Bolalipid membrane structure revealed by solid-state 2H NMR spectroscopy

Membranes made from three specifically deuterium-labeled ether-linked bolalipids, [1',1',20',20'-2H4]C20BAS-PC, [2',2',19',19'-2H4]C20BAS-PC, or [10',11'-2H2]C20BAS-PC, were analyzed by 2H NMR spectroscopy. Unlike more common monopolar, ester-linked phospholipids, C20BAS-PC exhibits a high degree of orientational order throughout the membrane and the sn-1 chain of the lipid initially penetrates the bilayer at an orientation different from that of the bilayer normal, resulting in inequivalent deuterium atoms at the C1 position. The approximate hydrophobic layer thickness and area per lipid are 18.4 A and 60.4 A2, respectively, at 25 degrees C, and their respective thermal expansion coefficients are within 20% of the monopolar phospholipid, DLPC.     

Rotor modulations and recoupling strategies in 13C solid-state magic-angle-spinning NMR spectroscopy: probing molecular orientation and dynamics.

Recoupling strategies for anisotropic interactions enable the investigation of molecular structure, order and dynamics in a sensitive and site-specific fashion by solid-state NMR spectroscopy. Whereas magic-angle spinning (MAS) efficiently averages anisotropic interactions and enhances spectral resolution, recoupling pulse sequences selectively restore certain parts of rotor-modulated dipole-dipole couplings or chemical shift anisotropies (CSA). More specifically, it is possible to recouple either the omegaR- or the 2omegaR-modulated terms of an interaction Hamiltonian, which exhibit different orientation dependencies and, in this way, provide a means of distinguishing whether the observed NMR spectra are affected by molecular motion or by molecular orientation. Sideband patterns generated by reconversion rotor encoding allow for a precise and selective determination of coupling constants and anisotropies, which contain site-specific information on structure, orientation and/or dynamics of individual molecular segments. Corresponding recoupling schemes are presented in a common context, and the possibilities of exploiting these effects for the determination of order parameters of oriented materials, such as oriented polymer chains or extruded fibres of a discotic mesogen, are discussed. The obtained orientational order parameters are compared to results from two-dimensional wide angle X-ray scattering (WAXS).     

Quantitative analysis of protein backbone dynamics in microcrystalline ubiquitin by solid-state NMR spectroscopy

Characterization of protein dynamics by solid-state NMR spectroscopy requires robust and accurate measurement protocols, which are not yet fully developed. In this study, we investigate the backbone dynamics of microcrystalline ubiquitin using different approaches. A rotational-echo double-resonance type (REDOR-type) methodology allows one to accurately measure (1)H-(15)N order parameters in highly deuterated samples. We show that the systematic errors in the REDOR experiment are as low as 1% or even less, giving access to accurate data for the amplitudes of backbone mobility. Combining such dipolar-coupling-derived order parameters with autocorrelated and cross-correlated (15)N relaxation rates, we are able to quantitate amplitudes and correlation times of backbone dynamics on picosecond and nanosecond time scales in a residue-resolved manner. While the mobility on picosecond time scales appears to have rather uniform amplitude throughout the protein, we unambiguously identify and quantitate nanosecond mobility with order parameters S(2) as low as 0.8 in some regions of the protein, where nanosecond dynamics has also been revealed in solution state. The methodology used here, a combination of accurate dipolar-coupling measurements and different relaxation parameters, yields details about dynamics on different time scales and can be applied to solid protein samples such as amyloid fibrils or membrane proteins.     

The lipidated membrane anchor of full length N-Ras protein shows an extensive dynamics as revealed by solid-state NMR spectroscopy

Many proteins involved in signal transduction are equipped with covalently attached lipid chains providing a hydrophobic anchor targeting these molecules to membranes. Despite the considerable biological significance of this membrane binding mechanism for 5-10% of all cellular proteins, to date very little is known about structural and dynamical features of lipidated membrane binding domains. Here we report the first comprehensive study of the molecular dynamics of the C-terminus of membrane-associated full-length lipidated Ras protein determined by solid-state NMR. Fully functional lipid-modified N-Ras protein was obtained by chemical-biological synthesis ligating the expressed water soluble N-terminus with a chemically synthesized (2)H or (13)C labeled lipidated heptapeptide. Dynamical parameters for the lipid chain modification at Cys 181 were determined from static (2)H NMR order parameter and relaxation measurements. Order parameters describing the amplitude of motion in the protein backbone and the side chain were determined from site-specific measurements of (1)H-(13)C dipolar couplings for all seven amino acids in the membrane anchor of Ras. Finally, the correlation times of motion were determined from temperature dependent relaxation time measurements and analyzed using a modified Lipari Szabo approach. Overall, the C-terminus of Ras shows a versatile dynamics with segmental fluctuations and axially symmetric overall motions on the membrane surface. In particular, the lipid chain modifications are highly flexible in the membrane.     

Principles of spin-echo modulation by J-couplings in magic-angle-spinning solid-state NMR.

In magic-angle-spinning solid-state NMR, the homonuclear J-couplings between pairs of spin-1/2 nuclei may be determined by studying the modulation of the spin echo induced by a pi-pulse, as a function of the echo duration. We present the theory of J-induced spin-echo modulation in magic-angle-spinning solids, and derive a set of modulation regimes which apply under different experimental conditions. In most cases, the dominant spin-echo modulation frequency is exactly equal to the J-coupling. Somewhat surprisingly, the chemical shift anisotropies and dipole-dipole couplings tend to stabilise--rather than abscure--the J-modulation. The theoretical conclusions are supported by numerical simulations and experimental results obtained for three representative samples containing 13C spin pairs.     

Protonation, tautomerization, and rotameric structure of histidine: a comprehensive study by magic-angle-spinning solid-state NMR

Histidine structure and chemistry lie at the heart of many enzyme active sites, ion channels, and metalloproteins. While solid-state NMR spectroscopy has been used to study histidine chemical shifts, the full pH dependence of the complete panel of (15)N, (13)C, and (1)H chemical shifts and the sensitivity of these chemical shifts to tautomeric structure have not been reported. Here we use magic-angle-spinning solid-state NMR spectroscopy to determine the (15)N, (13)C, and (1)H chemical shifts of histidine from pH 4.5 to 11. Two-dimensional homonuclear and heteronuclear correlation spectra indicate that these chemical shifts depend sensitively on the protonation state and tautomeric structure. The chemical shifts of the rare π tautomer were observed for the first time, at the most basic pH used. Intra- and intermolecular hydrogen bonding between the imidazole nitrogens and the histidine backbone or water was detected, and N-H bond length measurements indicated the strength of the hydrogen bond. We also demonstrate the accurate measurement of the histidine side-chain torsion angles χ(1) and χ(2) through backbone-side chain (13)C-(15)N distances; the resulting torsion angles were within 4° of the crystal structure values. These results provide a comprehensive set of benchmark values for NMR parameters of histidine over a wide pH range and should facilitate the study of functionally important histidines in proteins.     

Structure, topology, and dynamics of membrane peptides and proteins from solid-state NMR spectroscopy

The high-resolution structure of membrane proteins is notoriously difficult to determine due to the hydrophobic nature of the protein-membrane complexes. Solid-state NMR spectroscopy is a unique and powerful atomic-resolution probe of the structure and dynamics of these important biological molecules. A number of new solid-state NMR methods for determining the depth of insertion, orientation, oligomeric structure, and long-range (10-15 A) distances of membrane proteins are summarized. Membrane protein depths can now be determined using several complementary techniques with varying site-specificity, distance precision, and mobility requirement on the protein. Membrane protein orientation can now be determined with or without macroscopic alignment, the latter providing a novel alternative for orientation determination of intrinsically curvature-inducing proteins. The novel analyses of beta-sheet membrane protein orientation are described. The quaternary structure of membrane peptide assemblies can now be elucidated using a 19F spin diffusion technique that simultaneously yields the oligomeric number and intermolecular distances up to 15 A. Finally, long-range distances up to approximately 10 A can now be measured using 1H spins with an accuracy of better than 1 A. These methods are demonstrated on several beta-sheet membrane peptides with antimicrobial activities and on two alpha-helical ion-channel proteins. Finally, we show that the nearly ubiquitous dynamics of membrane proteins can be readily examined using 2D correlation experiments. An intimate appreciation of molecular motion in these systems not only leads to important insights into the specific function of these membrane proteins but also may be exploited for other purposes such as orientation determination.     

Heteronuclear recoupling in solid-state magic-angle-spinning NMR via overtone irradiation

A heteronuclear dipolar recoupling scheme applicable to I-S spin pairs undergoing magic-angle-spinning (MAS) is introduced, based on the overtone irradiation of one of the coupled nuclei. It is shown that when I is a quadrupole, for instance (14)N, irradiating this spin at a multiple of its Larmor frequency prevents the formation of MAS dipolar echoes. The ensuing S-spin signal dephasing is significant and dependent on a number of parameters, including the I-S dipolar coupling, the magnitude of I's quadrupolar coupling, and the relative orientations between these two coupling tensors. When applied to a spin-1 nucleus, this overtone recoupling method differs from hitherto proposed recoupling strategies in that it involves only the +/-1> I(z) eigenstates. Its dephasing efficiency becomes independent of first-order quadrupolar effects yet shows a high sensitivity to second-order offsets. A constant-time/variable-offset recoupling sequence thus provides a simple route to acquire, in an indirect fashion, (14)N overtone spectra from rotating powders. The principles underlying this kind of S-(14)N experiments and different applications involving S = (13)C, (59)Co sites are presented.     

Characterization of folding intermediates of a domain-swapped protein by solid-state NMR spectroscopy

We have employed two-dimensional solid-state NMR to study structure and dynamics of insoluble folding states of the domain-swapped protein Crh. Starting from the protein precipitated at its pI, conformational changes due to a modest temperature increase were investigated at the level of individual residues and in real-time. As compared to the crystalline state, Crh pI-precipitates exhibited a higher degree of molecular mobility for several regions of the protein. A rigidly intact center was observed including a subset of residues of the hydrophobic core. Raising the temperature by 13 K to 282 K created a partially unfolded intermediate state that was converted into beta-sheet-rich aggregates that are mostly of spherical character according to electron microscopy. Residue-by-residue analysis indicated that two out of three alpha-helices in aggregated Crh underwent major structural rearrangements while the third helix was preserved. Residues in the hinge region exhibited major chemical-shift changes, indicating that the domain swap was not conserved in the aggregated form. Our study provides direct evidence that protein aggregates of a domain-swapped protein retain a significant fraction of native secondary structure and demonstrates that solid-state NMR can be used to directly monitor slow molecular folding events.     

Toward the characterization of peptidoglycan structure and protein-peptidoglycan interactions by solid-state NMR spectroscopy

Solid-state NMR spectroscopy is applied to intact peptidoglycan sacculi of the Gram-negative bacterium Escherichia coli. High-quality solid-state NMR spectra allow atom-resolved investigation of the peptidoglycan structure and dynamics as well as the study of protein-peptidoglycan interactions.     

Structure determination of a membrane protein with two trans-membrane helices in aligned phospholipid bicelles by solid-state NMR spectroscopy

The structure of the membrane protein MerFt was determined in magnetically aligned phospholipid bicelles by solid-state NMR spectroscopy. With two trans-membrane helices and a 10-residue inter-helical loop, this truncated construct of the mercury transport membrane protein MerF has sufficient structural complexity to demonstrate the feasibility of determining the structures of polytopic membrane proteins in their native phospholipid bilayer environment under physiological conditions. PISEMA, SAMMY, and other double-resonance experiments were applied to uniformly and selectively (15)N-labeled samples to resolve and assign the backbone amide resonances and to measure the associated (15)N chemical shift and (1)H-(15)N heteronuclear dipolar coupling frequencies as orientation constraints for structure calculations. (1)H/(13)C/(15)N triple-resonance experiments were applied to selectively (13)C'- and (15)N-labeled samples to complete the resonance assignments, especially for residues in the nonhelical regions of the protein. A single resonance is observed for each labeled site in one- and two-dimensional spectra. Therefore, each residue has a unique conformation, and all protein molecules in the sample have the same three-dimensional structure and are oriented identically in planar phospholipid bilayers. Combined with the absence of significant intensity near the isotropic resonance frequency, this demonstrates that the entire protein, including the loop and terminal regions, has a well-defined, stable structure in phospholipid bilayers.     

Powder crystallography by proton solid-state NMR spectroscopy

The investigation of 1H-1H spin-diffusion build-up curves using a rate matrix analysis approach shows that high-resolution magic angle spinning NMR of protons, applied to powdered organic compounds, provides a method to probe crystalline arrangements. The comparison between experimental 1H data and simulation is shown to depend strongly on the parameters of the crystal structure, for example on the unit cell parameters or the orientation of the molecule in the unit cell, and those parameters are experimentally determined for a model organic compound.     

Determination of global structure from distance and orientation constraints in biological solids using solid-state NMR spectroscopy

We report the results from a new solid-state NMR experiment, DANTE-REDOR, which can determine global secondary structure in uniformly (13C,15N)-enriched systems by simultaneously measuring distance and orientation constraints. Following a heteronuclear spin-pair selection using a DANTE pulse train, the magnitude and orientation of the internuclear dipole vector, within the chemical shift anisotropy (CSA) frame of the observed nucleus, are determined by tracking the dephasing of individual spinning sidebands under magic angle spinning. The efficacy of the experiment is demonstrated by measuring the imidazole side-chain orientation in U-[13C6,15N3]-L-histidine x HCl x H2O.     

Longer-range distances by spinning-angle-encoding solid-state NMR spectroscopy

A new spinning-angle-encoding spin-echo solid-state NMR approach is used to accurately determine the dipolar coupling corresponding to a C-C distance over 4 ? in a fully labelled dipeptide. The dipolar coupling dependent spin-echo modulation was recorded off magic angle, switching back to the magic angle for the acquisition of the free-induction decay, so as to obtain optimum sensitivity. The retention of both ideal resolution and long-range distance sensitivity was achieved by redesigning a 600 MHz HX MAS NMR probe to provide fast angle switching during the NMR experiment: for 1.8 mm rotors, angle changes of up to ～5° in ～10 ms were achieved at 12 kHz MAS. A new experimental design that combines a reference and a dipolar-modulated experiment and a master-curve approach to data interpretation is presented.     

In situ structural characterization of a recombinant protein in native Escherichia coli membranes with solid-state magic-angle-spinning NMR

The feasibility of using solid-state magic-angle-spinning NMR spectroscopy for in situ structural characterization of the LR11 (sorLA) transmembrane domain (TM) in native Escherichia coli membranes is presented. LR11 interacts with the human amyloid precursor protein (APP), a central player in the pathology of Alzheimer's disease. The background signals from E. coli lipids and membrane proteins had only minor effects on the LR11 TM resonances. Approximately 50% of the LR11 TM residues were assigned by using (13)C PARIS data. These assignments allowed comparisons of the secondary structure of the LR11 TM in native membrane environments and commonly used membrane mimics (e.g., micelles). In situ spectroscopy bypasses several obstacles in the preparation of membrane proteins for structural analysis and offers the opportunity to investigate how membrane heterogeneity, bilayer asymmetry, chemical gradients, and macromolecular crowding affect the protein structure.