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Motion-adapted pulse sequences for oriented sample (OS) solid-state NMR of biopolymers.

ABSTRACT: One of the main applications of solid-state NMR is to study the structure and dynamics of biopolymers, such as membrane proteins, under physiological conditions where the polypeptides undergo global motions as they do in biological membranes. The effects of NMR radiofrequency irradiations on nuclear spins are strongly influenced by these motions. For example, we previously showed that the MSHOT-Pi4 pulse sequence yields spectra with resonance line widths about half of those observed using the conventional pulse sequence when applied to membrane proteins undergoing rapid uniaxial rotational diffusion in phospholipid bilayers. In contrast, the line widths were not changed in microcrystalline samples where the molecules did not undergo global motions. Here, we demonstrate experimentally and describe analytically how some Hamiltonian terms are susceptible to sample motions, and it is their removal through the critical π/2 Z-rotational symmetry that confers the "motion adapted" property to the MSHOT-Pi4 pulse sequence. This leads to the design of separated local field pulse sequence "Motion-adapted SAMPI4" and is generalized to an approach for the design of decoupling sequences whose performance is superior in the presence of molecular motions. It works by cancelling the spin interaction by explicitly averaging the reduced Wigner matrix to zero, rather than utilizing the 2π nutation to average spin interactions. This approach is applicable to both stationary and magic angle spinning solid-state NMR experiments.

SUBMITTER: Lu GJ

PROVIDER: S-EPMC3772948 | biostudies-other | 2013 Aug

REPOSITORIES: biostudies-other

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Project description:The validation of protein structures through functional assays has been the norm for many years. Functional assays perform this validation for water-soluble proteins very well, but they need to be performed in the same environment as that used for the structural analysis. This is difficult for membrane proteins that are often structurally characterized in detergent environments, although functional assays for these proteins are most frequently performed in lipid bilayers. Because the structure of membrane proteins is known to be sensitive to the membrane mimetic environment, such functional assays are appropriate for validating the protein construct, but not the membrane protein structure. Here, we compare oriented sample solid-state NMR spectral data of diacylglycerol kinase previously published with predictions of such data from recent structures of this protein. A solution NMR structure of diacylglycerol kinase has been obtained in detergent micelles and three crystal structures have been obtained in a monoolein cubic phase. All of the structures are trimeric with each monomer having three transmembrane and one amphipathic helices. However, the solution NMR structure shows typical perturbations induced by a micelle environment that is reflected in the predicted solid-state NMR resonances from the structural coordinates. The crystal structures show few such perturbations, especially for the wild-type structure and especially for the monomers that do not have significant crystal contacts. For these monomers the predicted and observed data are nearly identical. The thermostabilized constructs do show more perturbations, especially the A41C mutation that introduces a hydrophilic residue into what would be the middle of the lipid bilayer inducing additional hydrogen bonding between trimers. These results demonstrate a general technique for validating membrane protein structures with minimal data obtained from membrane proteins in liquid crystalline lipid bilayers by oriented sample solid-state NMR.

Project description:The finite-pulse radio frequency driven dipolar recoupling (fp-RFDR) pulse sequence is used in 2D homonuclear chemical shift correlation experiments under magic angle spinning (MAS). A recent study demonstrated the advantages of using a short phase cycle, XY4, and its super-cycle, XY4(1)4, for the fp-RFDR pulse sequence employed in 2D (1)H/(1)H single-quantum/single-quantum correlation experiments under ultrafast MAS conditions. In this study, we report a comprehensive analysis on the dipolar recoupling efficiencies of XY4, XY4(1)2, XY4(1)3, XY4(1)4, and XY8(1)4 phase cycles under different spinning speeds ranging from 10 to 100 kHz. The theoretical calculations reveal the presence of second-order terms (T(10)T(2,±2), T(1,±1)T(2,±1), etc.) in the recoupled homonuclear dipolar coupling Hamiltonian only when the basic XY4 phase cycle is utilized, making it advantageous for proton-proton magnetization transfer under ultrafast MAS conditions. It is also found that the recoupling efficiency of fp-RFDR is quite dependent on the duty factor (τ180/τR) as well as on the strength of homonuclear dipolar couplings. The rate of longitudinal magnetization transfer increases linearly with the duty factor of fp-RFDR for all the XY-based phase cycles investigated in this study. Examination of the performances of different phase cycles against chemical shift offset and RF field inhomogeneity effects revealed that XY4(1)4 is the most tolerant phase cycle, while the shortest phase cycle XY4 suppressed the RF field inhomogeneity effects most efficiently under slow spinning speeds. Our results suggest that the difference in the fp-RFDR recoupling efficiencies decreases with the increasing MAS speed, while ultrafast (>60 kHz) spinning speed is advantageous as it recouples a large amount of homonuclear dipolar couplings and therefore enable fast magnetization exchange. The effects of higher-order terms and cross terms between various interactions in the effective Hamiltonian of fp-RFDR are also analyzed using numerical simulations for various phase cycles. Results obtained via numerical simulations are in excellent agreement with ultrafast MAS experimental results from the powder samples of glycine and l-alanine.

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Motion-adapted pulse sequences for oriented sample (OS) solid-state NMR of biopolymers.

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