Complete resolution of the solid-state NMR spectrum of a uniformly 15N-labeled membrane protein in phospholipid bilayers

Complete resolution of the amide resonances in a three-dimensional solid-state NMR correlation spectrum of a uniformly 15N-labeled membrane protein in oriented phospholipid bilayers is demonstrated. The three orientationally dependent frequencies, 1H chemical shift, 1H–15N dipolar coupling, and 15N chemical shift, associated with each amide resonance are responsible for resolution among resonances and provide sufficient angular restrictions for protein structure determination. Because the protein is completely immobilized by the phospholipids on the relevant NMR time scales (10 kHz), the linewidths will not degrade in the spectra of larger proteins. Therefore, these results demonstrate that solid-state NMR experiments can overcome the correlation time problem and extend the range of proteins that can have their structures determined by NMR spectroscopy to include uniformly 15N-labeled membrane proteins in phospholipid bilayers.

Multiple quantum solid-state NMR indicates a parallel, not antiparallel, organization of β-sheets in Alzheimer's β-amyloid fibrils

Senile plaques associated with Alzheimer's disease contain deposits of fibrils formed by 39- to 43-residue β-amyloid peptides with possible neurotoxic effects. X-ray diffraction measurements on oriented fibril bundles have indicated an extended β-sheet structure for Alzheimer's β-amyloid fibrils and other amyloid fibrils, but the supramolecular organization of the β-sheets and other structural details are not well established because of the intrinsically noncrystalline, insoluble nature of amyloid fibrils. Here we report solid-state NMR measurements, using a multiple quantum (MQ) 13C NMR technique, that probe the β-sheet organization in fibrils formed by the full-length, 40-residue β-amyloid peptide (Aβ1–40). Although an antiparallel β-sheet organization often is assumed and is invoked in recent structural models for full-length β-amyloid fibrils, the MQNMR data indicate an in-register, parallel organization. This work provides site-specific, atomic-level structural constraints on full-length β-amyloid fibrils and applies MQNMR to a significant problem in structural biology.

Photochemically induced nuclear spin polarization in reaction centers of photosystem II observed by 13C-solid-state NMR reveals a strongly asymmetric electronic structure of the P680.+ primary donor chlorophyll

We report 13C magic angle spinning NMR observation of photochemically induced dynamic nuclear spin polarization (photo- CIDNP) in the reaction center (RC) of photosystem II (PS2). The light-enhanced NMR signals of the natural abundance 13C provide information on the electronic structure of the primary electron donor P680 (chlorophyll a molecules absorbing around 680 nm) and on the pz spin density pattern in its oxidized form, P680⨥. Most centerband signals can be attributed to a single chlorophyll a (Chl a) cofactor that has little interaction with other pigments. The chemical shift anisotropy of the most intense signals is characteristic for aromatic carbon atoms. The data reveal a pronounced asymmetry of the electronic spin density distribution within the P680⨥. PS2 shows only a single broad and intense emissive signal, which is assigned to both the C-10 and C-15 methine carbon atoms. The spin density appears shifted toward ring III. This shift is remarkable, because, for monomeric Chl a radical cations in solution, the region of highest spin density is around ring II. It leads to a first hypothesis as to how the planet can provide itself with the chemical potential to split water and generate an oxygen atmosphere using the Chl a macroaromatic cycle. A local electrostatic field close to ring III can polarize the electronic charge and associated spin density and increase the redox potential of P680 by stabilizing the highest occupied molecular orbital, without a major change of color. This field could be produced, e.g., by protonation of the keto group of ring V. Finally, the radical cation electronic structure in PS2 is different from that in the bacterial RC, which shows at least four emissive centerbands, indicating a symmetric spin density distribution over the entire bacteriochlorophyll macrocycle.

Natural abundance solid-state carbon NMR studies of photosynthetic reaction centers with photoinduced polarization.

Solid-state NMR spectra of natural abundance 13C in reaction centers from photosynthetic bacteria Rhodobacter sphaeroides R-26 was measured. When the quinone acceptors were removed and continuous visible illumination of the sample was provided, exceptionally strong nuclear spin polarization was observed in NMR lines with chemical shifts resembling those of the aromatic carbons in bacteriochlorophyll and bacteriopheophytin. The observation of spin polarized 15N nuclei in bacteriochlorophyll and bacteriopheophytin was previously demonstrated with nonspecifically 15N-labeled reaction centers. Both the carbon and the nitrogen NMR studies indicate that the polarization is developed on species that carry unpaired electrons in the early electron transfer steps, including the bacteriochlorophyll dimer donor P860 and probably the bacteriopheophytin acceptor. I. Both enhanced-absorptive and emissive polarization were seen in the carbon spectrum; most lines were absorptive but the methine carbons of the porphyrin ring (alpha, beta, gamma, ) exhibited emissive polarization. The change in the sign of the hyperfine coupling at these sites indicates the existence of nodes in the spin density distribution on the tetrapyrrole cofactors flanking each methine carbon bridge.

Solid-state synthesis and mechanical unfolding of polymers of T4 lysozyme

Recent advances in single molecule manipulation methods offer a novel approach to investigating the protein folding problem. These studies usually are done on molecules that are naturally organized as linear arrays of globular domains. To extend these techniques to study proteins that normally exist as monomers, we have developed a method of synthesizing polymers of protein molecules in the solid state. By introducing cysteines at locations where bacteriophage T4 lysozyme molecules contact each other in a crystal and taking advantage of the alignment provided by the lattice, we have obtained polymers of defined polarity up to 25 molecules long that retain enzymatic activity. These polymers then were manipulated mechanically by using a modified scanning force microscope to characterize the force-induced reversible unfolding of the individual lysozyme molecules. This approach should be general and adaptable to many other proteins with known crystal structures. For T4 lysozyme, the force required to unfold the monomers was 64 ± 16 pN at the pulling speed used. Refolding occurred within 1 sec of relaxation with an efficiency close to 100%. Analysis of the force versus extension curves suggests that the mechanical unfolding transition follows a two-state model. The unfolding forces determined in 1 M guanidine hydrochloride indicate that in these conditions the activation barrier for unfolding is reduced by 2 kcal/mol.

Diversity Arrays: a solid state technology for sequence information independent genotyping

Here we present the successful application of the microarray technology platform to the analysis of DNA polymorphisms. Using the rice genome as a model, we demonstrate the potential of a high-throughput genome analysis method called Diversity Array Technology, DArT‘. In the format presented here the technology is assaying for the presence (or amount) of a specific DNA fragment in a representation derived from the total genomic DNA of an organism or a population of organisms. Two different approaches are presented: the first involves contrasting two representations on a single array while the second involves contrasting a representation with a reference DNA fragment common to all elements of the array. The Diversity Panels created using this method allow genetic fingerprinting of any organism or group of organisms belonging to the gene pool from which the panel was developed. Diversity Arrays enable rapid and economical application of a highly parallel, solid-state genotyping technology to any genome or complex genomic mixtures.

A peptide that inhibits hydroxyapatite growth is in an extended conformation on the crystal surface

Proteins play an important role in the biological mechanisms controlling hard tissue development, but the details of molecular recognition at inorganic crystal interfaces remain poorly characterized. We have applied a recently developed homonuclear dipolar recoupling solid-state NMR technique, dipolar recoupling with a windowless sequence (DRAWS), to directly probe the conformation of an acidic peptide adsorbed to hydroxyapatite (HAP) crystals. The phosphorylated hexapeptide, DpSpSEEK (N6, where pS denotes phosphorylated serine), was derived from the N terminus of the salivary protein statherin. Constant-composition kinetic characterization demonstrated that, like the native statherin, this peptide inhibits the growth of HAP seed crystals when preadsorbed to the crystal surface. The DRAWS technique was used to measure the internuclear distance between two 13C labels at the carbonyl positions of the adjacent phosphoserine residues. Dipolar dephasing measured at short mixing times yielded a mean separation distance of 3.2 ± 0.1 Å. Data obtained by using longer mixing times suggest a broad distribution of conformations about this average distance. Using a more complex model with discrete α-helical and extended conformations did not yield a better fit to the data and was not consistent with chemical shift analysis. These results suggest that the peptide is predominantly in an extended conformation rather than an α-helical state on the HAP surface. Solid-state NMR approaches can thus be used to determine directly the conformation of biologically relevant peptides on HAP surfaces. A better understanding of peptide and protein conformation on biomineral surfaces may provide design principles useful for the modification of orthopedic and dental implants with coatings and biological growth factors that are designed to enhance biocompatibility with surrounding tissue.

Correlation of the structural and functional domains in the membrane protein Vpu from HIV-1

Vpu is an 81-residue membrane protein encoded by the HIV-1 genome. NMR experiments show that the protein folds into two distinct domains, a transmembrane hydrophobic helix and a cytoplasmic domain with two in-plane amphipathic α-helices separated by a linker region. Resonances in one-dimensional solid-state NMR spectra of uniformly 15N labeled Vpu are clearly segregated into two bands at chemical shift frequencies associated with NH bonds in a transmembrane α-helix, perpendicular to the membrane surface, and with NH bonds in the cytoplasmic helices parallel to the membrane surface. Solid-state NMR spectra of truncated Vpu2–51 (residues 2–51), which contains the transmembrane α-helix and the first amphipathic helix of the cytoplasmic domain, and of a construct Vpu28–81 (residues 28–81), which contains only the cytoplasmic domain, support this structural model of Vpu in the membrane. Full-length Vpu (residues 2–81) forms discrete ion-conducting channels of heterogeneous conductance in lipid bilayers. The most frequent conductances were 22 ± 3 pS and 12 ± 3 pS in 0.5 M KCl and 29 ± 3 pS and 12 ± 3 pS in 0.5 M NaCl. In agreement with the structural model, truncated Vpu2–51, which has the transmembrane helix, forms discrete channels in lipid bilayers, whereas the cytoplasmic domain Vpu28–81, which lacks the transmembrane helix, does not. This finding shows that the channel activity is associated with the transmembrane helical domain. The pattern of channel activity is characteristic of the self-assembly of conductive oligomers in the membrane and is compatible with the structural and functional findings.

Propagating structure of Alzheimer’s β-amyloid(10–35) is parallel β-sheet with residues in exact register

The pathognomonic plaques of Alzheimer’s disease are composed primarily of the 39- to 43-aa β-amyloid (Aβ) peptide. Crosslinking of Aβ peptides by tissue transglutaminase (tTg) indicates that Gln15 of one peptide is proximate to Lys16 of another in aggregated Aβ. Here we report how the fibril structure is resolved by mapping interstrand distances in this core region of the Aβ peptide chain with solid-state NMR. Isotopic substitution provides the source points for measuring distances in aggregated Aβ. Peptides containing a single carbonyl 13C label at Gln15, Lys16, Leu17, or Val18 were synthesized and evaluated by NMR dipolar recoupling methods for the measurement of interpeptide distances to a resolution of 0.2 Å. Analysis of these data establish that this central core of Aβ consists of a parallel β-sheet structure in which identical residues on adjacent chains are aligned directly, i.e., in register. Our data, in conjunction with existing structural data, establish that the Aβ fibril is a hydrogen-bonded, parallel β-sheet defining the long axis of the Aβ fibril propagation.

Dynamics and orientation of N+(CD3)3-bromoacetylcholine bound to its binding site on the nicotinic acetylcholine receptor

Dynamic and structural information has been obtained for an analogue of acetylcholine while bound to the agonist binding site on the nicotinic acetylcholine receptor (nAcChoR), using wide-line deuterium solid-state NMR. Analysis of the deuterium lineshape obtained at various temperatures from unoriented nAcChoR membranes labeled with deuterated bromoacetylcholine (BAC) showed that the quaternary ammonium group of the ligand is well constrained within the agonist binding site when compared with the dynamics observed in the crystalline solids. This motional restriction would suggest that a high degree of complementarity exists between the quaternary ammonium group of the ligand and the protein within the agonist binding site. nAcChoR membranes were uniaxially oriented by isopotential centrifugation as determined by phosphorous NMR of the membrane phospholipids. Analysis of the deuterium NMR lineshape of these oriented membranes enriched with the nAcChoR labeled with N+(CD3)3-BAC has enabled us to determine that the angle formed between the quaternary ammonium group of the BAC and the membrane normal is 42° in the desensitized form of the receptor. This measurement allows us to orient in part the bound ligand within the proposed receptor binding site.

Solid and liquid phase 59Co NMR studies of cobalamins and their derivatives

We describe the application of 59Co NMR to the study of naturally occurring cobalamins. Targets of these investigations included vitamin B12, the B12 coenzyme, methylcobalamin, and dicyanocobyrinic acid heptamethylester. These measurements were carried out on solutions and powders of different origins, and repeated at a variety of magnetic field strengths. Particularly informative were the solid-state central transition NMR spectra, which when combined with numerical line shape analyses provided a clear description of the cobalt coupling parameters. These parameters showed a high sensitivity to the type of ligands attached to the metal and to the crystallization history of the sample. 59Co NMR determinations also were carried out on synthetic cobaloximes possessing alkyl, cyanide, aquo, and nitrogenated axial groups, substituents that paralleled the coordination of the natural compounds. These analogs displayed coupling anisotropies comparable to those of the cobalamins, as well as systematic up-field shifts that can be rationalized in terms of their stronger binding affinity to the cobalt atom. Cobaloximes also displayed a higher regularity in the relative orientations of their quadrupole and shielding coupling tensors, reflecting a higher symmetry in their in-plane coordination. For the cobalamines, poor correlations were observed between the values measured for the quadrupole couplings in the solid and the line widths observed in the corresponding solution 59Co NMR resonances.