Improved 1H amide resonance line narrowing in oriented sample solid-state NMR of membrane proteins in phospholipid bilayers

Lu, George J.; Park, Sang Ho; Opella, Stanley J.

doi:10.1016/j.jmr.2012.04.008

Cited by 13 publications

(17 citation statements)

References 47 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Relatively high amide H N chemical shift resolution of ~0.4 ppm on bicelle-bound membrane proteins has recently been shown to be achievable under the static condition, without spinning, by using optimized 1 H- 1 H homonuclear decoupling sequences and by correlating H N chemical shifts with 15 N chemical shifts 70, 71 . The OMAS 1 H- 31 P correlation technique is complementary to that approach.…”

Section: Discussionmentioning

confidence: 99%

Investigation of the Curvature Induction and Membrane Localization of the Influenza Virus M2 Protein Using Static and Off-Magic-Angle Spinning Solid-State Nuclear Magnetic Resonance of Oriented Bicelles

Wang

Hong

2015

Biochemistry

View full text Add to dashboard Cite

A wide variety of membrane proteins induce membrane curvature for function, thus it is important to develop new methods to simultaneously determine membrane curvature and protein binding sites in membranes with multiple curvatures. We introduce solid-state NMR methods based on magnetically oriented bicelles and off-magic-angle spinning (OMAS) to measure membrane curvature and the binding site of proteins in mixed-curvature membranes. We demonstrate these methods on the influenza virus M2 protein, which not only acts as a proton channel but also mediates virus assembly and membrane scission. An M2 peptide encompassing the transmembrane (TM) domain and an amphipathic helix, M2(21-61), was studied and compared with the TM peptide (M2TM). Static 31P NMR spectra of magnetically oriented DMPC/DHPC bicelles exhibit a temperature-independent isotropic chemical shift in the presence of M2(21-61) but not M2TM, indicating that the amphipathic helix confers the peptide with the ability to generate a high-curvature phase. 2D 31P spectra indicate that this high-curvature phase is associated with the DHPC bicelle edges, suggestive of the structure of budding viruses from the host cell. 31P- and 13C-detected 1H relaxation times of the lipids indicate that the majority of M2(21-61) is bound to the high-curvature phase. Using OMAS experiments, we resolved the 31P signals of lipids with identical headgroups based on their distinct chemical shift anisotropies. Based on this resolution, 2D 1H-31P correlation spectra show that the amide protons in M2(21-61) correlate with the DMPC but not the DHPC 31P signal of the bicelle, indicating that a small percentage of M2(21-61) partitions into the planar region of the bicelles. These results show that the M2 amphipathic helix induces high membrane curvature and localizes the protein to this phase, in excellent agreement with the membrane-scission function of the protein. These bicelle-based relaxation and OMAS solid-state NMR techniques are generally applicable to curvature-inducing membrane proteins such as those involved in membrane trafficking, membrane fusion, and cell division.

show abstract

Section: Discussionmentioning

confidence: 99%

Investigation of the Curvature Induction and Membrane Localization of the Influenza Virus M2 Protein Using Static and Off-Magic-Angle Spinning Solid-State Nuclear Magnetic Resonance of Oriented Bicelles

Wang

Hong

2015

Biochemistry

View full text Add to dashboard Cite

show abstract

“…These advances were extended to the study of full-length MerF by oriented sample solid-state NMR (Lu & Opella, 2014), where a new round of technology improvement was made including the new MSHOT-Pi4 pulse sequence (Lu et al, 2012), new assignment strategies (Knox et al, 2010; Lu et al, 2011; Nevzorov, 2008), and new structure calculation methods (Marassi et al, 2011; Tian et al, 2012). Also, it was in the structure determination of MerFt (Das et al, 2012) that the general pulse sequences and sample conditions for rotationally aligned solid-state NMR were established (Das et al, 2012; Marassi et al, 2011).…”

Section: Examples Of Membrane Proteinsmentioning

confidence: 99%

NMR structures of membrane proteins in phospholipid bilayers

Radoicic

Opella

2014

Quart. Rev. Biophys.

Self Cite

View full text Add to dashboard Cite

Membrane proteins have always presented technical challenges for structural studies because of their requirement for a lipid environment. Multiple approaches exist including X-ray crystallography and electron microscopy that can give significant insights into their structure and function. However, nuclear magnetic resonance (NMR) is unique in that it offers the possibility of determining the structures of unmodified membrane proteins in their native environment of phospholipid bilayers under physiological conditions. Furthermore, NMR enables the characterization of the structure and dynamics of backbone and side chain sites of the proteins alone and in complexes with both small molecules and other biopolymers. The learning curve has been steep for the field as most initial studies were performed under non-native environments using modified proteins until ultimately progress in both techniques and instrumentation led to the possibility of examining unmodified membrane proteins in phospholipid bilayers under physiological conditions. This review aims to provide an overview of the development and application of NMR to membrane proteins. It highlights some of the most significant structural milestones that have been reached by NMR spectroscopy of membrane proteins; especially those accomplished with the proteins in phospholipid bilayer environments where they function.

show abstract

“…43,44 The underlying mechanism of the "motion-adapted" property was characterized theoretically and experimentally as the interference between the protein's rotational exchange motion and the radiofrequency pulses. Importantly, in recent work to apply multiple contact cross-polarization to enhance signals, Nevzorov and co-workers observed much more pronounced signal enhancement for membrane protein sample than for NAL single crystal, 45,46 and this technique was later adapted to enhance signals in MAS solid-state NMR on membrane proteins undergoing rotational diffusion.…”

Section: B Discussion Of Motion Interference and Spin Exchange Distancementioning

confidence: 99%

Mechanism of dilute-spin-exchange in solid-state NMR

Opella

2014

The Journal of Chemical Physics

Self Cite

View full text Add to dashboard Cite

In the stationary, aligned samples used in oriented sample (OS) solid-state NMR, 1 H-1 H homonuclear dipolar couplings are not attenuated as they are in magic angle spinning solid-state NMR; consequently, they are available for participation in dipolar coupling-based spin-exchange processes. Here we describe analytically the pathways of 15 N-15 N spin-exchange mediated by 1 H-1 H homonuclear dipolar couplings. The mixed-order proton-relay mechanism can be differentiated from the third spin assisted recoupling mechanism by setting the 1 H to an off-resonance frequency so that it is at the "magic angle" during the spin-exchange interval in the experiment, since the "magic angle" irradiation nearly quenches the former but only slightly attenuates the latter. Experimental spectra from a single crystal of N-acetyl leucine confirm that this proton-relay mechanism plays the dominant role in 15 N-15 N dilute-spin-exchange in OS solid-state NMR in crystalline samples. Remarkably, the "forbidden" spin-exchange condition under "magic angle" irradiation results in 15 N-15 N cross-peaks intensities that are comparable to those observed with on-resonance irradiation in applications to proteins. The mechanism of the proton relay in dilute-spin-exchange is crucial for the design of polarization transfer experiments.

show abstract

Improved 1H amide resonance line narrowing in oriented sample solid-state NMR of membrane proteins in phospholipid bilayers

Cited by 13 publications

References 47 publications

Investigation of the Curvature Induction and Membrane Localization of the Influenza Virus M2 Protein Using Static and Off-Magic-Angle Spinning Solid-State Nuclear Magnetic Resonance of Oriented Bicelles

Investigation of the Curvature Induction and Membrane Localization of the Influenza Virus M2 Protein Using Static and Off-Magic-Angle Spinning Solid-State Nuclear Magnetic Resonance of Oriented Bicelles

NMR structures of membrane proteins in phospholipid bilayers

Mechanism of dilute-spin-exchange in solid-state NMR

Contact Info

Product

Resources

About