33 research outputs found
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, N1âH1 vectors
in G and N3âH3 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 NâH 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
NâH 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
Liquid Crystalline Phase of G-Tetrad DNA for NMR Study of Detergent-Solubilized Proteins
Liquid Crystalline Phase of G-Tetrad DNA for NMR Study of Detergent-Solubilized Protein
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 Parkinsonâs disease, is common in mammals.
The impact of this modification on the proteinâs 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 <sup>3</sup><i>J</i><sub>HH</sub> 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 <sup>3</sup><i>J</i><sub>HH</sub> 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
âinitiationâelongationâ process of binding to
the membrane
Accurate Measurement of Residual Dipolar Couplings in Large RNAs by Variable Flip Angle NMR
NMR approaches using
nucleotide-specific deuterium labeling schemes
have enabled structural studies of biologically relevant RNAs of increasing
size and complexity. Although local structure is well-determined using
these methods, definition of global structural features, including
relative orientations of independent helices, remains a challenge.
Residual dipolar couplings, a potential source of orientation information,
have not been obtainable for large RNAs due to poor sensitivity resulting
from rapid heteronuclear signal decay. Here we report a novel multiple
quantum NMR method for RDC determination that employs flip angle variation
rather than a coupling evolution period. The accuracy of the method
and its utility for establishing interhelical orientations are demonstrated
for a 36-nucleotide RNA, for which comparative data could be obtained.
Applied to a 78 kDa Rev response element from the HIV-1 virus, which
has an effective rotational correlation time of ca. 160 ns, the method
yields sensitivity gains of an order of magnitude or greater over
existing approaches. Solution-state access to structural organization
in RNAs of at least 230 nucleotides is now possible
Accurate Measurement of Residual Dipolar Couplings in Large RNAs by Variable Flip Angle NMR
NMR approaches using
nucleotide-specific deuterium labeling schemes
have enabled structural studies of biologically relevant RNAs of increasing
size and complexity. Although local structure is well-determined using
these methods, definition of global structural features, including
relative orientations of independent helices, remains a challenge.
Residual dipolar couplings, a potential source of orientation information,
have not been obtainable for large RNAs due to poor sensitivity resulting
from rapid heteronuclear signal decay. Here we report a novel multiple
quantum NMR method for RDC determination that employs flip angle variation
rather than a coupling evolution period. The accuracy of the method
and its utility for establishing interhelical orientations are demonstrated
for a 36-nucleotide RNA, for which comparative data could be obtained.
Applied to a 78 kDa Rev response element from the HIV-1 virus, which
has an effective rotational correlation time of ca. 160 ns, the method
yields sensitivity gains of an order of magnitude or greater over
existing approaches. Solution-state access to structural organization
in RNAs of at least 230 nucleotides is now possible
Accurate Measurement of Residual Dipolar Couplings in Large RNAs by Variable Flip Angle NMR
NMR approaches using
nucleotide-specific deuterium labeling schemes
have enabled structural studies of biologically relevant RNAs of increasing
size and complexity. Although local structure is well-determined using
these methods, definition of global structural features, including
relative orientations of independent helices, remains a challenge.
Residual dipolar couplings, a potential source of orientation information,
have not been obtainable for large RNAs due to poor sensitivity resulting
from rapid heteronuclear signal decay. Here we report a novel multiple
quantum NMR method for RDC determination that employs flip angle variation
rather than a coupling evolution period. The accuracy of the method
and its utility for establishing interhelical orientations are demonstrated
for a 36-nucleotide RNA, for which comparative data could be obtained.
Applied to a 78 kDa Rev response element from the HIV-1 virus, which
has an effective rotational correlation time of ca. 160 ns, the method
yields sensitivity gains of an order of magnitude or greater over
existing approaches. Solution-state access to structural organization
in RNAs of at least 230 nucleotides is now possible
Nuclear Magnetic Resonance Observation of 뱉Synuclein 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 Parkinsonâs 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 proteinâs Lys C<sup>Δ</sup> 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 αSâbilayer interaction, including
the pivotal state in which the available lipid surface is limited.
Protection of Lys C<sup>Δ</sup> amino groups by αSâbilayer
interaction will strongly impact quantitative interpretation of DSG
cross-linking experiments
Monomeric α-Synuclein Binds Congo Red Micelles in a Disordered Manner
The histological dye Congo Red (CR) previously has been shown to inhibit α-synuclein (aS) fibrillation, but the mode of this inhibition remained unclear. Because of favorable exchange kinetics, interaction between CR and aS lends itself to a detailed nuclear magnetic resonance study, and relaxation dispersion measurements yield the bound fraction and time scales for the interaction of aS with CR. We find that at pH 6, CR exists as a micelle, and at a CR:aS molar ratio of âŒ1, only a small fraction of aS (âŒ2%) is bound to these micelles. Rapid exchange (<i>k</i><sub>ex</sub> ⌠3000 s<sup>â1</sup>) between the free and CR-bound states broadens and strongly attenuates resonances of aS by two processes: a magnetic field-dependent contribution, caused by the chemical shift difference between the two states, and a nearly field-independent contribution caused by slower tumbling of aS bound to the CR micelle. The salt dependence of the interaction suggests a predominantly electrostatic mechanism for the 60 N-terminal residues, while the weaker interaction between residues 61â100 and CR is mostly hydrophobic. Chemical shift and transferred NOE data indicate that aS becomes slightly more helical but remains largely disordered when bound to CR. Results indicate that inhibition of fibril formation does not result from binding of CR to free aS and, therefore, must result from interaction of aS fibrils or protofibrils with CR micelles
Accurate Measurement of Residual Dipolar Couplings in Large RNAs by Variable Flip Angle NMR
NMR approaches using
nucleotide-specific deuterium labeling schemes
have enabled structural studies of biologically relevant RNAs of increasing
size and complexity. Although local structure is well-determined using
these methods, definition of global structural features, including
relative orientations of independent helices, remains a challenge.
Residual dipolar couplings, a potential source of orientation information,
have not been obtainable for large RNAs due to poor sensitivity resulting
from rapid heteronuclear signal decay. Here we report a novel multiple
quantum NMR method for RDC determination that employs flip angle variation
rather than a coupling evolution period. The accuracy of the method
and its utility for establishing interhelical orientations are demonstrated
for a 36-nucleotide RNA, for which comparative data could be obtained.
Applied to a 78 kDa Rev response element from the HIV-1 virus, which
has an effective rotational correlation time of ca. 160 ns, the method
yields sensitivity gains of an order of magnitude or greater over
existing approaches. Solution-state access to structural organization
in RNAs of at least 230 nucleotides is now possible
Whole-Body Rocking Motion of a Fusion Peptide in Lipid Bilayers from Size-Dispersed <sup>15</sup>N NMR Relaxation
Biological membranes present a highly fluid environment, and integration of proteins within such membranes is itself highly dynamic: proteins diffuse laterally within the plane of the membrane and rotationally about the normal vector of this plane. We demonstrate that whole-body motions of proteins within a lipid bilayer can be determined from NMR <sup>15</sup>N relaxation rates collected for different-sized bicelles. The importance of membrane integration and interaction is particularly acute for proteins and peptides that function on the membrane itself, as is the case for pore-forming and fusion-inducing proteins. For the influenza hemagglutinin fusion peptide, which lies on the surface of membranes and catalyzes the fusion of membranes and vesicles, we found large-amplitude, rigid-body wobbling motions on the nanosecond time scale relative to the lipid bilayer. This behavior complements prior analyses where data were commonly interpreted in terms of a static oblique angle of insertion for the fusion peptide with respect to the membrane. Quantitative disentanglement of the relative motions of two interacting objects by systematic variation of the size of one is applicable to a wide range of systems beyond proteinâmembrane interactions