Screening molecular associations with lipid membranes using natural abundance 13C cross-polarization magic-angle spinning NMR and principal component analysis

We describe an NMR approach for detecting the interactions between phospholipid membranes and proteins, peptides, or small molecules. First, 1H-13C dipolar coupling profiles are obtained from hydrated lipid samples at natural isotope abundance using cross-polarization magic-angle spinning NMR methods. Principal component analysis of dipolar coupling profiles for synthetic lipid membranes in the presence of a range of biologically active additives reveals clusters that relate to different modes of interaction of the additives with the lipid bilayer. Finally, by representing profiles from multiple samples in the form of contour plots, it is possible to reveal statistically significant changes in dipolar couplings, which reflect perturbations in the lipid molecules at the membrane surface or within the hydrophobic interior.     

Mapping the orientation of helices in micelle-bound peptides by paramagnetic relaxation waves

Many antimicrobial peptides form alpha-helices when bound to a membrane. In addition, around 80% of residues in membrane-bound proteins are found in alpha-helical regions. The orientation and location of such helical peptides and proteins in the membrane are key factors determining their function and activity. Here we present a new solution state NMR method for obtaining the orientation of helical peptides in a membrane-mimetic environment (micelle-bound) without any chemical perturbation of the peptide-micelle system. By monitoring proton longitudinal relaxation rates upon addition of the freely water-soluble and inert paramagnetic probe Gd(DTPA-BMA) to an alpha-helical peptide, a wavelike pattern with a periodicity of 3.6 residues per turn is observed. The tilt and azimuth (rotation) angle of the helix determine the shape of this paramagnetic relaxation wave and can be obtained by least-square fitting of measured relaxation enhancements. Results are presented for the 15-residue antimicrobial peptide CM15 which forms an amphipathic helix almost parallel to the surface of the micelle. Thus, a few fast experiments enable the identification of helical regions and determination of the helix orientation within the micelle without the need for covalent modification, isotopic labeling, or sophisticated equipment. This approach opens a path toward the topology determination of alpha-helical membrane-proteins without the need for a complete NOE-based structure determination.     

NMR methods for studying the structure and dynamics of oncogenic and antihistaminic peptides in biomembranes

We present several applications of both wide-line and magic angle spinning (MAS) solid-state NMR of bicelles in which are embedded fragments of a tyrosine kinase receptor or enkephalins. The magnetically orientable bicelle membranes are shown to be of particular interest for studying the functional properties of lipids and proteins in a state that is very close to their natural environment. Quadrupolar, dipolar and chemical shielding interactions can be used to determine minute alterations of internal membrane dynamics and the orientation of peptides with respect to the membrane plane. MAS of bicelles can in turn lead to high-resolution proton spectra of hydrated membranes. Using deuterium-proton contrast methods one can then obtain pseudo-high-resolution proton spectra of peptides or proteins embedded in deuterated membranes and determine their atomic 3D structure using quasi-conventional liquid-state NMR methods.     

Nitrogen-14 solid-state NMR spectroscopy of aligned phospholipid bilayers to probe peptide-lipid interaction and oligomerization of membrane associated peptides

Characterization of the oligomerization of membrane-associated peptides is important to understand the folding and function of biomolecules like antimicrobial peptides, fusion peptides, amyloid peptides, toxins, and ion channels. However, this has been considered to be very difficult, because the amphipathic properties of the constituents of the cell membrane pose tremendous challenges to most commonly used biophysical techniques. In this study, we present the application of a simple (14)N solid-state NMR spectroscopy of aligned model membranes containing a phosphatidyl choline lipid to investigate the oligomerization of membrane-associated peptides. Since the near-symmetric nature of the choline headgroup of a phosphocholine lipid considerably reduces the (14)N quadrupole coupling, there are significant practical advantages in using (14)N solid-state NMR experiments to probe the interaction of peptide or protein with the surface of model membranes. Experimental results for several membrane-associated peptides are presented in this paper. Our results suggest that the experimentally measured (14)N quadrupole splitting of the lipid depends on the peptide-induced changes in the electrostatic potential of the lipid bilayer surface and therefore on the nature of the peptide, peptide-membrane interaction, and peptide-peptide interaction. It is inferred that the membrane orientation and oligomerization of the membrane-associated peptides can be measured using (14)N solid-state NMR spectroscopy.     

Determining the orientation of uniaxially rotating membrane proteins using unoriented samples: a 2H, 13C, AND 15N solid-state NMR investigation of the dynamics and orientation of a transmembrane helical bundle

Membrane protein orientation has traditionally been determined by NMR using mechanically or magnetically aligned samples. Here we show a new NMR approach that abolishes the need for preparing macroscopically aligned membranes. When the protein undergoes fast uniaxial rotation around the bilayer normal, the 0 degrees -frequency of the motionally averaged powder spectrum is identical to the frequency of the aligned protein whose alignment axis is along the magnetic field. Thus, one can use unoriented membranes to determine the orientation of the protein relative to the bilayer normal. We demonstrate this approach on the M2 transmembrane peptide (M2TMP) of influenza A virus, which is known to assemble into a proton-conducting tetrameric helical bundle. The fast uniaxial rotational diffusion of the M2TMP helical bundle around the membrane normal is characterized via 2H quadrupolar couplings, C-H and N-H dipolar couplings, 13C chemical shift anisotropies, and 1H T1rho relaxation times. We then show that 15N chemical shift anisotropy and N-H dipolar coupling measured on these powder samples can be analyzed to yield precise tilt angles and rotation angles of the helices. The data show that the tilt angle of the M2TMP helices depends on the membrane thickness to reduce the hydrophobic mismatch. Moreover, the orientation of a longer M2 peptide containing both the transmembrane domain and cytoplasmic residues is similar to the orientation of the transmembrane domain alone, suggesting that the transmembrane domain regulates the orientation of this protein and that structural information obtained from M2TMP may be extrapolated to the longer peptide. This powder-NMR approach for orientation determination is generally applicable and can be extended to larger membrane proteins.     

Determining the topology of integral membrane peptides using EPR spectroscopy

This paper reports on the development of a new structural biology technique for determining the membrane topology of an integral membrane protein inserted into magnetically aligned phospholipid bilayers (bicelles) using EPR spectroscopy. The nitroxide spin probe, 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC), was attached to the pore-lining transmembrane domain (M2delta) of the nicotinic acetylcholine receptor (AChR) and incorporated into a bicelle. The corresponding EPR spectra revealed hyperfine splittings that were highly dependent on the macroscopic orientation of the bicelles with respect to the static magnetic field. The helical tilt of the peptide can be easily calculated using the hyperfine splittings gleaned from the orientational dependent EPR spectra. A helical tilt of 14 degrees was calculated for the M2delta peptide with respect to the bilayer normal of the membrane, which agrees well with previous 15N solid-state NMR studies. The helical tilt of the peptide was verified by simulating the corresponding EPR spectra using the standardized MOMD approach. This new method is advantageous because: (1) bicelle samples are easy to prepare, (2) the helical tilt can be directly calculated from the orientational-dependent hyperfine splitting in the EPR spectra, and (3) EPR spectroscopy is approximately 1000-fold more sensitive than 15N solid-state NMR spectroscopy; thus, the helical tilt of an integral membrane peptide can be determined with only 100 microg of peptide. The helical tilt can be determined more accurately by placing TOAC spin labels at several positions with this technique.     

Uptake of Cell-Penetrating Peptide RL2 by Human Lung Cancer Cells: Monitoring by Electron Paramagnetic Resonance and Confocal Laser Scanning Microscopy

RL2 is a recombinant analogue of a human κ-casein fragment, capable of penetrating cells and inducing apoptosis of cancer cells with no toxicity to normal cells. The exact mechanism of RL2 penetration into cells remains unknown. In this study, we investigated the mechanism of RL2 penetration into human lung cancer A549 cells by a combination of electron paramagnetic resonance (EPR) spectroscopy and confocal laser scanning microscopy. EPR spectra of A549 cells incubated with RL2 (sRL2) spin-labeled by a highly stable 3-carboxy-2,2,5,5-tetraethylpyrrolidine-1-oxyl radical were found to contain three components, with their contributions changing with time. The combined EPR and confocal-microscopy data allowed us to assign these three forms of sRL2 to the spin-labeled protein sticking to the membrane of the cell and endosomes, to the spin-labeled protein in the cell interior, and to spin labeled short peptides formed in the cell because of protein digestion. EPR spectroscopy enabled us to follow the kinetics of transformations between different forms of the spin-labeled protein at a minimal spin concentration (3–16 μM) in the cell. The prospects of applications of spin-labeled cell-penetrating peptides to EPR imaging, DNP, and magnetic resonance imaging are discussed, as is possible research on an intrinsically disordered protein in the cell by pulsed dipolar EPR spectroscopy.     

NMR of membrane-associated peptides and proteins

In living cells, membrane proteins are essential to signal transduction, nutrient use, and energy exchange between the cell and environment. Due to challenges in protein expression, purification and crystallization, deposition of membrane protein structures in the Protein Data Bank lags far behind existing structures for soluble proteins. This review describes recent advances in solution NMR allowing the study of a select set of peripheral and integral membrane proteins. Surface-binding proteins discussed include amphitropic proteins, antimicrobial and anticancer peptides, the HIV-1 gp41 peptides, human alpha-synuclein and apolipoproteins. Also discussed are transmembrane proteins including bacterial outer membrane beta-barrel proteins and oligomeric alpha-helical proteins. These structural studies are possible due to solubilization of the proteins in membrane-mimetic constructs such as detergent micelles and bicelles. In addition to protein dynamics, protein-lipid interactions such as those between arginines and phosphatidylglycerols have been detected directly by NMR. These examples illustrate the unique role solution NMR spectroscopy plays in structural biology of membrane proteins.     

Determination of peptide oligomerization in lipid bilayers using 19F spin diffusion NMR

Aggregation or oligomerization is important for the function of many membrane peptides such as ion channels and antimicrobial peptides. However, direct proof of aggregation and the determination of the number of molecules in the aggregate have been difficult due to the lack of suitable high-resolution methods for membrane peptides. We propose a 19F spin diffusion magic-angle-spinning NMR technique to determine the oligomeric state of peptides bound to the lipid bilayer. Magnetization transfer between chemically equivalent but orientationally different 19F spins on different molecules reduces the 19F magnetization in an exchange experiment. At long mixing times, the equilibrium 19F magnetization is 1/M, where M is the number of orientationally different molecules in the aggregate. The use of the 19F spin increases the homonuclear dipolar coupling and thus the distance reach. We demonstrate this technique on crystalline model compounds with known numbers of molecules in the asymmetric unit cell, and show that 19F spin diffusion is more efficient than that of 13C by a factor of approximately 500. Application to a beta-hairpin antimicrobial peptide, protegrin-1, shows that the peptide is almost completely dimerized in POPC bilayers at a concentration of 7.4 mol %. Decreasing the peptide concentration reduced the dimer fraction. Using a monomer-dimer equilibrium model, we estimate the DeltaG for dimer formation to be -10.2 +/- 2.3 kJ/mol. This is in good agreement with the previously measured free energy reduction for partitioning and aggregating beta-sheet peptides into phospholipid membranes. This 19F spin diffusion technique opens the possibility of determining the oligomeric structures of membrane peptides.     

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.     

Probing local environments in paramagnetic europium-substituted Keggin solids by 31P magic angle spinning NMR spectroscopy

Paramagnetic Eu-substituted Keggin oxopolytungstates crystallize in different forms, determined by the nature of the counterions. The crystal packing is in turn responsible for the variations in the geometry of paramagnetic Eu sites with respect to the anion core. We probed the paramagnetic environments in a series of Eu-substituted Keggin solids, by 31P magic angle spinning NMR spectroscopy. 31P spinning sideband envelopes are dominated by the electron-nuclear dipolar interaction. For the compounds under investigation, both the magnitude and the asymmetry parameter of the electron-nuclear dipolar coupling tensor are sensitive to the mutual arrangements of paramagnetic Eu sites in the crystal lattice. and also report on the stoichiometry of the anion. The electron-nuclear dipolar coupling tensors were calculated from the crystallographic coordinates and the experimentally determined effective magnetic moments, assuming a point dipole approximation. The computed tensors are in very good agreement with the experimental spectra. Furthermore, the P-Eu distance estimates, accurate to within 0.06-0.12 A, can be obtained directly from the magnitude of the electron-nuclear dipolar coupling. This work demonstrates that 31P MAS NMR spectroscopy is a useful probe for investigating local environments in paramagnetic Keggin solids.     

Molecular dynamics in paramagnetic materials as studied by magic-angle spinning 2H NMR spectra

A magic-angle spinning (MAS) 2H NMR experiment was applied to study the molecular motion in paramagnetic compounds. The temperature dependences of 2H MAS NMR spectra were measured for paramagnetic [M(H2O)6][SiF6] (M=Ni2+, Mn2+, Co2+) and diamagnetic [Zn(H2O)6][SiF6]. The paramagnetic compounds exhibited an asymmetric line shape in 2H MAS NMR spectra because of the electron-nuclear dipolar coupling. The drastic changes in the shape of spinning sideband patterns and in the line width of spinning sidebands due to the 180 degrees flip of water molecules and the reorientation of [M(H2O)6]2+ about its C3 axis were observed. In the paramagnetic compounds, paramagnetic spin-spin relaxation and anisotropic g-factor result in additional linebroadening of each of the spinning sidebands. The spectral simulation of MAS 2H NMR, including the effects of paramagnetic shift and anisotropic spin-spin relaxation due to electron-nuclear dipolar coupling and anisotropic g-factor, was performed for several molecular motions. Information about molecular motions in the dynamic range of 10(2) s(-1)相似文献   

Bis‐Gadolinium Complexes for Solid Effect and Cross Effect Dynamic Nuclear Polarization

High‐spin complexes act as polarizing agents (PAs) for dynamic nuclear polarization (DNP) in solid‐state NMR spectroscopy and feature promising aspects towards biomolecular DNP. We present a study on bis(Gd‐chelate)s which enable cross effect (CE) DNP owing to spatial confinement of two dipolar‐coupled electron spins. Their well‐defined Gd⋅⋅⋅Gd distances in the range of 1.2–3.4 nm allowed us to elucidate the Gd⋅⋅⋅Gd distance dependence of the DNP mechanism and NMR signal enhancement. We found that Gd⋅⋅⋅Gd distances above 2.1 nm result in solid effect DNP while distances between 1.2 and 2.1 nm enable CE for ¹H, ¹³C, and ¹⁵N nuclear spins. We compare 263 GHz electron paramagnetic resonance (EPR) spectra with the obtained DNP field profiles and discuss possible CE matching conditions within the high‐spin system and the influence of dipolar broadening of the EPR signal. Our findings foster the understanding of the CE mechanism and the design of high‐spin PAs for specific applications of DNP.     

Triplet Fullerenes as Prospective Spin Labels for Nanoscale Distance Measurements by Pulsed Dipolar EPR Spectroscopy

Precise nanoscale distance measurements by pulsed electron paramagnetic resonance (EPR) spectroscopy play a crucial role in structural studies of biomolecules. The properties of the spin labels used in this approach determine the sensitivity limits, attainable distances, and proximity to biological conditions. Herein, we propose and validate the use of photoexcited fullerenes as spin labels for pulsed dipolar (PD) EPR distance measurements. Hyperpolarization and the narrower spectrum of fullerenes compared to other triplets (e.g., porphyrins) boost the sensitivity, and superior relaxation properties allow PD EPR measurements up to a near‐room temperature. This approach is demonstrated using fullerene–nitroxide and fullerene–triarylmethyl pairs, as well as a supramolecular complex of fullerene with nitroxide‐labeled protein. Photoexcited triplet fullerenes can be considered as new spin labels with outstanding spectroscopic properties for future structural studies of biomolecules.     

Binding orientation and activity determinants of the antimicrobial peptide cryptdin-4 revealed by potency of mutants

Cryptdin-4 is a β-sheet antimicrobial peptide of the defensin family that is found in the immune system of mice. Several structure–activity studies of this peptide have previously been conducted, but none have been based on residue–membrane interactions as part of an overall hypothesis on the peptide's orientation in the membrane. We pursue this valuable approach by first using previously reported NMR structural data to propose a membrane-bound orientation of the peptide. Four mutants are then strategically designed to modulate membrane perturbative activity in a manner consistent with the proposed binding orientation. Membrane perturbation is evaluated using a simple fluorescence-based vesicle leakage assay using POPG to form the model membrane. Effects of peptide mutations are found to be consistent with the suggested binding orientation. This approach is successfully used to create synthetic peptides with enhanced or diminished ability to perturb membranes and also yields insights on the nature of peptide–membrane interactions.     

The dynamics and orientation of a lipophilic drug within model membranes determined by 13C solid-state NMR

Methods for determining how a drug interacts with cellular membranes at the molecular level can give valuable insight into the mode of action of the drug and its absorption, distribution and metabolism profile. A procedure is described here to determine the orientation and location of the lipophilic drug trifluoperazine (TFP) intercalated into dimyristoylphosphatidylcholine (DMPC) bilayers, by using a novel combination of high-resolution solid-state nuclear magnetic resonance (SSNMR) methods to observe signals from (13)C within the drug at natural abundance. SSNMR measurements of (1)H-(13)C dipolar couplings for TFP and selective broadening of (13)C NMR peaks by paramagnetic Mn(2+) together suggest a model for the location, orientation and dynamics of the drug within lipid bilayers that offers an explanation for the lysoprotective effect of the drug at low concentrations. The experiments described are straightforward to implement and can be used for the routine analysis of drug-membrane interactions to provide useful information for drug design and structure refinement.     

Spin hamiltonian parameters for Cu(II)-prion peptide complexes from L-band electron paramagnetic resonance spectroscopy

Cu(II) is an essential element for life but is also associated with numerous and serious medical conditions, particularly neurodegeneration. Structural modeling of crystallization-resistant biological Cu(II) species relies on detailed spectroscopic analysis. Electron paramagnetic resonance (EPR) can, in principle, provide spin hamiltonian parameters that contain information on the geometry and ligand atom complement of Cu(II). Unfortunately, EPR spectra of Cu(II) recorded at the traditional X-band frequency are complicated by (i) strains in the region of the spectrum corresponding to the g(∥) orientation and (ii) potentially very many overlapping transitions in the g(⊥) region. The rapid progress of density functional theory computation as a means to correlate EPR and structure, and the increasing need to study Cu(II) associated with biomolecules in more biologically and biomedically relevant environments such as cells and tissue, have spurred the development of a technique for the extraction of a more complete set of spin hamiltonian parameters that is relatively straightforward and widely applicable. EPR at L-band (1-2 GHz) provides much enhanced spectral resolution and straightforward analysis via computer simulation methods. Herein, the anisotropic spin hamiltonian parameters and the nitrogen coordination numbers for two hitherto incompletely characterized Cu(II)-bound species of a prion peptide complex are determined by analysis of their L-band EPR spectra.     

Structure, assembly, and topology of the G185R mutant of the fourth transmembrane domain of divalent metal transporter

The mammalian iron transporter, divalent metal transporter (DMT1), is a 12-transmembrane domain integral protein, responsible for dietary iron uptake in the duodenum and iron acquisition from transferrin in peripheral tissues. Two disease-causing mutants in animals have been found and attributed to the same missense mutation (G185R), which occurs within the putative transmembrane domain 4 (TM4) of DMT1. We have characterized a synthetic 24-mer peptide, corresponding to the sequence of the TM4 of DMT1 with G185R mutation using circular dichroism (CD) and NMR spectroscopy and show that the G185R peptide assumes mainly alpha-helical conformations in various membrane-mimetic environments. Solution structures derived from NMR and molecular dynamics/simulated annealing calculations demonstrate that the peptide exhibits a highly defined alpha-helix in its middle portion, flanked by a highly flexible N-terminus and a relatively ordered C-terminus. Both the folding and location of the C-terminus in SDS micelles are regulated by pH values. Paramagnetic broadening on peptide NMR signals by spin-labeled 5- and 16-doxylstearic acids and Mn(2+) ion suggests that both the N-terminus and the helical region of the peptide are embedded in SDS micelles. Surprisingly, self-association of the peptides for both the wild type and the G185R mutant studied by CD, electrospray ionization mass spectrometry, and NMR diffusion-ordered spectroscopy demonstrated that mutation of the Gly185 to a bulky and positively charged arginine causes a different self-assembly of the peptide, e.g., from a trimer to a hexamer, which implies that the quaternary structure of integral DMT1 may be crucial for its function in vivo.     

Light-Induced Pulsed EPR Dipolar Spectroscopy on a Paradigmatic Hemeprotein

Light-induced pulsed EPR dipolar spectroscopic methods allow the determination of nanometer distances between paramagnetic sites. Here we employ orthogonal spin labels, a chromophore triplet state and a stable radical, to carry out distance measurements in singly nitroxide-labeled human neuroglobin. We demonstrate that Zn-substitution of neuroglobin, to populate the Zn(II) protoporphyrin IX triplet state, makes it possible to perform light-induced pulsed dipolar experiments on hemeproteins, extending the use of light-induced dipolar spectroscopy to this large class of metalloproteins. The versatility of the method is ensured by the employment of different techniques: relaxation-induced dipolar modulation enhancement (RIDME) is applied for the first time to the photoexcited triplet state. In addition, an alternative pulse scheme for laser-induced magnetic dipole (LaserIMD) spectroscopy, based on the refocused-echo detection sequence, is proposed for accurate zero-time determination and reliable distance analysis.     

Molecular insight into the electrostatic membrane surface potential by 14n/31p MAS NMR spectroscopy: nociceptin-lipid association

Exploiting naturally abundant (14)N and (31)P nuclei by high-resolution MAS NMR (magic angle spinning nuclear magnetic resonance) provides a molecular view of the electrostatic potential present at the surface of biological model membranes, the electrostatic charge distribution across the membrane interface, and changes that occur upon peptide association. The spectral resolution in (31)P and (14)N MAS NMR spectra is sufficient to probe directly the negatively charged phosphate and positively charged choline segment of the electrostatic P(-)-O-CH(2)-CH(2)-N(+)(CH(3))(3) headgroup dipole of zwitterionic DMPC (dimyristoylphosphatidylcholine) in mixed-lipid systems. The isotropic shifts report on the size of the potential existing at the phosphate and ammonium group within the lipid headgroup while the chemical shielding anisotropy ((31)P) and anisotropic quadrupolar interaction ((14)N) characterize changes in headgroup orientation in response to surface potential. The (31)P/(14)N isotropic chemical shifts for DMPC show opposing systematic changes in response to changing membrane potential, reflecting the size of the electrostatic potential at opposing ends of the P(-)-N(+) dipole. The orientational response of the DMPC lipid headgroup to electrostatic surface variations is visible in the anisotropic features of (14)N and (31)P NMR spectra. These features are analyzed in terms of a modified "molecular voltmeter" model, with changes in dynamic averaging reflecting the tilt of the C(beta)-N(+)(CH)(3) choline and PO(4)(-) segment. These properties have been exploited to characterize the changes in surface potential upon the binding of nociceptin to negatively charged membranes, a process assumed to proceed its agonistic binding to its opoid G-protein coupled receptor.