Tilt and Azimuthal Angles of a Transmembrane Peptide: A Comparison between Molecular Dynamics Calculations and Solid-State NMR Data of Sarcolipin in Lipid Membranes

Shi, Lei; Cembran, Alessandro; Gao, Jiali; Veglia, Gianluigi

doi:10.1016/j.bpj.2009.02.025

Cited by 33 publications

(45 citation statements)

References 118 publications

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“…Along with W23, the conserved residues R6, located near the cytosolic N-terminus, and R27, near the lumenal C-terminus, anchor SLN to the lipid membrane, modulating the tilt and rotation angles which seem to be only minimally affected in the different membrane mimetic systems (Mote et al 2011). The NMR structural ensembles determined by the hybrid method are in good agreement with molecular dynamics simulations (Shi et al 2009a) which show a similar orientation of the indole side chain of W23, and a previous oriented solid state NMR study (Buffy et al 2006b) carried out mechanically aligned DOPC/DOPE bilayers, which shows a similar the tilt angle of ∼23° with respect to the bilayer.…”

Section: Resultssupporting

confidence: 84%

Determination of structural topology of a membrane protein in lipid bilayers using polarization optimized experiments (POE) for static and MAS solid state NMR spectroscopy

2013

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The low sensitivity inherent to both the static and magic angle spinning techniques of solid-state NMR (ssNMR) spectroscopy has thus far limited the routine application of multidimensional experiments to determine the structure of membrane proteins in lipid bilayers. Here, we demonstrate the advantage of using a recently developed class of experiments, polarization optimized experiments (POE), for both static and MAS spectroscopy to achieve higher sensitivity and substantial time-savings for 2D and 3D experiments. We used sarcolipin, a single pass membrane protein, reconstituted in oriented bicelles (for oriented ssNMR) and multilamellar vesicles (for MAS ssNMR) as a benchmark. The restraints derived by these experiments are then combined into a hybrid energy function to allow simultaneous determination of structure and topology. The resulting structural ensemble converged to a helical conformation with a backbone RMSD ∼ 0.44 Å, a tilt angle of 24° ± 1°, and an azimuthal angle of 55° ± 6°. This work represents a crucial first step toward obtaining high-resolution structures of large membrane proteins using combined multidimensional O-ssNMR and MAS-ssNMR.

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Section: Resultssupporting

confidence: 84%

Determination of structural topology of a membrane protein in lipid bilayers using polarization optimized experiments (POE) for static and MAS solid state NMR spectroscopy

2013

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“…52 Left: distribution of the water molecules as mapped during the molecular dynamics trajectories. Right: structure of SLN in lipid membranes.…”

Section: Figurementioning

confidence: 99%

Probing Residue-Specific Water–Protein Interactions in Oriented Lipid Membranes via Solid-State NMR Spectroscopy

et al. 2016

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Water plays a central role in membrane protein folding and function. It not only catalyzes lipid membrane self-assembly but also affects the structural integrity and conformational dynamics of membrane proteins. Magic angle spinning (MAS) solid-state NMR (ssNMR) is the technique of choice for measuring water accessibility of membrane proteins, providing a measure for membrane protein topology and insertion within lipid bilayers. However, the sensitivity and resolution of membrane protein samples for MAS experiments are often dictated by hydration levels, which affect the structural dynamics of membrane proteins. Oriented-sample ssNMR (OS-ssNMR) is a complementary technique to determine both structure and topology of membrane proteins in liquid crystalline bilayers. Recent advancements in OS-ssNMR involve the use of oriented bicellar phases that have improved both sensitivity and resolution. Importantly, for bicelle formation and orientation, lipid bilayers must be well organized and hydrated, resulting in the protein's topology being similar to that found in native membranes. Under these conditions, the NMR resonances become relatively narrow, enabling a better separation of 1H–15N dipolar couplings and anisotropic 15N chemical shifts with separated local field (SLF) experiments. Here, we report a residue-specific water accessibility experiment for a small membrane protein, sarcolipin (SLN), embedded in oriented lipid bicelles as probed by new water-edited SLF (WE-SLF) experiments. We show that SLN's residues belonging to the juxtamembrane region are more exposed to the water–lipid interface than the corresponding membrane-embedded residues. The information that can be obtained from the WE-SLF experiments can be interpreted using a simple theoretical model based on spin-diffusion theory and offers a complete characterization of membrane proteins in realistic membrane bilayer systems.

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“…Similarly, the addition and subtraction of two data sets for the second acquisition will give magnetization resulting from NN and CAN pathways, respectively. Figure 3B shows the polarization build-up and decay of four pathways as a function of specific-CP contact period τ, on U- 13 C, 15 N microcrystalline ubiquitin and a single-pass membrane protein, sarcolipin[16, 23-26]. At optimal polarization transfer (∼2800 or 3100 μs) from N to CA and vice versa, the residual polarization of 13 C and 15 N is approximately 55 and 35%, respectively.…”

Section: Bidirectional Specific-cp and Residual Polarizationmentioning

confidence: 99%

Multiple acquisition of magic angle spinning solid-state NMR experiments using one receiver: Application to microcrystalline and membrane protein preparations

Gopinath

Veglia

2015

Journal of Magnetic Resonance

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Solid-State NMR spectroscopy of proteins is a notoriously low-throughput technique. Relatively low-sensitivity and poor resolution of protein samples require long acquisition times for multidimensional NMR experiments. To speed up data acquisition, we developed a family of experiments called Polarization Optimized Experiments (POE), in which we utilized the orphan spin operators that are discarded in classical multidimensional NMR experiments, recovering them to allow simultaneous acquisition of multiple 2D and 3D experiments, all while using conventional probes with spectrometers equipped with one receiver. POEs allow the concatenation of multiple 2D or 3D pulse sequences into a single experiment, thus potentially combining all of the aforementioned advances, boosting the capability of ssNMR spectrometers at least two-fold without the addition of any hardware. In this Perspective, we describe the first generation of POEs, such as dual acquisition MAS (or DUMAS) methods, and then illustrate the evolution of these experiments into MEIOSIS, a method that enables the simultaneous acquisition of multiple 2D and 3D spectra. Using these new pulse schemes for the solid-state NMR investigation of biopolymers makes it possible to obtain sequential resonance assignments, as well as distance restraints, in about half the experimental time. While designed for acquisition of heteronuclei, these new experiments can be easily implemented for proton detection and coupled with other recent advancements, such as dynamic polarization, to improve signal to noise. Finally, we illustrate the application of these methods to microcrystalline protein preparations as well as single and multi-span membrane proteins reconstituted in lipid membranes.

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Tilt and Azimuthal Angles of a Transmembrane Peptide: A Comparison between Molecular Dynamics Calculations and Solid-State NMR Data of Sarcolipin in Lipid Membranes

Cited by 33 publications

References 118 publications

Determination of structural topology of a membrane protein in lipid bilayers using polarization optimized experiments (POE) for static and MAS solid state NMR spectroscopy

Determination of structural topology of a membrane protein in lipid bilayers using polarization optimized experiments (POE) for static and MAS solid state NMR spectroscopy

Probing Residue-Specific Water–Protein Interactions in Oriented Lipid Membranes via Solid-State NMR Spectroscopy

Multiple acquisition of magic angle spinning solid-state NMR experiments using one receiver: Application to microcrystalline and membrane protein preparations

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