PISA Wheels Research Articles

ChemInform Abstract: Determining the Orientation and Localization of Membrane‐Bound Peptides

AbstractReview: 121 refs.

Determining the orientation and localization of membrane-bound peptides.

Many naturally occurring bioactive peptides bind to biological membranes. Studying and elucidating the mode of interaction is often an essential step to understand their molecular and biological functions. To obtain the complete orientation and immersion depth of such compounds in the membrane or a membrane-mimetic system, a number of methods are available, which are separated in this review into four main classes: solution NMR, solid-state NMR, EPR and other methods. Solution NMR methods include the Nuclear Overhauser Effect (NOE) between peptide and membrane signals, residual dipolar couplings and the use of paramagnetic probes, either within the membrane-mimetic or in the solvent. The vast array of solid state NMR methods to study membrane-bound peptide orientation and localization includes the anisotropic chemical shift, PISA wheels, dipolar waves, the GALA, MAOS and REDOR methods and again the use of paramagnetic additives on relaxation rates. Paramagnetic additives, with their effect on spectral linewidths, have also been used in EPR spectroscopy. Additionally, the orientation of a peptide within a membrane can be obtained by the anisotropic hyperfine tensor of a rigidly attached nitroxide label. Besides these magnetic resonance techniques a series of other methods to probe the orientation of peptides in membranes has been developed, consisting of fluorescence-, infrared- and oriented circular dichroism spectroscopy, colorimetry, interface-sensitive X-ray and neutron scattering and Quartz crystal microbalance.

Membrane Alignment of the Pore-Forming Component TatAd of the Twin-Arginine Translocase from Bacillus subtilis Resolved by Solid-State NMR Spectroscopy

The twin-arginine translocase (Tat) provides protein export in bacteria and plant chloroplasts and is capable of transporting fully folded proteins across the membrane. We resolved the conformation and membrane alignment of the pore-forming subunit TatA(d) from Bacillus subtilis using solid-state NMR spectroscopy. The relevant structured part of the protein, TatA(2-45), contains a transmembrane segment (TMS) and an amphiphilic helix (APH). It was reconstituted in planar bicelles, which represent the lipid environment of a bacterial membrane. The SAMMY solid-state NMR experiment was used to correlate (15)N chemical shifts and (1)H-(15)N dipolar couplings in the backbone and side chains of the (15)N-labeled protein. The observed wheel-like patterns ("PISA wheels") in the resulting 2-dimensional spectra confirm the α-helical character of the two segments and reveal their alignment in the lipid bilayer. Helix tilt angles (τ(TMS) = 13°, τ(APH) = 64°) were obtained from uniformly labeled protein, and azimuthal rotations (ρ(Val15) = 235°, ρ(Ile29) = 25°) were obtained from selective labels. These constraints define two distinct families of allowed structures for TatA in the membrane-bound state. The manifold of solutions could be narrowed down to a unique structure by using input from a liquid-state NMR study of TatA in detergent micelles, as recently described [Hu, Y.; Zhao, E.; Li, H.; Xia, B.; Jin, C. J. Am. Chem. Soc. 2010, DOI: 10.1021/ja1053785]. Interestingly, the APH showed an unexpectedly slanted alignment in the protein, different from that of the isolated APH peptide. This finding implies that the amphiphilic region of TatA is not just a flexible attachment to the transmembrane anchor but might be able to form intra- or even intermolecular salt-bridges, which could play a key role in pore assembly.

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

We report molecular dynamics simulations in the explicit membrane environment of a small membrane-embedded protein, sarcolipin, which regulates the sarcoplasmic reticulum Ca-ATPase activity in both cardiac and skeletal muscle. In its monomeric form, we found that sarcolipin adopts a helical conformation, with a computed average tilt angle of 28 ± 6° and azymuthal angles of 66 ± 22°, in reasonable accord with angles determined experimentally (23 ± 2° and 50 ± 4°, respectively) using solid-state NMR with separated-local-field experiments. The effects of time and spatial averaging on both 15N chemical shift anisotropy and 1H/ 15N dipolar couplings have been analyzed using short-time averages of fast amide out-of-plane motions and following principal component dynamic trajectories. We found that it is possible to reproduce the regular oscillatory patterns observed for the anisotropic NMR parameters (i.e., PISA wheels) employing average amide vectors. This work highlights the role of molecular dynamics simulations as a tool for the analysis and interpretation of solid-state NMR data.

Solid State NMR of Membrane Proteins: Towards Complex Structural and Functional Information for Bacterial ABC Class Importers

Despite the wealth of protein structural data available today, membrane protein structural characterization continues to pose a significant challenge in structural biology. Choosing the membrane mimetic is a challenge and often detergent micelles are employed. However, detergents are prone to induce distortions in the protein structure. An emerging technique, solid state nuclear magnetic resonance (NMR), provides a path to structural characterization using an environment similar to the native one, liquid crystalline lipid bilayers.Static solid state NMR experiments on proteins determine the orientation of the peptide planes with respect to the magnetic field. All 15N-1H dipolar couplings and anisotropic 15N chemical shifts observed in two dimensional separated local field experiments (PISEMA) lie within a butterfly shape in the spectrum. Special patterns called PISA wheels arise for uniformly aligned protein samples which directly reflect the orientation of protein secondary structure. Using these wheels the tilt angle of each helical axis from the magnetic field and membrane normal can be determined without complete structure determination. Consequently, a single data set allows for characterization of secondary structure in the membrane mimetic as well as providing a set of high resolution peptide plane orientations that can be used directly in structural refinement.Uniform alignment has been achieved for multiple proteins in our laboratory. Still, studies on larger membrane proteins are required to make solid state NMR a generally applicable technique for membrane protein structure characterization. One excellent example is SugAB, the transmembrane domain of an ABC importer from Mycobacterium tuberculosis. These transport proteins contain at least 10 transmembrane helices. This makes SugAB an excellent target for determining the utility of solid state NMR to structurally characterize large membrane proteins.

Uniformly aligned full-length membrane proteins in liquid crystalline bilayers for structural characterization.

High-resolution solid-state NMR spectra of three full-length membrane proteins uniformly aligned in lipid bilayers between glass slides are observed at high magnetic field. The resolution of the specific amino acid labeled samples shows promise for large membrane protein structure determination utilizing aligned samples and shows resonance patterns known as PISA wheels. The tilt angles of the transmembrane helices are extracted from the resonance patterns in PISEMA spectra.

Three-dimensional Structure of the Channel-forming Trans-membrane Domain of Virus Protein “u” (Vpu) from HIV-1

The three-dimensional structure of the channel-forming trans-membrane domain of virus protein “u” (Vpu) of HIV-1 was determined by NMR spectroscopy in micelle and bilayer samples. Vpu 2–30+ is a 36-residue polypeptide that consists of residues 2–30 from the N terminus of Vpu and a six-residue “solubility tag” at its C terminus that facilitates the isolation, purification, and sample preparation of this highly hydrophobic minimal channel-forming domain. Nearly all of the resonances in the two-dimensional 1H/ 15N HSQC spectrum of uniformly 15N labeled Vpu 2–30+ in micelles are superimposable on those from the corresponding residues in the spectrum of full-length Vpu, which indicates that the structure of the trans-membrane domain is not strongly affected by the presence of the cytoplasmic domain at its C terminus. The two-dimensional 1H/ 15N PISEMA spectrum of Vpu 2–30+ in lipid bilayers aligned between glass plates has been fully resolved and assigned. The “wheel-like” pattern of resonances in the spectrum is characteristic of a slightly tilted membrane-spanning helix. Experiments were also performed on weakly aligned micelle samples to measure residual dipolar couplings and chemical shift anisotropies. The analysis of the PISA wheels and Dipolar Waves obtained from both weakly and completely aligned samples show that Vpu 2–30+ has a trans-membrane α-helix spanning residues 8–25 with an average tilt of 13°. The helix is kinked slightly at Ile17, which results in tilts of 12° for residues 8–16 and 15° for residues 17–25. A structural fit to the experimental solid-state NMR data results in a three-dimensional structure with precision equivalent to an RMSD of 0.4 Å. Vpu 2–30+ exists mainly as an oligomer on PFO-PAGE and forms ion-channels, a most frequent conductance of 96(±6) pS in lipid bilayers. The structural features of the trans-membrane domain are determinants of the ion-channel activity that may be associated with the protein's role in facilitating the budding of new virus particles from infected cells.

Simultaneous assignment and structure determination of a membrane protein from NMR orientational restraints.

A solid-state NMR approach for simultaneous resonance assignment and three-dimensional structure determination of a membrane protein in lipid bilayers is described. The approach is based on the scattering, hence the descriptor "shotgun," of (15)N-labeled amino acids throughout the protein sequence (and the resulting NMR spectra). The samples are obtained by protein expression in bacteria grown on media in which one type of amino acid is labeled and the others are not. Shotgun NMR short-circuits the laborious and time-consuming process of obtaining complete sequential assignments prior to the calculation of a protein structure from the NMR data by taking advantage of the orientational information inherent to the spectra of aligned proteins. As a result, it is possible to simultaneously assign resonances and measure orientational restraints for structure determination. A total of five two-dimensional (1)H/(15)N PISEMA (polarization inversion spin exchange at the magic angle) spectra, from one uniformly and four selectively (15)N-labeled samples, were sufficient to determine the structure of the membrane-bound form of the 50-residue major pVIII coat protein of fd filamentous bacteriophage. Pisa (polarity index slat angle) wheels are an essential element in the process, which starts with the simultaneous assignment of resonances and the assembly of isolated polypeptide segments, and culminates in the complete three-dimensional structure of the protein with atomic resolution. The principles are also applicable to weakly aligned proteins studied by solution NMR spectroscopy. [The structure we determined for the membrane-bound form of the Fd bacteriophage pVIII coat protein has been deposited in the Protein Data Bank as PDB file 1MZT.]

Specification and Visualization of Anisotropic Interaction Tensors in Polypeptides and Numerical Simulations in Biological Solid-State NMR

Software facilitating numerical simulation of solid-state NMR experiments on polypeptides is presented. The Tcl-controlled SIMMOL program reads in atomic coordinates in the PDB format from which it generates typical or user-defined parameters for the chemical shift, J coupling, quadrupolar coupling, and dipolar coupling tensors. The output is a spin system file for numerical simulations, e.g., using SIMPSON (Bak, Rasmussen, and Nielsen, J. Magn. Reson. 147, 296 (2000)), as well as a 3D visualization of the molecular structure, or selected parts of this, with user-controlled representation of relevant tensors, bonds, atoms, peptide planes, and coordinate systems. The combination of SIMPSON and SIMMOL allows straightforward simulation of the response of advanced solid-state NMR experiments on typical nuclear spin interactions present in polypeptides. Thus, SIMMOL may be considered a "sample changer" to the SIMPSON "computer spectrometer" and proves to be very useful for the design and optimization of pulse sequences for application on uniformly or extensively isotope-labeled peptides where multiple-spin interactions need to be considered. These aspects are demonstrated by optimization and simulation of novel DCP and C7 based 2D N(CO)CA, N(CA)CB, and N(CA)CX MAS correlation experiments for multiple-spin clusters in ubiquitin and by simulation of PISA wheels from PISEMA spectra of uniaxially oriented bacteriorhodopsin and rhodopsin under conditions of finite RF pulses and multiple spin interactions.

Using pisa pies to resolve ambiguities in angular constraints from PISEMA spectra of aligned proteins.

The structures of proteins are mapped onto the patterns of resonances in NMR spectra of aligned samples. This is most clearly illustrated with Pisa wheels of helical membrane proteins, where the distinctive 'wheel-like' patterns of resonances reflect the tilt and rotation of the helices in the bilayers. These patterns contain both structural and assignment information. This Communication describes a simple way of using this information to resolve angular ambiguities inherent in orientational constraints derived from NMR data. This contributes to the use of solid-state NMR of aligned samples for protein structure determination.

PISEMA powder patterns and PISA wheels.

Resonance patterns observed in 2D PISEMA (polarization inversion spin exchange at magic angle) spectra from a transmembrane alpha-helix have been demonstrated to yield structural details of the protein. This paper presents a mathematical discussion of the PISEMA powder spectrum as the image in the frequency plane of a quadratic function from the sphere of unit vectors. The simplicity of this function allows easy calculation of the powder spectrum. Based on this analysis of powder patterns, four degeneracies are discussed which arise in determining possible orientations associated with PISA spectra. This paper also gives parametric equations for PISA wheels, which are specific patterns observed in PISEMA spectra of oriented peptides. These wheels are useful both in assigning the resonances and in determining the orientation of the helix with respect to the magnetic field. The union of these PISA wheels gives the entire powder spectrum.

A Simple Approach to Membrane Protein Secondary Structure and Topology based on NMR Spectroscopy

This paper describes a simple, qualitative approach for the determination of membrane protein secondary structure and topology in lipid bilayer membranes. The approach is based on the observation of wheel-like resonance patterns observed in the NMR 1H- 15N/ 15N polarization inversion with spin exchange at the magic angle (PISEMA) and 1H/ 15N heteronuclear correlation (HETCOR) spectra of membrane proteins in oriented lipid bilayers. These patterns, named Pisa wheels, have been previously shown to reflect helical wheel projections of residues that are characteristic of α-helices associated with membranes. This study extends the analysis of these patterns to β-strands associated with membranes and demonstrates that, as for the case of α-helices, Pisa wheels are extremely sensitive to the tilt, rotation, and twist of β-strands in the membrane. Therefore, the Pisa wheels provide a sensitive, visually accessible, qualitative index of membrane protein secondary structure and topology.