Solid-state (13)C NMR reveals effects of temperature and hydration on elastin.

Elastin is the principal protein component of the elastic fiber in vertebrate tissue. The waters of hydration in the elastic fiber are believed to play a critical role in the structure and function of this largely hydrophobic, amorphous protein. (13)C CPMAS NMR spectra are acquired for elastin samples with different hydration levels. The spectral intensities in the aliphatic region undergo significant changes as 70% of the water in hydrated elastin is removed. In addition, dramatic differences in the CPMAS spectra of hydrated, lyophilized, and partially dehydrated elastin samples over a relatively small temperature range (-20 degrees C to 37 degrees C) are observed. Results from other experiments, including (13)C T(1) and (1)H T(1 rho) measurements, direct polarization with magic-angle spinning, and static CP of the hydrated and lyophilized elastin preparations, also support the model that there is significant mobility in fully hydrated elastin. Our results support models in which water plays an integral role in the structure and proper function of elastin in vertebrate tissue.     

Conformational exchange on the microsecond time scale in alpha-helix and beta-hairpin peptides measured by 13C NMR transverse relaxation

13C-NMR relaxation experiments (T(1), T(2), T(1)(rho), and NOE) were performed on selectively enriched residues in two peptides, one hydrophobic staple alpha-helix-forming peptide GFSKAELAKARAAKRGGY and one beta-hairpin-forming peptide RGITVNGKTYGR, in water and in water/trifluoroethanol (TFE). Exchange contributions, R(ex), to spin-spin relaxation rates for (13)C(alpha) and (13)C(beta) groups were derived and were ascribed to be mainly due to peptide folding-unfolding. To evaluate the exchange time, tau(ex), from R(ex), the chemical shift difference between folded and unfolded states, Deltadelta, and the populations of these states, p(i), were determined from the temperature dependence of (13)C chemical shifts. For both peptides, values for tau(ex) fell in the 1 micros to 10 micros range. Under conditions where the peptides are most folded (water/TFE, 5 degrees C), tau(ex) values for all residues in each respective peptide were essentially the same, supporting the presence of a global folding-unfolding exchange process. Rounded-up average tau(ex) values were 4 micros for the helix peptide and 9 micros for the hairpin peptide. This 2-3-fold difference in exchange times between helix and hairpin peptides is consistent with that observed for folding-unfolding of other small peptides.     

Solid state NMR and X-ray diffraction studies of alpha-D-galacturonic acid monohydrate

Crystalline alpha-D-galacturonic acid monohydrate has been studied by 13C CPMAS NMR and X-ray crystallography. The molecular dynamics were investigated by evaluating 13C spin-lattice relaxation in the rotating frame (T1rho) and chemical-shift-anisotropy properties of each carbon. Only limited molecular motions can be detected in the low frequency (< 10(4) Hz) range by 13C relaxation time measurements (T1rho) and changes of chemical shift anisotropy properties as a function of temperature. X-ray analysis (at both ambient temperature and 150 K) shows that the acid has the usual chair-shaped, pyranose ring conformation, and that the acid and water molecules are linked, through all their O-H groups, in an extensively hydrogen-bonded lattice.     

19F NMR investigation of molecular motion and packing in sonicated phospholipid vesicles

Dimyristoylphosphatidylcholine (DMPC) labeled with a C19F2 group in the 4-, 8-, or 12-position of the 2-acyl chain has been investigated in sonicated unilamellar vesicles (SUV) by fluorine-19 nuclear magnetic resonance (NMR) at 282.4 MHz from 26 to 42 degrees C. The 19F NMR spectra exhibit two overlapping resonances with different line widths. Spin-lattice relaxation time measurements have been performed in both the laboratory frame (T1) and the rotating frame (T1 rho) in order to investigate the packing and dynamics of phospholipids in lipid bilayers. Quantitative line-shape and relaxation analyses are possible by using the experimental chemical shift anisotropy (delta nu CSA) and the internuclear F-F vector order parameter (SFF) values obtained from the 19F powder spectra of multilamellar liposomes. The following conclusions can be made: The 19F chemical shift difference between the inside and outside leaflets of SUV can be used to monitor the lateral packing of the phospholipid in the two SUV monolayers. The hydrocarbon chains in the outer layer are found to be more tightly packed than those of the inner one, and the differences between them become smaller near the chain terminals. The effective correlation time [(1-4) x 10(-7) s] obtained from either the motional narrowing of the line widths or off-resonance T1 rho measurements is shorter than that estimated from the Stokes-Einstein diffusion model (10(-6) s), on the basis of a hydrodynamic radius of 110 A for SUV.(ABSTRACT TRUNCATED AT 250 WORDS)     

13C n.m.r. study of L-alanine peptides

The di-, tri-, and tetrapeptides of L-alanine have been studied in aqueous solution by 13C n.m.r. spectroscopy at 25 and 50 MHz. By using selectively 13C enriched analogs containing either 90% 13C methyl or carbonyl carbons and measurements as a function of pH, assignment of the chemical shifts of the peptides has been made. T1 and NOE measurements of the peptides in their cationic, anionic, and zwitterionic states have been recorded as a function of concentration. The results show considerable segmental motion along the backbone carbons of the peptides, with only small changes occurring in the dynamic motions of the peptides as their charge states are altered. The lack of concentration dependence of the chemical shift and T1 values, as well as the similarity of T1 values for individual peptides in the three charge states, indicate that the peptides do not self-associate in aqueous solution.     

A high-resolution solid-state 13C-NMR study on crystalline bovine heart cytochrome-c oxidase and lysozyme. Dynamic behavior of protein and detergent in the complex.

We have recorded 100.6-MHz high-resolution solid-state 13C-NMR spectra of crystalline cytochrome-c oxidase from bovine heart muscle and hen egg-white lysozyme, to compare conformation and dynamics of a typical membrane-protein complex with those of lysozyme. The absence of severe interference with the solid-state 13C-NMR spectra, from both the line broadenings from paramagnetic centers and overlapping of intense detergent signals, provided spectral resolution of 13C-NMR feature of cytochrome-c oxidase crystals comparable to that of lysozyme crystal and better than that of dissolved or lyophilized samples. In fact, the observed peak intensities of the polar heads of the detergents BL8SY and Brij 35 were only about 10% and 3% of the anticipated values, respectively. The dynamic behavior of the backbone and side chains of cytochrome-c oxidase was compared with that of lysozyme on the basis of the 13C spin-lattice relaxation times (T1): the backbone of the cytochrome-c oxidase turned out to be more flexible than that of lysozyme. Molecular motions of the detergent molecules attached to the proteins are found to be highly heterogeneous. Detergent molecules undergo rapid tumbling motions in the crystals in about 10 ns as detected by T1. In addition to rapid motions, slow motions were detected by 1H spin-lattice relaxation time in the rotating frame (TH1 rho) and cross-polarization time (TCH), together with data from static spectra, indicating that the aliphatic portion of the detergent interacts more strongly with hydrophobic protein surfaces than do the polar heads.     

Magic angle spinning carbon-13 NMR of tobacco mosaic virus. An application of the high-resolution solid-state NMR spectroscopy to very large biological systems.

Magic angle spinning 13C NMR was used to study tobacco mosaic virus (TMV) in solution. Well-resolved 13C NMR spectra were obtained, in which several carbon resonances of amino acids of the TMV coat protein subunits that are not observable by conventional high-resolution NMR spectroscopy can be designed. RNA resonance were absent, however, in the magic angle spinning 13C NMR spectra. Since three different binding sites are available for each nucleotide of the RNA, this is probably due to a line broadening caused by distributions of isotropic chemical shift values. In 13C-enriched TM 13C-13C dipolar interactions also gave rise to line broadening. By suitable pulse techniques that discriminate carbon resonances on the basis of their T1 and T1 rho values, it was possible to select particular groups of carbon nuclei with characteristic motional properties. Magic angle spinning 13C NMR spectra obtained with these pulse techniques are extremely well resolved.     

NMR characterization of hydration and thermal stress in tomato fruit cuticles

In its natural environment, the plant cuticle, which is composed of the biopolymer cutin and a mixture of surface and embedded cuticular waxes, experiences a wide variety of temperatures and hydration states. Consequently, a complete understanding of cuticular function requires study of its thermal and mechanical properties as a function of hydration. Herein, we report the results of a comprehensive 13C nuclear magnetic resonance (NMR) relaxation study of hydrated tomato fruit cuticle. Cross-polarization and direct-polarization experiments serve to measure the solid-like and liquid-like components, respectively, of hydrated cuticle. Localized, high-frequency motions are probed by T1(C) spin relaxation measurements, whereas T1rho(H) and T1rho(C) experiments reflect low-frequency, lower amplitude polymer-chain motions. In addition, variable-temperature measurements of T1(C) and T1rho(C) for dry tomato cuticles are used to evaluate the impact of temperature stress. Results of these experiments are interpreted in terms of changes occurring in individual polymer motions of the cutin/wax components of tomato cuticle and in the interaction of these components within intact cuticle, both of which are expected to influence the functional integrity of this protective plant covering.     

Activities of daily living influence tibial cartilage T1rho relaxation times

Quantitative T1rho magnetic resonance imaging (MRI) can potentially help identify early-stage osteoarthritis (OA) by non-invasively assessing proteoglycan concentration in articular cartilage. T1rho relaxation times are negatively correlated with proteoglycan concentration. Cartilage compresses in response to load, resulting in water exudation, a relative increase in proteoglycan concentration, and a decrease in the corresponding T1rho relaxation times. To date, there is limited information on changes in cartilage composition resulting from daily activity. Therefore, the objective of this study was to quantify changes in tibial cartilage T1rho relaxation times in healthy human subjects following activities of daily living. It was hypothesized that water exudation throughout the day would lead to decreased T1rho relaxation times. Subjects underwent MR imaging in the morning and afternoon on the same day and were free to go about their normal activities between scans. Our findings confirmed the hypothesis that tibial cartilage T1rho relaxation times significantly decreased (by 7%) over the course of the day with loading, which is indicative of a relative increase in proteoglycan concentration. Additionally, baseline T1rho values varied with position within the cartilage, supporting a need for site-specific measurements of T1rho relaxation times. Understanding how loading alters the proteoglycan concentration in healthy cartilage may hold clinical significance pertaining to cartilage homeostasis and potentially help to elucidate a mechanism for OA development. These results also indicate that future studies using T1rho relaxation times as an indicator of cartilage health should control the loading history prior to image acquisition to ensure the appropriate interpretation of the data.     

Supramolecular structure in full-length Alzheimer's beta-amyloid fibrils: evidence for a parallel beta-sheet organization from solid-state nuclear magnetic resonance

We report constraints on the supramolecular structure of amyloid fibrils formed by the 40-residue beta-amyloid peptide associated with Alzheimer's disease (A beta(1-40)) obtained from solid-state nuclear magnetic resonance (NMR) measurements of intermolecular dipole-dipole couplings between (13)C labels at 11 carbon sites in residues 2 through 39. The measurements are carried out under magic-angle spinning conditions, using the constant-time finite-pulse radiofrequency-driven recoupling (fpRFDR-CT) technique. We also present one-dimensional (13)C magic-angle spinning NMR spectra of the labeled A beta(1-40) samples. The fpRFDR-CT data reveal nearest-neighbor intermolecular distances of 4.8 +/- 0.5 A for carbon sites from residues 12 through 39, indicating a parallel alignment of neighboring peptide chains in the predominantly beta-sheet structure of the amyloid fibrils. The one-dimensional NMR spectra indicate structural order at these sites. The fpRFDR-CT data and NMR spectra also indicate structural disorder in the N-terminal segment of A beta(1-40), including the first nine residues. These results place strong constraints on any molecular-level structural model for full-length beta-amyloid fibrils.     

Use of relaxation-edited one-dimensional and two dimensional nuclear magnetic resonance spectroscopy to improve detection of small metabolites in blood plasma

The 1H nuclear magnetic resonance (NMR) spectra of biological samples, such as blood plasma and tissues, are information rich but data complex owing to superposition of the resonances from a multitude of different chemical entities in multiple-phase compartments, hampering detection and subsequent resonance assignments. To overcome these problems, several spectral-editing NMR experiments are described here, combining spin-relaxation filters (based on T(1), T(rho), and T(2)) with both one-dimensional and two-dimensional (2D) NMR spectroscopy. These techniques enable the separation of NMR resonances based on their relaxation times and allow simplification of the complex spectra. In this paper, the approach is exemplified using a control human blood plasma, which is a complex mixture of proteins, lipoproteins, and small-molecule metabolites. In the case of T(1rho)- and T(2)-edited 2D NMR experiments, a "flip-back" pulse was introduced after the relaxation editing to make the phase cycling of the "relaxation filter" and the 2D NMR part independent, thus enabling easy implementation of the phase-sensitive 2D NMR experiments. These methods also permit much higher receiver gains to be used to reduce digitization error, in particular, for the small resonances, which are sometimes vitally important for metabonomics studies. Both pulse sequences and experimental results are discussed for T(1)-, T(1rho)-, and T(2)-filtered COSY, T(2)-filtered phase-sensitive DQF-COSY, and T(1), T(1rho)-, and T(2)-filtered TOCSY NMR.     

Localization of binding sites for carboxyl terminal specific anti-rhodopsin monoclonal antibodies using synthetic peptides

The binding sites for four monoclonal antibodies, rho 1D4, rho 3C2, rho 3A6, and rho 1C5, have been localized within the C-terminal region of bovine rhodopsin: Asp18'-Glu-Ala16'-Ser-Thr-Thr-Val12'-Ser-Lys-Thr-Gl u8'-Thr-Ser-Gln-Val4'-Ala-Pr o -Ala1'. Antibody binding sites were localized by using synthetic C-terminal peptides in conjunction with solid-phase competitive inhibition assays and limited proteolytic digestion of rhodopsin in conjunction with electrophoretic immunoblotting techniques. Binding of the rho 1D4 and rho 3C2 antibodies to immobilized rhodopsin was inhibited with peptides of length 1'-8' and longer. Antibody rho 1D4 binding was not inhibited by peptides 2'-13' or 3'-18', indicating that the C-terminal alanine residue of rhodopsin was required. Similar competitive inhibition studies indicated that the antibody rho 3A6 required peptides of length 1'-12' and longer whereas rho 1C5 required peptide 1'-18'. Peptide 3'-18' was as effective as 1'-18' in inhibiting rho 3A6 binding to rhodopsin, but replacement of glutamic acid in position 8' with glutamine abolished competition. This substitution had little effect on the binding of antibody rho 1C5. Thus, Glu8' was essential for rho 3A6 binding but not for the binding of the rho 1C5 antibody. Cleavage of the seven amino acid C-terminus from rhodopsin and further cleavage to F1 (Mr 25 000) and F2 (Mr 12 000) fragments with Staphylococcus aureus V8 protease abolished binding of rho 1D4 antibody to the membrane-bound rhodopsin fragments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Orientation dependence of NMR relaxation time, T(1rho), in lipid bilayers

The orientation dependence of the low frequency NMR relaxation time, T(1rho), of protons in aligned phospholipid bilayers was measured using 13C cross polarisation and direct proton experiments. The contribution of intra- and inter-molecular interactions to proton T(1rho) was determined by using dimyristoyl phosphatidylcholine (DMPC) with one hydrocarbon chain deuterated and dispersed in perdeuterated DMPC. The results indicated that intramolecular motions on the kHz timescale were the major cause of T(1rho) relaxation in phospholipid bilayers.     

Slow motion in the CAA*TTG sequence of a DNA decamer duplex studied by NMR

In DNA duplexes, pyrimidine-purine steps are believed to be flexible or conformationally unstable. Indeed, several DNA crystal structures exhibit a multitude of conformations for CpA*TpG steps. The question arises of whether this structural flexibility is accompanied by dynamical flexibility, i.e., a question pertaining to the energy barrier between conformations. Except for TpA steps, slow motions on the microsecond-to-millisecond time scale have not been detected in duplexes until now. In the present study, such slow motion was investigated by 1H, 13C, and 15N NMR relaxation measurements on a DNA decamer d(CATTTGCATC)*d(GATGCAAATG). The DNA decamer was enriched with 15% 13C and 98% 15N isotopes for each adenosine and guanosine residue. Three lines of evidence support the notion of slow motion in the CAA*TTG moiety. Analysis of (15)N relaxation showed that the order parameter, S2, of guanosine imino NH groups was about 0.8, similar to that of CH groups for this oligomer. The strong temperature dependence of guanosine NH S2 in the CAA*TTG sequence indicated the presence of a large-amplitude motion. Signals of adenosine H8 protons in the CAA*TTG sequence were broadened in 2D 1H NOESY spectra, which also suggested the existence of slow motion. As well as being smaller than for other adenine residues, the 1H T2 values exhibited a magnetic field strength dependence for all adenosine H8 signals in the ATTTG*CAAAT region, suggesting slow motions more pronounced at the first adenosine in the CAA*TTG sequence but extending over the CAAAT*ATTTG region. This phenomenon was further examined by the pulse field strength dependence of the 1H, 13C, and 15N T1rho values. 1H and 13C T1rho values showed a pulse field strength dependence, but 15N T1rho did not. Assuming a two-site exchange process, an exchange time constant of 20-300 micros was estimated for the first adenosine in the CAA sequence. The exact nature of this motion remains unknown.     

Study of high molecular weight wheat glutenin subunit 1Dx5 by 13C and 1H solid-state NMR spectroscopy. I. Role of covalent crosslinking

This work describes a carbon and proton solid-state NMR study of the hydration of a high molecular weight wheat glutenin subunit, 1Dx5. The effect of the presence of disulfide bonds on the hydration behavior of the subunit is investigated by a comparison of the unalkylated and alkylated forms of the protein. Hydration induces partial plasticization of the protein so that some segments become more mobile than others. The 13C cross-polarization and magic-angle spinning (MAS) spectra of the samples in the dry state and at two hydration levels (approximately 40 and approximately 65% D2O) were used to monitor the protein fraction resisting plasticization (trains). Conversely, 13C single pulse excitation and 1H-MAS experiments were used to gain information on the more plasticized segments (loops). The molecular motion of the two protein dynamic populations was further characterized by 13C T1 and 1H T(1rho), T2, and T1 relaxation times. The results suggest that hydration leads to the formation of a network held by a cooperative action of hydrogen bonded glutamines and some hydrophobic interactions. The looser protein segments are suggested to be glycine- and glutamine-rich segments. The primary structure is therefore expected to significantly determine the proportion of trains and loops in the network. The presence of disulfide bonds was observed to promote easier plasticization of the protein and the formation of a more mobile network, probably involving a higher number of loops and/or larger loops.     

Mitochondrial deoxyribonucleic acid from Tetrahymena pyriformis and its kinetic complexity

1. Mitochondrial DNA from Tetrahymena pyriformis strain T has a buoyant density (rho) of 1.685 compared with rho1.688 for whole cell DNA. Mitochondrial preparations from T. pyriformis strain W show an enrichment of a light satellite (rho1.686), although this is not obtained free from nuclear DNA (rho1.692). 2. T. pyriformis mitochondrial DNA renatures rapidly and the kinetics of this process indicate a complexity of approx. 3x10(7) daltons. 3. The base-pairing in the renaturation product is of a precise nature, since the ;melting' temperature (80.5 degrees C) is indistinguishable from that of the native DNA (80.5 degrees C). 4. Centrifugation of mitochondrial DNA in an alkaline caesium chloride density gradient gives two bands, implying the separation of the complementary strands.     

Use of (13)c MAS NMR to study domain structure and dynamics of polysaccharides in the native starch granules

To investigate the domain structure and dynamics of polysaccharides in the native starch granules, a variety of high resolution, solid-state (13)C NMR techniques have been applied to all three (A-, B-, and C-) types of starch with different water content. Both single-pulse-excitation magic-angle-spinning (SPEMAS) and cross-polarization-magic-angle-spinning (CPMAS) methods have been employed together with the PRISE (proton relaxation induced spectral-editing) techniques to distinguish polysaccharide fractions in different domains and having distinct dynamics. It has been found that, for all three types of dry starch granules, there are two sets of NMR signals corresponding to two distinct ordered polysaccharides. Hydration leads to substantial mobilization of the polysaccharides in the amorphous regions, but no fundamental changes in the rigidity of the polysaccharides in the crystalline (double) helices. Full hydration also leads to limited mobility changes to the polysaccharides in the amorphous lamellae (branching zone) within the amylopectin clusters and in the gaps between the arrays of the amylopectin clusters. Under magic-angle spinning, proton relaxation-time measurements showed a single component for T(1), two components for T(1rho), and three components for T(2). PRISE experiments permitted the neat separation of the (13)C resonances of polysaccharides in the crystalline lamellae from those in the amorphous lamellae and the amylose in the gaps between amylopectin clusters. It has been found that the long (1)H T(1rho) component ( approximately 30 ms) is associated with polysaccharides in the crystalline lamellae in the form of double helices, whereas the short T(1rho) component (2-4 ms) is associated with amylose in the gaps between amylopectin clusters. The short (1)H T(2) component ( approximately 14 micros) is associated with polysaccharides in the crystalline lamellae; the intermediate component (300-400 micros) is associated with polysaccharides in the amorphous lamellae and amylose in the gaps between amylopectin clusters. The long T(2) component is associated with both mobile starch protons and the residue water protons.     

Proton exchange as a relaxation mechanism for T1 in the rotating frame in native and immobilized protein solutions.

T1 relaxation in the rotating frame (T1rho) is a sensitive magnetic resonance imaging (MRI) contrast for acute brain insults. Biophysical mechanisms affecting T1rho relaxation rate (R1rho) and R1rho dispersion (dependency of R1rho on the spin-lock field) were studied in protein solutions by varying their chemical environment and pH in native, heat-denatured, and glutaraldehyde (GA) cross-linked samples. Low pH strongly reduced R1rho in heat-denatured phantoms displaying proton resonances from a number of side-chain chemical groups in high-resolution 1H NMR spectra. At pH of 5.5, R1rho dispersion was completely absent. In contrast, in the GA-treated phantoms with very few NMR visible side chain groups, acidic pH showed virtually no effect on R1rho. The present data point to a crucial role of proton exchange on R1rho and R1rho dispersion in immobilized protein solution mimicking tissue relaxation properties.     

Solid-state nuclear magnetic resonance evidence for an extended beta strand conformation of the membrane-bound HIV-1 fusion peptide.

Solid-state nuclear magnetic resonance (NMR) spectroscopy was applied to the membrane-bound form of a synthetic peptide representing the 23-residue N-terminal fusion peptide domain of the HIV-1 gp41 envelope glycoprotein. 1D solid-state NMR line width measurements of singly 13C carbonyl labeled peptides showed that a significant population of the membrane-bound peptide is well-structured in its N-terminal and central regions while the C-terminus has more disorder. There was some dependence of line width on lipid composition, with narrower line widths and hence greater structural order observed for a lipid composition comparable to that found in the virus and its target T cells. In the more ordered N-terminal and central regions of the peptide, the 13C carbonyl chemical shifts are consistent with a nonhelical membrane-bound conformation. Additional evidence for a beta strand membrane-bound conformation was provided by analysis of 2D rotor-synchronized magic angle spinning NMR spectra of doubly 13C carbonyl labeled peptides. Lipid mixing and aqueous contents leakage assays were applied to demonstrate the fusogenicity of the peptide under conditions comparable to those used for the solid-state NMR sample preparation.