Homonuclear and heteronuclear NMR studies of a statherin fragment bound to hydroxyapatite crystals

Acidic proteins found in mineralized tissues act as nature's crystal engineers, where they play a key role in promoting or inhibiting the growth of minerals such as hydroxyapatite (HAP), Ca10(PO4)6(OH)2, the main mineral component of bone and teeth. Key to understanding the structural basis of protein-crystal recognition and protein control of hard tissue growth is the nature of interactions between the protein side chains and the crystal surface. In an earlier work we have measured the proximity of the lysine (K6) side chain in an SN-15 peptide fragment of the salivary protein statherin adsorbed to the Phosphorus-rich surface of HAP using solid-state NMR recoupling experiments. 15N{31P} rotational echo double resonance (REDOR) NMR data on the side-chain nitrogen in K6 gave rise to three different models of protein-surface interaction to explain the experimental data acquired. In this work we extend the analysis of the REDOR data by examining the contribution of interactions between surface phosphorus atoms to the observed 15N REDOR decay. We performed 31P-31P recoupling experiments in HAP and (NH4)2HPO4 (DHP) to explore the nature of dipolar coupled 31P spin networks. These studies indicate that extensive networks of dipolar coupled 31P spins can be represented as stronger effective dipolar couplings, the existence of which must be included in the analysis of REDOR data. We carried out 15N{31P} REDOR in the case of DHP to determine how the size of the dephasing spin network influences the interpretation of the REDOR data. Although use of an extended 31P coupled spin network simulates the REDOR data well, a simplified 31P dephasing system composed of two spins with a larger dipolar coupling also simulates the REDOR data and only perturbs the heteronuclear couplings very slightly. The 31P-31P dipolar couplings between phosphorus nuclei in HAP can be replaced by an effective dipolar interaction of 600 Hz between two 31P spins. We incorporated this coupling and applied the above approach to reanalyze the 15N{31P} REDOR of the lysine side chain approaching the HAP surface and have refined the binding models proposed earlier. We obtain 15N-31P distances between 3.3 and 5 A from these models that are indicative of the possibility of a lysine-phosphate hydrogen bond.     

A solid-state NMR study of the dynamics and interactions of phenylalanine rings in a statherin fragment bound to hydroxyapatite crystals

Extracellular matrix proteins regulate hard tissue growth by acting as adhesion sites for cells, by triggering cell signaling pathways, and by directly regulating the primary and/or secondary crystallization of hydroxyapatite, the mineral component of bone and teeth. Despite the key role that these proteins play in the regulation of hard tissue growth in humans, the exact mechanism used by these proteins to recognize mineral surfaces is poorly understood. Interactions between mineral surfaces and proteins very likely involve specific contacts between the lattice and the protein side chains, so elucidation of the nature of interactions between protein side chains and their corresponding inorganic mineral surfaces will provide insight into the recognition and regulation of hard tissue growth. Isotropic chemical shifts, chemical shift anisotropies (CSAs), NMR line-width information, (13)C rotating frame relaxation measurements, as well as direct detection of correlations between (13)C spins on protein side chains and (31)P spins in the crystal surface with REDOR NMR show that, in the peptide fragment derived from the N-terminal 15 amino acids of salivary statherin (i.e., SN-15), the side chain of the phenylalanine nearest the C-terminus of the peptide (F14) is dynamically constrained and oriented near the surface, whereas the side chain of the phenylalanine located nearest to the peptide's N-terminus (F7) is more mobile and is oriented away from the hydroxyapatite surface. The relative dynamics and proximities of F7 and F14 to the surface together with prior data obtained for the side chain of SN-15's unique lysine (i.e., K6) were used to construct a new picture for the structure of the surface-bound peptide and its orientation to the crystal surface.     

Boltzmann statistics rotational-echo double-resonance analysis

A new approach to rotational-echo double-resonance (REDOR) data analysis, analogous to Boltzmann maximum entropy statistics, is reported. This Boltzmann statistics REDOR (BS-REDOR) approach is useful for reconstructing an unbiased internuclear distance distribution for multiple internuclear distances from experimentally limited REDOR data sets on isolated spin pairs. The analysis is characterized by exploring reconstructions on model data and applied to both [1-(13)C,15N]-glycine and a long intramolecular distance in Abeta (16-22) peptide nanotubes. The approach also provides insight into the minimal number of REDOR data points required to allow faithful determination of dipolar couplings in systems with multiple internuclear distances.     

Revealing the configuration and crystal packing of organic compounds by solid-state NMR spectroscopy: methoxycarbonylurea, a case study

The molecular configuration and intermolecular arrangement of polycrystalline methoxycarbonylurea (MCU) has been studied by a combination of chemical editing, rotational echo double resonance (REDOR) spectroscopy and ab initio calculations. From the multispin IS(n) REDOR experiments several dipolar couplings were determined and converted into distance constraints. Intra- and intermolecular dipolar couplings were distinguished by isotope dilution. The configuration of the MCU molecule can be determined from three torsion angles Psi1, Psi2, and Psi3. Ab initio calculations showed that these angles are either 0 degrees or 180 degrees (Z or E). From the REDOR experiments, the E configuration was found for Psi1 and Psi2 and the Z configuration for Psi3. Thus the configuration of MCU in the solid state was determined to be EEZ. Distance constraints for the intermolecular arrangement of MCU were obtained by performing REDOR experiments on 13C15N2 MCU with different degrees of isotope dilution and on a cocrystallized 1:1 mixture of 13C(urea) MCU and 15N(amide) MCU. By combining these distance constraints with molecular modeling, three different possible packing motifs for MCU molecules were found. The molecules in these motifs are arranged as linear chains with methoxy groups at the borders of the chains. All the intermolecular hydrogen bond donors and acceptors in the interior of the chain are saturated.     

Frequency selective heteronuclear dipolar recoupling in rotating solids: accurate (13)C-(15)N distance measurements in uniformly (13)C,(15)N-labeled peptides

We describe a magic-angle spinning NMR experiment for selective (13)C-(15)N distance measurements in uniformly (13)C,(15)N-labeled solids, where multiple (13)C-(15)N and (13)C-(13)C interactions complicate the accurate measurement of structurally interesting, weak (13)C-(15)N dipolar couplings. The new experiment, termed FSR (frequency selective REDOR), combines the REDOR pulse sequence with a frequency selective spin-echo to recouple a single (13)C-(15)N dipolar interaction in a multiple spin system. Concurrently the remaining (13)C-(15)N dipolar couplings and all (13)C-(13)C scalar couplings to the selected (13)C are suppressed. The (13)C-(15)N coupling of interest is extracted by a least-squares fit of the experimentally observed modulation of the (13)C spin-echo intensity to the analytical expression describing the dipolar dephasing in an isolated heteronuclear spin pair under conventional REDOR. The experiment is demonstrated in three uniformly (13)C,(15)N-labeled model systems: asparagine, N-acetyl-L-Val-L-Leu and N-formyl-L-Met-L-Leu-L-Phe; in N-formyl-[U-(13)C,(15)N]L-Met-L-Leu-L-Phe we have determined a total of 16 internuclear distances in the 2.5-6 A range.     

Long-range 1H-19F distance measurement in peptides by solid-state NMR

A new NMR technique for determining long-range 1H-19F distances in solids is demonstrated. Using a modified rotational-echo double resonance (REDOR) sequence involving 1H homonuclear decoupling and composite 19F pulses, we show that it is possible to determine 1H-19F distances to approximately 8 A. The detrimental effect of the large 19F chemical shift to REDOR dephasing is partially compensated for by the composite pulse, 90 degrees 225 degrees 315 degrees . The 1HNLeu-19FPhe distance in the peptide f-MLF-OH was found to be 7.7 A. This was used to refine the Phe side chain conformation. The 1H-19F REDOR technique should be useful for restraining the three-dimensional structure of proteins.     

Direct observation of phenylalanine orientations in statherin bound to hydroxyapatite surfaces

Extracellular biomineralization proteins such as salivary statherin control the growth of hydroxyapatite (HAP), the principal component of teeth and bones. Despite the important role that statherin plays in the regulation of hard tissue formation in humans, the surface recognition mechanisms involved are poorly understood. The protein-surface interaction likely involves very specific contacts between the surface atoms and the key protein side chains. This study demonstrates for the first time the power of combining near-edge X-ray absorption fine structure (NEXAFS) spectroscopy with element labeling to quantify the orientation of individual side chains. In this work, the 15 amino acid N-terminal binding domain of statherin has been adsorbed onto HAP surfaces, and the orientations of phenylalanine rings F7 and F14 have been determined using NEXAFS analysis and fluorine labels at individual phenylalanine sites. The NEXAFS-derived phenylalanine tilt angles have been verified with sum frequency generation spectroscopy.     

Investigation of the binding of epimer A of the covalent hydrate of 6,7-bis(trifluoromethyl)-8-D-ribityllumazine to a recombinant F22W Bacillus subtilis lumazine synthase mutant by (15)N[(19)F] REDOR NMR

The two epimeric covalent hydrates A and B of 6,7-bis(trifluoromethyl)-8-D-ribityllumazine are metabolically stable analogues of hypothetical intermediates proposed in the reactions catalyzed by riboflavin synthase and lumazine synthase. To confirm the stereochemical assignments previously based solely on results for epimer B, a (15)N[(19)F] REDOR NMR study was performed on the complex formed from epimer A and a recombinant, uniformly (15)N-labeled F22W mutant of Bacillus subtilis lumazine synthase. The results indicate that the fluorines of the ligands are closer to the side chain nitrogens of Arg127 and farther away from the side chain nitrogens of Lys135 in epimer B than in epimer A. These results are consistent with the assignment of the earlier 7R configuration of epimer A and the 7S configuration of epimer B.     

Dielectric relaxation of aqueous solutions of hydrophilic versus amphiphilic peptides

We report on molecular dynamics simulations of the frequency-dependent dielectric relaxation spectra at room temperature for aqueous solutions of a hydrophilic peptide and an amphiphilic peptide at two concentrations. We find that only the high-concentration amphiphilic peptide solution exhibits an anomalous dielectric increment over that of pure water, while the hydrophilic peptide exhibits a significant dielectric decrement. The dielectric component analysis carried out by decomposing these peptide solutions into peptide, hydration layer, and outer layer(s) of water clearly shows the presence of a unique dipolar component with a relaxation time scale on the order of approximately 25 ps (compared to the bulk water time scale of approximately 11 ps) that originates from the interaction between the hydration layer water and the outer layer(s) of water. Results obtained from the dielectric component analysis further show the emergence of a distinct and much lower frequency relaxation process for the high-concentration amphiphilic peptide compared to the hydrophilic peptide due to strong peptide dipolar couplings to all constituents, accompanied by a slowing of the structural relaxation in all water layers, giving rise to time scales close to approximately 1 ns. We suggest that the molecular origin of the dielectric relaxation anomalies is due to frustration in the water network arising from the amphiphilic chemistry of the peptide that does not allow it to reorient on the picosecond time scale of bulk water motions. This explanation is consistent with the idea of the "slaving" of residue side chain motions to protein surface water, and furthermore offers the possibility that the anomalous dynamics observed from a number of spectroscopies arises at the interface of hydrophobic and hydrophilic domains on the protein surface.     

Dipolar double-quantum filtered rotational-echo double resonance

The homonuclear dipolar coupling of a directly bonded (13)C-(13)C pair has been used to create a dipolar double-quantum filter (D-DQF) to remove the natural-abundance (13)C background in (13)C[(2)H] rotational-echo double-resonance (REDOR) experiments. The most efficient version of this experiment has the D-DQF excitation and reconversion preceding the REDOR evolution period. Calculated and observed (13)C[(2)H]D-DQF-REDOR dephasings were in agreement for a test sample of mixed recrystallized labeled alanines.     

13C/15N-19F intermolecular REDOR NMR study of the interaction of TAR RNA with Tat peptides

The complex of the HIV TAR RNA with the viral regulatory protein Tat is of considerable interest, but the plasticity of this interaction has made it impossible so far to establish the structure of that complex. In order to explore a new approach to obtain structural information on protein-RNA complexes, we performed (13)C/(15)N-(19)F REDOR NMR experiments in the solid state on TAR bound to a peptide comprising the RNA-binding section of Tat. A critical arginine in the peptide was uniformly (13)C and (15)N labeled, and 5-fluorouridine was incorporated at the U23 position of TAR. REDOR irradiation resulted in dephasing of the (13)C and (15)N resonances, indicating the proximity of the U23(5F)-C and U23(5F)-N spin pairs. Best fits to the REDOR data show the U23(5F)-C distances and the U23(5F)-N distances are in good agreement with the distances obtained from solution NMR structures of partial complexes of Tat with TAR. These results demonstrate that it is possible to study protein-RNA complexes using solid-state REDOR NMR measurements, adding to a growing list of solid state techniques for studying protein-nucleic acid complexes.     

Structural diversity of amyloid fibril formed in human calcitonin as revealed by site-directed 13C solid-state NMR spectroscopy

Fibril formation in human calcitonin (hCT) from aqueous solution at pH 4.1 was examined and compared with those at pH 3.3 and 7.5 corresponding to three different net charges by means of site-directed (13)C solid-state NMR spectroscopy. Notably, the observed (13)C chemical shifts and lineshapes of the (13)C CP/MAS spectra differed substantially among fibrils prepared at different pHs. It was found that antiparallel beta-sheet structures were formed at pH 7.5 and 4.1 in the central core regions. In the C-terminal region, random coils were formed at both pH 7.5 and 4.1, although the random coil region at pH 4.1 was larger than that at pH 7.5. Fibrillation kinetics analyzed by a two-step autocatalytic reaction mechanism showed that the rate constants k(1) and k(2) for nucleation and maturation reactions of the fibril formation, respectively, were separately determined and the values correlated well with the net positive charges of Lys(18) and His(20) rather than the existence of a negative charge of Asp(15). Further, an attempt was made to assess interatomic distances between amide nitrogen and carbonyl carbon of neighboring chains of (13)C, (15)N-labeled hCT and a model pentapeptide by (13)C REDOR measurements by taking into account its dipolar interaction analyzed by the 3 spin system proposed previously. A unique chain packing of the antiparallel beta-sheets was proposed as a dominant fibril structure, although the possibility of a contribution of chain packing consisting of sliding one or two residues perpendicular to the fibril direction cannot be ruled out. In addition, it appears that the phenyl rings of Phe(16) are aligned on the same side of the beta-sheet and make the beta-sheet stable by forming pi-pi interactions between the beta-strands.     

Probing the local environments of phosphorus in aluminophosphate-based mesostructured lamellar materials by solid-state NMR spectroscopy

We have characterized a series of mesostructured aluminophosphate (AlPO)-based lamellar materials by several solid-state NMR techniques. In particular, we were able to estimate the average number of Al atoms and identify the nature of other ancillary groups in the second coordination sphere for each P site. Our work has shown that a combination of several dipolar coupling-based (31)P/(27)Al double-resonance techniques such as transfer of population in double-resonance (TRAPDOR), rotational echo double-resonance (REDOR), and heteronuclear correlation spectroscopy (HETCOR) as well as (1)H --> (31)P cross polarization (CP) can provide more detailed structural information regarding the local environments of P and Al atoms in AlPO-based mesostructured materials, which is not readily available from straightforward (31)P and (27)Al magic-angle spinning (MAS) experiments.     

Measurements of carbon to amide-proton distances by C-H dipolar recoupling with 15N NMR detection

A new magic-angle spinning NMR method for measuring internuclear distances between a 13C-labeled site and amide protons is described. The magnetization of the protons evolves under homonuclear decoupling and the recoupled 13C-1H dipolar interaction, which provides simple spin-pair REDOR curves if only one 13C-labeled site is present. The modulation of the amide proton HN is detected via short 1H-15N cross polarization followed by 15N detection. The method is demonstrated on two specifically 13C- and 15N-labeled peptides, with 13C-HN distances from 2.2 to ca. 6 A. This technique promises to be particularly useful for measuring distances between 13C=O and H-15N groups, to identify hydrogen bonds in peptides and proteins.     

Band-selective carbonyl to aliphatic side chain 13C-13C distance measurements in U-13C,15N-labeled solid peptides by magic angle spinning NMR

We describe three-dimensional magic angle spinning NMR experiments that enable simultaneous band-selective measurement of the multiple distance constraints between carbonyl and side chain carbons in uniformly 13C,15N-labeled peptides. The approaches are designed to circumvent the dipolar truncation and to allow experimental separation of the multiple quantum (MQ) relaxation and dipolar effects. The pulse sequences employ the double quantum (DQ) rotational resonance in the tilted frame (R2TR) to perform selective polarization transfers that reintroduce the 13C'-13Cgamma,delta dipolar interactions. The scheme avoids recoupling of the strongly coupled C'-Calpha and C'-Cbeta spin pairs, therefore minimizing dipolar truncation effects. The experiment is performed in a constant time fashion as a function of the radio frequency irradiation intensity and measures the line shape of the DQ transition. The width and the intensity of this line shape are analyzed in terms of the DQ relaxation and dipolar coupling. The attenuation of the multispin effects in the presence of relaxation enables a two-spin approximation to be employed for the analysis of the experimental data. The systematic error introduced by this approximation is estimated by comparing the results with a three-spin simulation. The contributions of B1-inhomogeneity, CSA orientation effects, and the effects of inhomogeneous line broadening are also estimated. The experiments are demonstrated in model U-13C,15N-labeled peptides, N-acetyl-L-Val-L-Leu and N-formyl-L-Met-L-Leu-L-Phe, where 10 and 6 distances, ranging between 3 and 6 A, were measured, respectively.     

The link between sequence and conformation in protein structures appears to be stereochemically established

In search of the link between sequence and conformation in protein structures, we perform molecular dynamics analysis of the effect of stereochemical mutation in end-protected octa-alanine Ac-Ala8-NHMe from poly-L to an alternating-L,D structure. The mutation has a dramatic effect, transforming the peptide from a condition of extreme sensitivity to one of extreme insensitivity to solvent. Examining the molecular folds of poly-L and alternating-L,D structure in atomistic detail, we find them to differ in the relationship between peptide dipolar interactions at the local and nonlocal levels, either conflicting or harmonious depending upon the chain stereochemistry. The stereochemical transformation of interpeptide electrostatics from a condition of conflict to one of harmony explains the long-standing puzzle of why poly-L and alternating-L,D peptides strongly differ in properties such as "stiffness" and solvent sensitivity. Furthermore, it is possible that poly-L stereochemistry is also the fulcrum of protein sensitivity to the effects of amino acid side-chain structures via dielectric arbitrations in interpeptide electrostatics. Indeed the evidence is accumulating that the amino acid side chains differing in alpha-helix and beta-sheet propensities also differ in their desolvating effects in the adjacent and nearest-neighbor peptides and thus possibly in the solvent screening of peptide dipolar interactions.     

Solid state NMR investigation of intact human bone quality: balancing issues and insight into the structure at the organic-mineral interface

Age-related bone fragility fractures present a significant problem for public health. Measures of bone quality are increasingly recognized to complement the conventional bone mineral density (BMD) based assessment of fracture risk. The ability to probe and understand bone quality at the molecular level is desirable in order to unravel how the structure of organic matrix and its association with mineral contribute to the overall mechanical properties. The (13)C{(31)P} REDOR MAS NMR (Rotational Echo Double Resonance Magic Angle Spinning Nuclear Magnetic Resonance) technique is uniquely suited for the study of the structure of the organic-mineral interface in bone. For the first time, we have applied it successfully to analyze the structure of intact (non-powdered) human cortical bone samples, from young healthy and old osteoporotic donors. Loading problems associated with the rapid rotation of intact bone were solved using a Finite Element Analysis (FEA) approach, and a method allowing osteoporotic samples to be balanced and spun reproducibly is described. REDOR NMR parameters were set to allow insight into the arrangement of the amino acids at the mineral interface to be accessed, and SVD (Singular Value Decomposition) was applied to enhance the signal to noise ratio and enable a better analysis of the data. From the REDOR data, it was found that carbon atoms belonging to citrate/glucosaminoglycans (GAGs) are closest to the mineral surface regardless of age or site. In contrast, the arrangement of the collagen backbone at the interface varied with site and age. The relative proximity of two of the main amino acids in bone matrix proteins, hydroxyproline and alanine, with respect to the mineral phase was analyzed in more detail, and discussed in view of glycation measurements which were carried out on the tissues. Overall, this work shows that the (13)C{(31)P} REDOR NMR approach could be used as a complementary technique to assess a novel aspect of bone quality, the organic-mineral interface structure.     

Conformational constraints in solid-state NMR of uniformly labeled polypeptides from double single-quantum-filtered rotational echo double resonance

A solid-state nuclear magnetic resonance (NMR) technique is described for obtaining constraints on the backbone conformation of a protein or peptide that is prepared with uniform (15)N,(13)C labeling of consecutive pairs of amino acids or of longer segments. The technique, called double single-quantum-filtered rotational echo double resonance (DSQ-REDOR), uses frequency-selective REDOR to prepare DSQ coherences involving directly bonded backbone (13)CO and (15)NH sites, to dephase these coherences under longer-range (15)NH-(13)CO dipole-dipole couplings in a conformationally dependent manner, and to convert the remaining DSQ coherences to detectable transverse (13)C-spin polarization. The efficacy of DSQ-REDOR is demonstrated in experiments on two isotopically labeled samples, the helical peptide MB(i + 4)EK and the amyloid-forming peptide Abeta(11-25).     

Theoretical study of a simple rotational‐echo double‐resonance NMR for homonuclear spin‐1/2 pairs

We investigate theoretically intriguing aspects of a simple rotational‐echo double‐resonance (REDOR) NMR technique for homonuclear spin‐1/2 pairs undergoing MAS. The simple technique sets Gaussian soft π pulses at every half MAS rotational period in the pulse sequence. The reduction in rotational echo amplitude (the REDOR echo reduction) is observed at the end of the evolution period t_e = (n + 1)T_r, where T_r is a MAS rotational period. The exact average Hamiltonians for the homonuclear REDOR (hm‐REDOR) technique are calculated by dividing the evolution period into four periods. We show theoretically and experimentally that the hm‐REDOR technique produces the REDOR echo reductions for homonuclear spin‐1/2 pairs. In addition, the theoretical results reveal that the REDOR echo reductions are independent of the chemical‐shift difference, δ, under a simple condition of κ = δ/ω_r ≥ 6 and t_e < 10 ? (1/d′), where ω_r is the sample spinning frequency and d′ is the dipolar coupling constant expressed in Hz. We call this simple condition the master condition. This means that the REDOR echo reductions for a homonuclear spin‐1/2 pair can be calculated under the master condition by considering only d′ and ω_r, which is the case for a heteronuclear spin pair. Finally, we demonstrate that four‐phase cycling yields the multiple‐quantum filtered hm‐REDOR experiment, where the appearance of the REDOR echo reductions shows that the echo reductions are definitely attributable to the homonuclear dipolar interaction even if there is a slight unwanted effect from the recovered chemical‐shift anisotropy in these reductions. Copyright © 2015 John Wiley & Sons, Ltd.     

The ATCUN domain as a probe of intermolecular interactions: application to calmodulin-peptide complexes

The utility of a three-residue Cu2+ binding motif (ATCUN domain) for studying intermolecular interactions is demonstrated. By comparing a set of 1H-15N correlation spectra recorded on complexes of calmodulin (CaM) and peptides with the ATCUN tag in the presence and absence of Cu2+ the two possible canonical binding orientations of the peptide can be rapidly distinguished. The methodology is confirmed with studies of complexes of CaM and peptides from myosin light chain kinase and CaM kinase kinase, for which high-resolution structures are available, and then applied to a complex with CaM kinase I for which structural data has not been obtained. The orientation of the CaM kinase I and myosin light chain kinase peptides are shown to be identical. In the case of a complex of CaM with a peptide for which structural information is not available, the present methodology, in combination with 1H-15N residual dipolar couplings measured on CaM, and the database of existing CaM-peptide structures, allows a homology model to be built rapidly and with confidence.