilayer interaction, including the pivotal state in which the available lipid surface is limited. Protection of Lys C^ε amino groups by αS–bilayer interaction will strongly impact quantitative interpretation of DSG cross-linking experiments

Side Chain Conformational Distributions of a Small Protein Derived from Model-Free Analysis of a Large Set of Residual Dipolar Couplings

Accurate quantitative measurement of structural dispersion in proteins remains a prime challenge to both X-ray crystallography and NMR spectroscopy. Here we use a model-free approach based on measurement of many residual dipolar couplings (RDCs) in differentially orienting aqueous liquid crystalline solutions to obtain the side chain χ₁ distribution sampled by each residue in solution. Applied to the small well-ordered model protein GB3, our approach reveals that the RDC data are compatible with a single narrow distribution of side chain χ₁ angles for only about 40% of the residues. For more than half of the residues, populations greater than 10% for a second rotamer are observed, and four residues require sampling of three rotameric states to fit the RDC data. In virtually all cases, sampled χ₁ values are found to center closely around ideal <i>g</i>^–, <i>g</i>⁺ and <i>t</i> rotameric angles, even though no rotamer restraint is used when deriving the sampled angles. The root-mean-square difference between experimental ³J_HαHβ couplings and those predicted by the Haasnoot-parametrized, motion-adjusted Karplus equation reduces from 2.05 to 0.75 Hz when using the new rotamer analysis instead of the 1.1-Å X-ray structure as input for the dihedral angles. A comparison between observed and predicted ³J_HαHβ values suggests that the root-mean-square amplitude of χ₁ angle fluctuations within a given rotamer well is ca. 20°. The quantitatively defined side chain rotamer equilibria obtained from our study set new benchmarks for evaluating improved molecular dynamics force fields, and also will enable further development of quantitative relations between side chain chemical shift and structure

Monomeric Aβ^1–40 and Aβ^1–42 Peptides in Solution Adopt Very Similar Ramachandran Map Distributions That Closely Resemble Random Coil

The pathogenesis of Alzheimer’s disease is characterized by the aggregation and fibrillation of amyloid peptides Aβ^1–40 and Aβ^1–42 into amyloid plaques. Despite strong potential therapeutic interest, the structural pathways associated with the conversion of monomeric Aβ peptides into oligomeric species remain largely unknown. In particular, the higher aggregation propensity and associated toxicity of Aβ^1–42 compared to that of Aβ^1–40 are poorly understood. To explore in detail the structural propensity of the monomeric Aβ^1–40 and Aβ^1–42 peptides in solution, we recorded a large set of nuclear magnetic resonance (NMR) parameters, including chemical shifts, nuclear Overhauser effects (NOEs), and <i>J</i> couplings. Systematic comparisons show that at neutral pH the Aβ^1–40 and Aβ^1–42 peptides populate almost indistinguishable coil-like conformations. Nuclear Overhauser effect spectra collected at very high resolution remove assignment ambiguities and show no long-range NOE contacts. Six sets of backbone <i>J</i> couplings (³<i>J</i>_HNHα, ³<i>J</i>_C′C′, ³<i>J</i>_C′Hα, ¹<i>J</i>_HαCα, ²<i>J</i>_NCα, and ¹<i>J</i>_NCα) recorded for Aβ^1–40 were used as input for the recently developed MERA Ramachandran map analysis, yielding residue-specific backbone ϕ/ψ torsion angle distributions that closely resemble random coil distributions, the absence of a significantly elevated propensity for β-conformations in the C-terminal region of the peptide, and a small but distinct propensity for α_L at K28. Our results suggest that the self-association of Aβ peptides into toxic oligomers is not driven by elevated propensities of the monomeric species to adopt β-strand-like conformations. Instead, the accelerated disappearance of Aβ NMR signals in D₂O over H₂O, particularly pronounced for Aβ^1–42, suggests that intermolecular interactions between the hydrophobic regions of the peptide dominate the aggregation process