Solid-state NMR Approach Research Articles

New tools for G-protein coupled receptor (GPCR) drug discovery: combination of baculoviral expression system and solid state NMR

Biotechnology using molecular biology, biochemistry, biophysics, and computational approaches provides an alternative approach for classical pharmacological screening to look at ligand-receptor interactions and receptor specificity, which should support the design of selective drugs based on detailed structural principles. This review addresses specific approaches to study function, structure and relevance of a major pharmaceutical target, namely the G-Protein Coupled Receptors (GPCRs). The main aim of this review has been to exploit and combine GPCR over-expression in a baculoviral expression system with solid-state MAS NMR (ssNMR) approaches for the elucidation of electronic structures of the coordinating ligands/drugs and their modes of interactions with the GPCRs. This review summarizes the approaches, possible future experiments and developments using the above combination of tools for GPCR drug discovery.

A high-resolution solid-state NMR approach for the structural studies of bicelles.

Bicelles are increasingly being used as membrane mimicking systems in NMR experiments to investigate the structure of membrane proteins. In this study, we demonstrate the effectiveness of a 2D solid-state NMR approach that can be used to measure the structural constraints, such as heteronuclear dipolar couplings between 1H, 13C, and 31P nuclei, in bicelles without the need for isotopic enrichment. This method does not require a high radio frequency power unlike the presently used rotating-frame separated-local-field (SLF) techniques, such as PISEMA. In addition, multiple dipolar couplings can be measured accurately, and the presence of a strong dipolar coupling does not suppress the weak couplings. High-resolution spectra obtained from magnetically aligned DMPC:DHPC bicelles even in the presence of peptides suggest that this approach will be useful in understanding lipid-protein interactions that play a vital role in shaping up the function of membrane proteins.

Solid-state 31P CP/MAS and static 65Cu NMR characterization of polycrystalline copper(I) dialkyldithiophosphate clusters

Polycrystalline tetra-nuclear Cu4[S2P(O-i-C3H7)2]4, hexa-nuclear Cu6[S2P(OC2H5)2]6, and octa-nuclear Cu8[S2P(O-i-C4H9)2]6(S) complexes were synthesized and analyzed by means of solid-state 31P CP/MAS and 65Cu static NMR spectroscopy. The symmetries of the electronic environments around each P-site were estimated from the 31P chemical shift anisotropy (CSA) parameters, Deltaaniso and eta. The 65Cu chemical shift and quadrupolar splitting parameters obtained from the experimental 65Cu NMR spectra of the polycrystalline Cu(I)-complexes are presented. A solid-state NMR approach for the elucidation of the stereochemistry of poly-nuclear Cu(I) dithiophosphate complexes, when the structural analysis of the systems by single-crystal X-ray diffraction is not readily available, is proposed.

Determining the Geometry of Hydrogen Bonds in Solids with Picometer Accuracy by Quantum‐Chemical Calculations and NMR Spectroscopy

The structure of multiply hydrogen-bonded systems is determined with picometer accuracy by a combined solid-state NMR and quantum-chemical approach. On the experimental side, advanced 1H-15N dipolar recoupling NMR techniques are capable of providing proton-nitrogen distances of up to about 250 pm with an accuracy level of +/-1 pm for short distances (i.e., around 100 pm) and +/-5 pm for longer ones (i.e., 180 to 250 pm). The experiments were performed under fast magic-angle spinning, which ensures sufficient dipolar decoupling and spectral resolution of the 1H resonance lines. On the quantum-chemical side, the structures of the hydrogen-bonded systems were computationally optimised, yielding complete sets of nitrogen-proton and proton-proton distances, which are essential for correctly interpreting the experimental NMR data. In this way, nitrogen-proton distances were determined with picometer accuracy, so that vibrational averaging effects on dipole-dipole couplings need to be considered. The obtained structures were finally confirmed by the complete agreement of computed and experimental 'H and '5N chemical shifts. This demonstrates that solid-state NMR and quantum-chemical methods ideally complement each other and, in a combined manner, represent a powerful approach for reliable, high-precision structure determination whenever scattering techniques are inapplicable.

Adiabatic TOBSY in rotating solids.

A MAS solid state NMR approach for achieving efficient scalar coupling mediated through-bond (13)C chemical shift correlations of the aliphatic carbons in uniformly labelled peptides/proteins is described. The method involves the application of a continuous train of adiabatic inversion pulses, as in the adiabatic TOCSY experiments carried out in solution state NMR studies. While rotor synchronised application of adiabatic inversion pulses leads to dipolar correlations, it is shown here via numerical simulations and experimental measurements that asynchronous application of adiabatic pulses can facilitate the mapping of through-bond connectivities. The method employs a suitable phasing scheme for generating the desired isotropic mixing Hamiltonian and requires moderate (13)C RF field strength only.

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.]

Stereochemical analysis by solid-state NMR: structural predictions in ambuic acid.

Relative stereochemistry is predicted for ambuic acid using a novel solid-state NMR approach. This NMR technique entails a comparison of measured shift tensor principal values with computed values for all diastereomers, allowing the selection of a best-fit structure. The proposed method extends previous solution NMR structural data by simultaneously modeling with high statistical probability hydrogen-bonding arrangements and molecular conformation at two positions. A dimeric structure is proposed for ambuic acid based on the initial poor fit of the carboxyl carbon tensors to a monomeric model. The dimer model, consisting of hydrogen bonding between pairs of neighboring carboxyl groups, reduces the root mean square error at the carboxy tensor by a factor of 2.7. Lattice details are thus also described by the proposed approach. The structural characterization method presented is of general applicability and may be especially useful for characterizing difficult to crystallize or hydrogen-poor materials.

Structure determination of membrane proteins by NMR spectroscopy

Current strategies for determining the structures of membrane proteins in lipid environments by NMR spectroscopy rely on the anisotropy of nuclear spin interactions, which are experimentally accessible through experiments performed on weakly and completely aligned samples. Importantly, the anisotropy of nuclear spin interactions results in a mapping of structure to the resonance frequencies and splittings observed in NMR spectra. Distinctive wheel-like patterns are observed in two-dimensional 1H-15N heteronuclear dipolar/15N chemical shift PISEMA (polarization inversion spin-exchange at the magic angle) spectra of helical membrane proteins in highly aligned lipid bilayer samples. One-dimensional dipolar waves are an extension of two-dimensional PISA (polarity index slant angle) wheels that map protein structures in NMR spectra of both weakly and completely aligned samples. Dipolar waves describe the periodic wave-like variations of the magnitudes of the heteronuclear dipolar couplings as a function of residue number in the absence of chemical shift effects. Since weakly aligned samples of proteins display these same effects, primarily as residual dipolar couplings, in solution NMR spectra, this represents a convergence of solid-state and solution NMR approaches to structure determination.

Dipolar waves as NMR maps of protein structure.

The anisotropy of nuclear spin interactions results in a unique mapping of structure to the resonance frequencies and split tings observed in NMR spectra, however, the determination of molecular structure from experimentally measured spectral parameters is complicated by angular ambiguities resulting from the symmetry properties of dipole-dipole and chemical shift interactions. This issue can be addressed through the periodicity inherent in secondary structure elements, which can be used as an index of topology. Distinctive wheel-like patterns are observed in two-dimensional 1H-15N heteronuclear dipolar/15N chemical shift PISEMA (polarization inversion spin-exchange at the magic angle) spectra of helical membrane proteins in highly aligned lipid bilayer samples. One-dimensional dipolar waves are an extension of two-dimensional PISA (polarity index slant angle) wheels to map protein structure in NMR spectra of both highly and weakly aligned samples. Dipolar waves describe the periodic wavelike variations of the magnitudes of the static heteronuclear dipolar couplings as a function of residue number in the absence of chemical shift effects. Weakly aligned samples of proteins display these same effects, primarily as residual dipolar couplings (RDCs), in solution NMR spectra. The corresponding properties of the RDCs in solution NMR spectra of weakly aligned helices represent a convergence of solid-state and solution NMR approaches to structure determination.

Studies of surfaces through molecular probe adsorption and solid-state NMR.

The interaction between the basic probe trimethylphosphine oxide and the Brønsted acid sites of a silica-alumina has been spectroscopically resolved for the first time using a new solid-state NMR approach that opens the possibilities for the investigation of surfaces.

Block Copolymer−Ceramic Hybrid Materials from Organically Modified Ceramic Precursors

The study of amphiphilic polymer based functional organic−inorganic hybrid materials is an emerging research area offering enormous scientific and technological promise. Here, we show that employing poly(isoprene-block-ethylene oxide) block copolymers (PI-b-PEO) and a silicon precursor, which contains a polymerizable organic moiety, unprecedented morphology control on the nanoscale is obtained. This control is based on a unique polymer−ceramic interface, which is characterized using a multi-nuclei solid-state NMR approach. The results show that the hydrophilic parts of the polymer are completely integrated into the ceramic phase, thereby leading to a quasi “two-phase system”, allowing for a more rational hybrid morphology design based on the current understanding of the phase behavior of block copolymers and copolymer−homopolymer mixtures. Examination of the full phase space of the hybrid materials reveals the existence of a new bicontinuous cubic structure that was not known to exist in polymer systems. SAXS and TEM data of this structure are consistent with a so-called “Plumber's nightmare” morphology. Selective solvent swelling of the hydrophobic parts of the hybrids leads to isolated nano-objects of different shapes, sizes, and compositions, while heat treatment generates mesoporous ceramic materials in which the mesostructure of the precursor material is preserved. Potential applications of these materials ranging from nanobiotechnology to catalysis are discussed.

Localization of 23Na+ in a DNA Quadruplex by High-Field Solid-State NMR

Monovalent cations such as Na+, K+, and NH4+ are known to stabilize DNA quadruplexes formed of guanine-quartets. Such structures readily form from the guanine-rich repeat sequences found in telomeres, the physical ends of eukaryotic chromosomes. We present a solid-state NMR approach for studying ions associated with G-quartets based on the direct NMR observation of 23Na+ ion resonances and report new methods for studying quadrupolar nuclei in biological solids. In the tetraplex forming oligonucleotide d(TG4T), high-field (17.6 T) NMR spectra cleanly resolve three distinct classes of sodium ions. A high-resolution 2D-MQMAS spectrum established the assignment of an amorphously broadened signal at −19 ppm (relative to 0.1 M NaCl) to surface-bound Na+ ions. Two-dimensional nutation spectroscopy was used to indicate the relative size of the quadrupole coupling for each line, while a standard exchange experiment established a correlation between surface (−19 ppm) and channel-bound (6.8 ppm) Na+ ions. Finally, f...

Lipid Matrix Properties in Cationic Membranes Interacting with Anionic Polyelectrolytes: A Solid-State NMR Approach

A combined solid-state NMR and small-angle X-ray scattering approach was employed to investigate morphology and lateral packing properties of mixed zwitterionic and cationic lipid membranes in the presence of an anionic polyelectrolyte. Binary mixtures of zwitterionic DMPC and cationic DODAP form stable lipid bilayers over a wide range of mixing ratios. Addition of negatively charged dextran sulfate (DS) leads to the formation of densely packed lipid bilayer stacks with the polyelectrolyte sandwiched between opposing lipid surfaces. Bridging of polyelectrolyte strands between bilayer surfaces provides attractive forces to overcompensate electrostatic and hydration repulsion, resulting in close approach of apposing membranes. The equilibrium surface distance is controlled by a balance of electrostatic, hydration, bridging, and entropic forces. Lipid molecules in the mixed membranes remain homogeneously distributed in both the absence and presence of DS. DS binds exclusively to cationic DODAP. As a result o...

A Solid State NMR Approach to the Structure-Activity Relationship of the Nucleoside Transport Protein NupC of Escherichia coli

A Solid State NMR Approach to the Structure-Activity Relationship of the Nucleoside Transport Protein NupC of <i>Escherichia coli</i>

Structural and orientational information of the membrane embedded M13 coat protein by 13C-MAS NMR spectroscopy

Oriented and unoriented M13 coat protein, incorporated into dimyristoyl phosphatidylcholine bilayers, has been studied by 13C-magic angle spinning nuclear magnetic resonance (MAS NMR) spectroscopy. Rotational resonance experiments provided two distance constraints between Cα and CO positions of the labelled residues Val-29/Val-30 (0.4±0.5nm) and Val-29/Val-31 (0.45±0.5nm) in its hydrophobic domain. The derived dihedral angles ( Φ, Ψ) for Val-30 revealed a local α-helical conformation. 13C-CP-MAS experiments on uniformly aligned samples (MAOSS experiments) using the 13CO labelled site of Val-30 allowed the determination of the helix tilt (20°±10°) in the membrane. It is shown that one uniform MAS high-resolution solid state NMR approach can be used to obtain structural and orientational data.

Determination of Torsion Angles in Proteins and Peptides Using Solid State NMR

A combination of Solid State NMR (SSNMR) dipolar recoupling and double quantum CSA−CSA correlation experiments are used to determine the Ramachandran angles φ and ι in a crystalline tripeptide (AGG) and a 14 amino acid peptide designed to be helical. The advantage of the SSNMR approach described herein is the ability to measure both φ and ψ to high resolution using a single noncrystalline, doubly carbonyl labeled peptide. It is also shown that DRAWS and DQDRAWS data are insensitive to 14N−13C and 15N−13C dipolar couplings making corrections for these effects unnecessary. Extremes in secondary structure (e.g., α-helix vs β-sheet) can be discerned by simple inspection of DQDRAWS spectra. Subtleties in secondary structure (α-helix vs 310-helix) can be distinguished by simulation of the DQDRAWS spectrum.

Deuterium-MAS NMR Spectroscopy on Oriented Membrane Proteins: Applications to Photointermediates of Bacteriorhodopsin

We present the first application of MAOSS (magic angle oriented sample spinning) NMR spectroscopy to a large membrane protein. This new solid-state NMR approach is used to study the orientation of the deuterated methyl group in [18-CD3]-retinal in oriented bacteriorhodopsin in both the photocycle ground state (bR568) and in the photo intermediate state M412. Deuterium MAS spectra consist of a set of narrow spinning sidebands if the sample spinning rate does not exceed the anisotropy of the quadrupole interaction. In ordered systems, such as proteins in oriented membranes, each sideband intensity is orientationally dependent. The observed MAS sideband pattern is modulated in a highly sensitive way by changes in the molecular orientation of the CD3 group during the transition from all-trans- to 13-cis-retinal upon photoactivation. The significant improvement in spectral sensitivity and resolution, compared to static NMR on oriented samples, allows a reliable and precise data analysis even from lower spin concentrations and has more general consequences for studying oriented membrane proteins by NMR. MAOSS NMR is shown to be a feasible method for the accurate determination of local molecular orientations in large molecular systems which are currently a challenge for crystallography.

Membrane protein structure determination by solid state NMR.

In the last 3–5 years, solid state NMR has matured as a method for defining structural details of membrane-embedded proteins and peptides. It is still not, and may never be, a method of choice for complete structural resolution, but the power of the approach for certain systems, and in resolving details for binding sites of ligands and specific parts of proteins, is being proved. As with solution state NMR, stable NMR isotope (2H, 13C, 15N, 19F) incorporation into such large and complex biomolecules has made a tremendous impact in the area, aiding spectral assignments, increasing the amount and value of the information gained, and permitting identification of specific functional intermediates. The practical details of handling membrane proteins, which has for so long dogged every structural and biochemical study of membrane proteins, including crystallography, are not absent in the solid state NMR approach. It is simply that the protein can be studied in situ, or at least in reconstituted systems when the purified or isolated proteins can be reintroduced into a membrane for study. In addition, the molecular weight limit (Mr < 30 000) of solution state NMR (due to the slow molecular tumbling rate with respect to the applied field), is not limiting with solid state methods. Solid state NMR methods can now be used to study membrane proteins and peptides, but support from chemical, molecular biological and biochemical approaches are required to produce systems suitable for giving the desired molecular information. Specialized instrumentation is also required (high power NMR probes with spinning or static capability, specific pulse methods, good temperature stability and wide bore magnets) and some applications can present a challenge experimentally in setting up the NMR instrument. However, the method is emerging as a useful addition for the structural biologist.

Identification and mobility of deuterated residues in peptides and proteins by – solid-state NMR

Abstract We present a solid-state NMR approach for 13 C chemical-shift identification and 2 H lineshape characterization of amino-acid residues in peptides and proteins deuterated by amide hydrogen/deuteron exchange. The technique exploits heteronuclear 13 C – 2 H dipolar couplings to correlate 13 C nuclei and nearby deuterons. Magic-angle spinning provides high sensitivity and 13 C chemical-site resolution. The simplest version of the experiment, which is closely related to REDOR, yields a 13 C spectrum permitting identification of the deuterated residues. In the full two-dimensional experiment, segmental dynamics are characterized in terms of 2 H -NMR lineshapes. The technique is demonstrated on dipeptides and a 14-kDa protein, with 13 C in natural abundance.

Structural descriptions of ligands in their binding site of integral membrane proteins at near physiological conditions using solid-state NMR

Using solid-state NMR approaches, it is now possible to define the structure and dynamics of binding for a small, isotopically (2H, 13C, 19F, 15N) labelled ligand, prosthetic group or solute in its binding site of a membrane-bound protein at near physiological conditions in natural membrane fragments or in reconstituted complexes. Studies of oriented membranes permit the orientational bond vectors of labelled groups to be determined to good precision, as shown for retinal in bacteriorhodopsin and bovine rhodopsin. Using novel magic angle spinning NMR methods on membrane dispersions, high-resolution NMR spectra can be obtained. Dipolar couplings can be reintroduced into the spectrum of labelled ligands in their binding sites of membrane-bound proteins to give interatomic distances to high precision (±0.5 A). Relaxation and cross-polarization data give estimates for the kinetics for on-off rates for binding. In addition, chemical shifts can be measured directly to help provide details of the binding environment for a bound ligand, as shown for analogues of drugs used in peptic ulcer treatment in the gastric ATPase, and for acetylcholine in the acetylcholine receptor.