Local and global dynamics of the G protein-coupled receptor CXCR1

The local and global dynamics of the chemokine receptor CXCR1 are characterized using a combination of solution NMR and solid-state NMR experiments. In isotropic bicelles (q = 0.1), only 13% of the expected number of backbone amide resonances is observed in (1)H/(15)N HSQC solution NMR spectra of uniformly (15)N-labeled samples; extensive deuteration and the use of TROSY made little difference in the 800 MHz spectra. The limited number of observed amide signals is ascribed to mobile backbone sites and assigned to specific residues in the protein; 19 of the signals are from residues at the N-terminus and 25 from residues at the C-terminus. The solution NMR spectra display no evidence of local backbone motions from residues in the transmembrane helices or interhelical loops of CXCR1. This finding is reinforced by comparisons of solid-state NMR spectra of both magnetically aligned and unoriented bilayers containing either full-length or doubly N- and C-terminal truncated CXCR1 constructs. CXCR1 undergoes rapid rotational diffusion about the normal of liquid crystalline phospholipid bilayers; reductions in the frequency span and a change to axial symmetry are observed for both carbonyl carbon and amide nitrogen chemical shift powder patterns of unoriented samples containing (13)C- and (15)N-labeled CXCR1. In contrast, when the phospholipids are in the gel phase, CXCR1 does not undergo rapid global reorientation on the 10(4) Hz time scale defined by the carbonyl carbon and amide nitrogen chemical shift powder patterns.     

Solid-state NMR structure determination

Biological applications of solid-state NMR (SS-NMR) have been predominantly in the area of model membrane systems. Increasingly the focus has been membrane peptides and proteins. SS-NMR is able to provide information about how the peptides or proteins interact with the lipids or other peptides/proteins in the membrane, their effect on the membrane and the location of the peptides or proteins relative to the membrane surface. Recent developments in biological SS-NMR have been made possible by improvements in labelling and NMR techniques. This review discusses aligned systems and magic angle spinning techniques, bilayers and bicelles, and measurement of chemical shift anisotropy and dipolar coupling. A number of specific experiments such as cross polarization, rotational resonance, REDOR, PISEMA, MAOSS and multidimensional experiments are described. In addition to 2H, 13C and 15N, recent solid-sate 1H, 19F and 17O NMR work is discussed. Several examples of the use of SS-NMR to determine the structure of membrane peptides and proteins are given.     

The structural properties of the transmembrane segment of the integral membrane protein phospholamban utilizing (13)C CPMAS, (2)H, and REDOR solid-state NMR spectroscopy

Solid-state NMR spectroscopic techniques were used to investigate the secondary structure of the transmembrane peptide phospholamban (TM-PLB), a sarcoplasmic Ca(2+) regulator. (13)C cross-polarization magic angle spinning spectra of (13)C carbonyl-labeled Leu39 of TM-PLB exhibited two peaks in a pure 1-palmitoyl-2-oleoyl-phosphocholine (POPC) bilayer, each due to a different structural conformation of phospholamban as characterized by the corresponding (13)C chemical shift. The addition of a negatively charged phospholipid (1-palmitoyl-2-oleoylphosphatidylglycerol (POPG)) to the POPC bilayer stabilized TM-PLB to an alpha-helical conformation as monitored by an enhancement of the alpha-helical carbonyl (13)C resonance in the corresponding NMR spectrum. (13)C-(15)N REDOR solid-state NMR spectroscopic experiments revealed the distance between the (13)C carbonyl carbon of Leu39 and the (15)N amide nitrogen of Leu42 to be 4.2+/-0.2A indicating an alpha-helical conformation of TM-PLB with a slight deviation from an ideal 3.6 amino acid per turn helix. Finally, the quadrupolar splittings of three (2)H labeled leucines (Leu28, Leu39, and Leu51) incorporated in mechanically aligned DOPE/DOPC bilayers yielded an 11 degrees +/-5 degrees tilt of TM-PLB with respect to the bilayer normal. In addition to elucidating valuable TM-PLB secondary structure information, the solid-state NMR spectroscopic data indicates that the type of phospholipids and the water content play a crucial role in the secondary structure and folding of TM-PLB in a phospholipid bilayer.     

GFT NMR Experiments for Polypeptide Backbone and 13Cβ Chemical Shift Assignment

(4,3)D, (5,3)D and (5,2)D GFT triple resonance NMR experiments are presented for polypeptide backbone and (13)C(beta) resonance assignment of (15)N/(13)C labeled proteins. The joint sampling of m = 2, 3 or 4 indirect chemical shift evolution periods of 4D and 5D NMR experiments yields the measurement of 2(m) - 1 linear combinations of shifts. To obtain sequential assignments, these are matched in corresponding experiments delineating either intra or interresidue correlations. Hence, an increased set of matches is registered when compared to conventional approaches, and the 4D or 5D information allows one to efficiently break chemical shift degeneracy. Moreover, comparison of single-quantum chemical shifts obtained after a least squares fit using either the intra or the interresidue data demonstrates that GFT NMR warrants highly accurate shift measurements. The new features of GFT NMR based resonance assignment strategies promise to be of particular value for establishing automated protocols.     

Mars - robust automatic backbone assignment of proteins

MARS a program for robust automatic backbone assignment of (13)C/(15)N labeled proteins is presented. MARS does not require tight thresholds for establishing sequential connectivity or detailed adjustment of these thresholds and it can work with a wide variety of NMR experiments. Using only (13)C(alpha)/(13)C(beta) connectivity information, MARS allows automatic, error-free assignment of 96% of the 370-residue maltose-binding protein. MARS can successfully be used when data are missing for a substantial portion of residues or for proteins with very high chemical shift degeneracy such as partially or fully unfolded proteins. Other sources of information, such as residue specific information or known assignments from a homologues protein, can be included into the assignment process. MARS exports its result in SPARKY format. This allows visual validation and integration of automated and manual assignment.     

Solid-state NMR study and assignments of the KcsA potassium ion channel of S. lividans

The extraordinary efficiency and selectivity of potassium channels have made them ideal systems for biophysical and functional studies of ion conduction. We carried out solid-state NMR studies of the selectivity filter region of the protein. Partial site-specific assignments of the NMR signals were obtained based on high field multidimensional solid-state NMR spectra of uniformly (13)C, (15)N enriched KcsA potassium channel from Streptomyces lividans. Both backbone and sidechain atoms were assigned for residues V76-D80 and P83-L90, in and near the selectivity filter region of the protein; this region exhibits good dispersion and useful chemical shift fingerprints. This study will enable structure, dynamic and mechanistic studies of ion conduction by NMR.     

Structure of an elastin-mimetic polypeptide by solid-state NMR chemical shift analysis

The conformation of an elastin-mimetic recombinant protein, [(VPGVG)4(VPGKG)]39, is investigated using solid-state NMR spectroscopy. The protein is extensively labeled with 13C and 15N, and two-dimensional 13C-13C and 15N-13C correlation experiments were carried out to resolve and assign the isotropic chemical shifts of the various sites. The Pro 15N, 13Calpha, and 13Cbeta isotropic shifts, and the Gly-3 Calpha isotropic and anisotropic chemical shifts support the predominance of type-II beta-turn structure at the Pro-Gly pair but reject a type-I beta-turn. The Val-1 preceding Pro adopts mostly beta-sheet torsion angles, while the Val-4 chemical shifts are intermediate between those of helix and sheet. The protein exhibits a significant conformational distribution, shown by the broad line widths of the 15N and 13C spectra. The average chemical shifts of the solid protein are similar to the values in solution, suggesting that the low-hydration polypeptide maintains the same conformation as in solution. The ability to measure these conformational restraints by solid-state NMR opens the possibility of determining the detailed structure of this class of fibrous proteins through torsion angles and distances.     

Analysis of the structure of synthetic and natural melanins by solid-phase NMR

The structures of one synthetic and two natural melanins are examined by solid-state NMR using cross polarization, magic angle sample spinning, and high-power proton decoupling. The structural features of synthetic dopa melanin are compared to those of melanin from malignant melanoma cells grown in culture and sepia melanin from squid ink. Natural abundance 13C and 15N spectra show resonances consistent with known pyrrolic and indolic structures within the heterogeneous biopolymer; 13C spectra indicate the presence of aliphatic residues in all three materials. These solid-phase experiments illustrate the promise of solid-phase NMR for elucidating structural information from insoluble biomaterials.     

The membrane interactions of antimicrobial peptides revealed by solid-state NMR spectroscopy

Solid-state NMR spectroscopic techniques provide valuable information about the structure, dynamics and topology of membrane-inserted polypeptides. In particular antimicrobial peptides (or 'host defence peptides') have early on been investigated by solid-state NMR spectroscopy and many technical innovations in this domain have been developed with the help of these compounds when reconstituted into oriented phospholipid bilayers. Using solid-state NMR spectroscopy it could be shown for the first time that magainins or derivatives thereof exhibit potent antimicrobial activities when their cationic amphipathic helix is oriented parallel to the bilayer surface, a configuration found in later years for many other linear cationic amphipathic peptides. In contrast transmembrane alignments or lipid-dependent tilt angles have been found for more hydrophobic sequences such as alamethicin or β-hairpin antimicrobials. This review presents various solid-state NMR approaches and develops the basic underlying concept how angular information can be obtained from oriented samples. It is demonstrated how this information is used to calculate structures and topologies of peptides in their native liquid-disordered phospholipid bilayer environment. Special emphasis is given to discuss which NMR parameters provide the most complementary information, the minimal number of restraints needed and the effect of motions on the analysis of the NMR spectra. Furthermore, recent (31)P and (2)H solid-state NMR measurements of lipids are presented including some unpublished data which aim at investigating the morphological and structural changes of oriented or non-oriented phospholipids. Finally the structural models that have been proposed for the mechanisms of action of these peptides will be presented and discussed in view of the solid-state NMR and other biophysical experiments.     

Solid-state NMR studies of the structure and mechanisms of proteins

Magic-angle spinning solid-state NMR experiments are well suited to investigating the structures and mechanisms of important proteins that are inaccessible to X-ray crystallography and solution NMR spectroscopy, including membrane proteins and disease-related protein aggregates. Good progress has been made in the development of methods for the complete structure determination of small (<20 kDa) solid proteins using uniformly 13C, 15N-labeled samples. Studies of selectively labeled proteins focusing on labeled active sites have yielded insights into the mechanisms of enzymes and of membrane proteins involved in energy and signal transduction. Studies of selectively labeled synthetic peptides have yielded structural models for biomedically important systems, including amyloid fibrils and surface-associated peptides involved in biomineralization and cell adhesion. Novel NMR and biochemical methods are being developed to target solid-state NMR experiments within large proteins and whole cells. These approaches are being used to investigate mechanisms of transmembrane signaling by membrane receptors and to characterize binding interactions between antibiotics and bacterial cell walls. Thus, solid-state NMR is proving to be a valuable biophysical tool for probing structure and dynamics in a wide range of biomolecules.     

Structure of tightly membrane-bound mastoparan-X, a G-protein-activating peptide, determined by solid-state NMR

The structure of mastoparan-X (MP-X), a G-protein activating peptide from wasp venom, in the state tightly bound to anionic phospholipid bilayers was determined by solid-state NMR spectroscopy. Carbon-13 and nitrogen-15 NMR signals of uniformly labeled MP-X were completely assigned by multidimensional intraresidue C-C, N-CalphaCbeta, and N-Calpha-C', and interresidue Calpha-CalphaCbeta, N-CalphaCbeta, and N-C'-Calpha correlation experiments. The backbone torsion angles were predicted from the chemical shifts of 13C', 13Calpha, 13Cbeta, and 15N signals with the aid of protein NMR database programs. In addition, two 13C-13C and three 13C-15N distances between backbone nuclei were precisely measured by rotational resonance and REDOR experiments, respectively. The backbone structure of MP-X was determined from the 26 dihedral angle restraints and five distances with an average root-mean-square deviation of 0.6 A. Peptide MP-X in the bilayer-bound state formed an amphiphilic alpha-helix for residues Trp3-Leu14 and adopted an extended conformation for Asn2. This membrane-bound conformation is discussed in relation to the peptide's activities to form pores in membranes and to activate G-proteins. This study demonstrates the power of multidimensional solid-state NMR of uniformly isotope-labeled molecules and distance measurements for determining the structures of peptides bound to lipid membranes.     

Structure and protein environment of the retinal chromophore in light- and dark-adapted bacteriorhodopsin studied by solid-state NMR

Our previous solid-state 13C NMR studies on bR have been directed at characterizing the structure and protein environment of the retinal chromophore in bR568 and bR548, the two components of the dark-adapted protein. In this paper, we extend these studies by presenting solid-state NMR spectra of light-adapted bR (bR568) and examining in more detail the chemical shift anisotropy of the retinal resonances near the ionone ring and Schiff base. Magic angle spinning (MAS) 13C NMR spectra were obtained of bR568, regenerated with retinal specifically 13C labeled at positions 12-15, which allowed assignment of the resonances observed in the dark-adapted bR spectrum. Of particular interest are the assignments of the 13C-13 and 13C-15 resonances. The 13C-15 chemical resonance for bR568 (160.0 ppm) is upfield of the 13C-15 resonance for bR548 (163.3 ppm). This difference is attributed to a weaker interaction between the Schiff base and its associated counterion in bR568. The 13C-13 chemical shift for bR568 (164.8 ppm) is close to that of the all-trans-retinal protonated Schiff base (PSB) model compound (approximately 162 ppm), while the 13C-13 resonance for bR548 (168.7 ppm) is approximately 7 ppm downfield of that of the 13-cis PSB model compound. The difference in the 13C-13 chemical shift between bR568 and bR548 is opposite that expected from the corresponding 15N chemical shifts of the Schiff base nitrogen and may be due to conformational distortion of the chromophore in the C13 = C14-C15 bonds.(ABSTRACT TRUNCATED AT 250 WORDS)     

The folding pathway of an FF domain: characterization of an on-pathway intermediate state under folding conditions by (15)N, (13)C(alpha) and (13)C-methyl relaxation dispersion and (1)H/(2)H-exchange NMR spectroscopy

The FF domain from the human protein HYPA/FBP11 folds via a low-energy on-pathway intermediate (I). Elucidation of the structure of such folding intermediates and denatured states under conditions that favour folding are difficult tasks. Here, we investigated the millisecond time-scale equilibrium folding transition of the 71-residue four-helix bundle wild-type protein by (15)N, (13)C(alpha) and methyl(13)C Carr-Purcell-Meiboom-Gill (CPMG) NMR relaxation dispersion experiments and by (1)H/(2)H-exchange measurements. The relaxation data for the wild-type protein fitted a simple two-site exchange process between the folded state (F) and I. Destabilization of F in mutants A17G and Q19G allowed the detection of the unfolded state U by (15)N CPMG relaxation dispersion. The dispersion data for these mutants fitted a three-site exchange scheme, U<-->I<-->F, with I populated higher than U. The kinetics and thermodynamics of the folding reaction were obtained via temperature and urea-dependent relaxation dispersion experiments, along with structural information on I from backbone (15)N, (13)C(alpha) and side-chain methyl (13)C chemical shifts, with further information from protection factors for the backbone amide groups from (1)H/(2)H-exchange. Notably, helices H1-H3 are at least partially formed in I, while helix H4 is largely disordered. Chemical shift differences for the methyl (13)C nuclei suggest a paucity of stable, native-like hydrophobic interactions in I. These data are consistent with Phi-analysis of the rate-limiting transition state between I and F. The combination of relaxation dispersion and Phi data can elucidate whole experimental folding pathways.     

Expression and initial structural insights from solid-state NMR of the M2 proton channel from influenza A virus

The M2 protein from influenza A virus has been expressed, purified, and reconstituted into DMPC/DMPG liposomes. SDS-PAGE analysis of reconstituted M2 protein in DMPC/DMPG liposomes demonstrates a stable tetrameric preparation. Circular dichroism spectra of the intact M2 protein in detergent indicate 67% alpha-helix. The uniformly (15)N-labeled M2 protein and both (15)N-Val- and (15)N-Leu-labeled M2 protein have been expressed from defined M9 media. The (1)H-(15)N HSQC (heteronuclear single quantum correlation) solution NMR experiments have been performed on the amino acid specific labeled protein in 300 mM SDS-d(25) micelles, and the data indicate a homogeneous preparation. The reconstituted M2/DMPC/DMPG proteoliposomes were used for preparing uniformly aligned solid-state NMR samples for (15)N-(1)H dipolar/(15)N chemical shift correlation experiments. The spectra support a transmembrane helix in M2 protein having a tilt angle of approximate 25 degrees, quantitatively similar to results obtained on the isolated M2 transmembrane peptide reconstituted in DMPC bilayers (38 degrees ). In addition, the spectra suggest that the tetrameric protein forms a symmetric or at least pseudosymmetric bundle consistent with data obtained by other research groups based on electrophysiological measurements and substituted cysteine scanning mutagenesis experiments that characterize a tetrameric structure.     

1H, 15N, and 13C NMR signal assignments of IIIGlc, a signal-transducing protein of Escherichia coli, using three-dimensional triple-resonance techniques

IIIGlc is an 18.1-kDa signal-transducing phosphocarrier protein of the phosphoenolpyruvate:glycose phosphotransferase system (PTS) of Escherichia coli. Virtually complete (98%) backbone 1H, 15N, and 13C nuclear magnetic resonance (NMR) signal assignments were determined by using a battery of triple-resonance three-dimensional (3D) NMR pulse sequences. In addition, nearly complete (1H, 95%; 13C, 85%) side-chain 1H and 13C signal assignments were obtained from an analysis of 3D 13C HCCH-COSY and HCCH-TOCSY spectra. These experiments rely almost exclusively upon one- and two-bond J couplings to transfer magnetization and to correlate proton and heteronuclear NMR signals. Hence, essentially complete signal assignments of this 168-residue protein were made without any assumptions regarding secondary structure and without the aid of a crystal structure, which is not yet available. Moreover, only three samples, one uniformly 15N-enriched, one uniformly 15N/13C-enriched, and one containing a few types of amino acids labeled with 15N and/or 13C, were needed to make the assignments. The backbone assignments together with the 3D 15N NOESY-HMQC and 13C NOESY-HMQC data have provided extensive information about the secondary structure of this protein [Pelton, J.G., Torchia, D.A., Meadow, N.D., Wong, C.-Y., & Roseman, S (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 3479-3488]. The nearly complete set of backbone and side-chain atom assignments reported herein provide a basis for studies of the three-dimensional structure and dynamics of IIIGlc as well as its interactions with a variety of membrane and cytoplasmic proteins.     

MAS solid-state NMR studies on the multidrug transporter EmrE

We study the uniformly 13C,15N isotopically enriched Escherichia coli multidrug resistance transporter EmrE using MAS solid-state NMR. Solid-state NMR can provide complementary structural information as the method allows studying membrane proteins in their native environment as no detergent is required for reconstitution. We compare the spectra obtained from wildtype EmrE to those obtained from the mutant EmrE-E14C. To resolve the critical amino acid E14, glutamic/aspartic acid selective experiments are carried out. These experiments allow to assign the chemical shift of the carboxylic carbon of E14. In addition, spectra are analyzed which are obtained in the presence and absence of the ligand TPP+.     

Comparative structure analysis of tyrosine and valine residues in unprocessed silk fibroin (silk I) and in the processed silk fiber (silk II) from Bombyx mori using solid-state (13)C,(15)N, and (2)H NMR

The solid-state (13)C CP-MAS NMR spectra of biosynthetically labeled [(13)C(alpha)]Tyr, [(13)C(beta)]Tyr, and [(13)C(alpha)]Val silk fibroin samples of Bombyx mori, in silk I (the solid-state structure before spinning) and silk II (the solid-state structure after spinning) forms, have been examined to gain insight into the conformational preferences of the semicrystalline regions. To establish the relationship between the primary structure of B. mori silk fibroin and the "local" structure, the conformation-dependent (13)C chemical shift contour plots for Tyr C(alpha), Tyr C(beta), and Val C(alpha) carbons were generated from the atomic coordinates of high-resolution crystal structures of 40 proteins and their characteristic (13)C isotropic NMR chemical shifts. From comparison of the observed Tyr C(alpha) and Tyr C(beta) chemical shifts with those predicted by the contour plots, there is strong evidence in favor of an antiparallel beta-sheet structure of the Tyr residues in the silk fibroin fibers. On the other hand, Tyr residues take a random coil conformation in the fibroin film with a silk I form. The Val residues are likely to assume a structure similar to those of Tyr residues in silk fiber and film. Solid-state (2)H NMR measurements of [3,3-(2)H(2)]Tyr-labeled B. mori silk fibroin indicate that the local mobility of the backbone and the C(alpha)-C(beta) bond is essentially "static" in both silk I and silk II forms. The orientation-dependent (i.e., parallel and perpendicular to the magnetic field) solid-state (15)N NMR spectra of biosynthetically labeled [(15)N]Tyr and [(15)N]Val silk fibers reveal the presence of highly oriented semicrystalline regions.     

NMR-based stable isotope resolved metabolomics in systems biochemistry

An important goal of metabolomics is to characterize the changes in metabolic networks in cells or various tissues of an organism in response to external perturbations or pathologies. The profiling of metabolites and their steady state concentrations does not directly provide information regarding the architecture and fluxes through metabolic networks. This requires tracer approaches. NMR is especially powerful as it can be used not only to identify and quantify metabolites in an unfractionated mixture such as biofluids or crude cell/tissue extracts, but also determine the positional isotopomer distributions of metabolites derived from a precursor enriched in stable isotopes such as (13)C and (15)N via metabolic transformations. In this article we demonstrate the application of a variety of 2-D NMR editing experiments to define the positional isotopomers of compounds present in polar and non-polar extracts of human lung cancer cells grown in either [U-(13)C]-glucose or [U-(13)C,(15)N]-glutamine as source tracers. The information provided by such experiments enabled unambiguous reconstruction of metabolic pathways, which is the foundation for further metabolic flux modeling.     

The structural properties of the transmembrane segment of the integral membrane protein phospholamban utilizing C CPMAS, H, and REDOR solid-state NMR spectroscopy

Solid-state NMR spectroscopic techniques were used to investigate the secondary structure of the transmembrane peptide phospholamban (TM-PLB), a sarcoplasmic Ca²⁺ regulator. ¹³C cross-polarization magic angle spinning spectra of ¹³C carbonyl-labeled Leu39 of TM-PLB exhibited two peaks in a pure 1-palmitoyl-2-oleoyl-phosphocholine (POPC) bilayer, each due to a different structural conformation of phospholamban as characterized by the corresponding ¹³C chemical shift. The addition of a negatively charged phospholipid (1-palmitoyl-2-oleoylphosphatidylglycerol (POPG)) to the POPC bilayer stabilized TM-PLB to an α-helical conformation as monitored by an enhancement of the α-helical carbonyl ¹³C resonance in the corresponding NMR spectrum. ¹³C-¹⁵N REDOR solid-state NMR spectroscopic experiments revealed the distance between the ¹³C carbonyl carbon of Leu39 and the ¹⁵N amide nitrogen of Leu42 to be 4.2 ± 0.2Å indicating an α-helical conformation of TM-PLB with a slight deviation from an ideal 3.6 amino acid per turn helix. Finally, the quadrupolar splittings of three ²H labeled leucines (Leu28, Leu39, and Leu51) incorporated in mechanically aligned DOPE/DOPC bilayers yielded an 11° ± 5° tilt of TM-PLB with respect to the bilayer normal. In addition to elucidating valuable TM-PLB secondary structure information, the solid-state NMR spectroscopic data indicates that the type of phospholipids and the water content play a crucial role in the secondary structure and folding of TM-PLB in a phospholipid bilayer.