Adiabatic compressibility and intrinsic viscosity studies on peptide aggregates

Density and sound velocity measurements and ¹H NMR investigations were carried out in aqueous solution at various temperatures for determining the adiabatic compressibility () and hydration of the tetrapeptide, TFA. Tyr-Gly-Phe-Ala-Obz I. The present investigation showed changes in the temperature coefficient of adiabatic compressibility at 40 °C. ¹H NMR studies indicated the inverse temperature transition in the concentration range studied.     

Compressibility and specific volume of actin decrease upon G to F transformation

We measured the densities as well as the sound velocities in solutions of G-actin, F-actin and the reconstituted thin filament. Using the data obtained, we determined their partial specific volumes and partial specific adiabatic compressibilities. The objectives were to investigate the volume change of actin upon polymerization and to detect the conformational change associated with the Ca²⁺-binding to the reconstituted thin filament. The partial specific volume and the partial specific adiabatic compressibility of G-actin were 0.749 cm³/g and 9.3 · 10⁻¹² cm²/dyne, respectively. The results suggest that G-actin is a rather soft protein compared with other globular proteins. The partial specific volumes of F-actin were in a range of 0.63–0.66 cm³/g depending on the solvent conditions. The partial specific adiabatic compressibilities of F-actin were negative (−(7–13) · 10⁻¹² cm³/dyne). These data indicate that the amount of hydration may increase by several times upon polymerization assuming that the size of the cavity remains constant. We detected little difference between the partial specific adiabatic compressibility of the reconstituted thin filament in a Ca²⁺-bound state and that in a Ca²⁺-unbound state. This suggests that the Ca²⁺ binding affected not the subunit itself but the inter-subunit junction.     

Study of the interaction of an α-helical transmembrane peptide with phosphatidylcholine bilayer membranes by means of densimetry and ultrasound velocimetry

We applied precise densimetry and ultrasound velocimetry methods to study the interaction of a synthetic α-helical transmembrane peptide, acetyl-K₂-L₂₄-K₂-amide (L₂₄), with model bilayer lipid membranes. The large unilamellar vesicles (LUVs) utilized were composed of a homologous series of n-saturated diacylphosphatidylcholines (PCs). PCs whose hydrocarbon chains contained from 13 to 16 carbon atoms, thus producing phospholipid bilayers of different thicknesses and gel to liquid-crystalline phase transition temperatures. This allowed us to analyze how the difference between the hydrophobic length of the peptide and the hydrophobic thickness of the lipid bilayer influences the thermodynamical and mechanical properties of the membranes. We showed that the incorporation of L₂₄ decreases the temperature and cooperativity of the main phase transition of all LUVs studied. The presence of L₂₄ in the bilayer also caused an increase of the specific volume and of the volume compressibility in the gel state bilayers. In the liquid crystalline state, the peptide decreases the specific volume at relatively higher peptide concentration (mole ratio L₂₄:PC = 1:50). The overall volume compressibility of the peptide-containing lipid bilayers in the liquid-crystalline state was in general higher in comparison with pure membranes. There was, however, a tendency for the volume compressibility of these lipid bilayers to decrease with higher peptide content in comparison with bilayers of lower peptide concentration. For one lipid composition, we also compared the thermodynamical and mechanical properties of LUVs and large multilamellar vesicles (MLVs) with and without L₂₄. As expected, a higher cooperativity of the changes of the thermodynamical and mechanical parameters took place for MLVs in comparison with LUVs. These results are in agreement with previously reported DSC and ²H NMR spectroscopy study of the interaction of the L₂₄ and structurally related peptides with phosphatidylcholine bilayers. An apparent discrepancy between ²H NMR spectroscopy and compressibility data in the liquid crystalline state may be connected with the complex and anisotropic nature of macroscopic mechanical properties of the membranes. The observed changes in membrane mechanical properties induced by the presence of L₂₄ suggest that around each peptide a distorted region exists that involves at least 2 layers of lipid molecules.     

Protein Linewidth and Solvent Dynamics in Frozen Solution NMR

Solid-state NMR of proteins in frozen aqueous solution is a potentially powerful technique in structural biology, especially if it is combined with dynamic nuclear polarization signal enhancement strategies. One concern regarding NMR studies of frozen solution protein samples at low temperatures is that they may have poor linewidths, thus preventing high-resolution studies. To learn more about how the solvent shell composition and temperature affects the protein linewidth, we recorded ¹H, ²H, and ¹³C spectra of ubiquitin in frozen water and frozen glycerol-water solutions at different temperatures. We found that the ¹³C protein linewidths generally increase with decreasing temperature. This line broadening was found to be inhomogeneous and independent of proton decoupling. In pure water, we observe an abrupt line broadening with the freezing of the bulk solvent, followed by continuous line broadening at lower temperatures. In frozen glycerol-water, we did not observe an abrupt line broadening and the NMR lines were generally narrower than for pure water at the same temperature. ¹H and ²H measurements characterizing the dynamics of water that is in exchange with the protein showed that the ¹³C line broadening is relatively independent from the arrest of isotropic water motions.     

Structural characterization of homogalacturonan by NMR spectroscopy-assignment of reference compounds

Complete assignment of ¹H and ¹³C NMR of six hexagalactopyranuronic acids with varying degree and pattern of methyl esterification is reported. The NMR experiments were run at room temperature using approximately 2 mg of sample making this method convenient for studying the structure of homogalacturonan oligosaccharides.     

Broadband homonuclear chemical shift correlation at high MAS frequencies: a study of tanh/tan adiabatic RF pulse schemes without $${^{{\bf 1}}\hbox{{\bf H}}}$$ decoupling during mixing

At high magic angle spinning (MAS) frequencies the potential of tanh/tan adiabatic RF pulse schemes for ¹³C chemical shift correlation without ¹H decoupling during mixing has been evaluated. It is shown via numerical simulations that a continuous train of adiabatic ¹³C inversion pulses applied at high RF field strengths leads to efficient broadband heteronuclear decoupling. It is demonstrated that this can be exploited effectively for generating through-bond and through-space, including double-quantum, correlation spectra of biological systems at high magnetic fields and spinning speeds with no ¹H decoupling applied during the mixing period. Experiments carried out on a polycrystalline sample of histidine clearly suggest that an improved signal to noise ratio can be realised by eliminating ¹H decoupling during mixing.     

Efficient solvent suppression with adiabatic inversion for 1H-detected solid-state NMR

This study introduces a conceptually new solvent suppression scheme with adiabatic inversion pulses for ¹H-detected multidimensional solid-state NMR (SSNMR) of biomolecules and other systems, which is termed “Solvent suppression of Liquid signal with Adiabatic Pulse” (SLAP). ¹H-detected 2D ¹³C/¹H SSNMR data of uniformly ¹³C- and ¹⁵N-labeled GB1 sample using ultra-fast magic angle spinning at a spinning rate of 60 kHz demonstrated that the SLAP scheme showed up to 3.5-fold better solvent suppression performance over a traditional solvent-suppression scheme for SSNMR, MISSISSIPPI (Zhou and Rienstra, J Magn Reson 192:167–172, 2008) with 2/3 of the average RF power.

     

Robust refocusing of 13C magnetization in multidimensional NMR experiments by adiabatic fast passage pulses

We show that adiabatic fast passage (AFP) pulses are robust refocusing elements of transverse ¹³C magnetization in multidimensional NMR experiments. A pair of identical AFP pulses can refocus selected parts or a complete ¹³ C chemical shift range in ¹³C spectra. In the constant time ¹³C-¹H HSQC, replacement of attenuated rectangular pulses by selective AFP pulses results in a sensitivity enhancement of up to a factor of 1.8. In the 3D CBCA(CO)NH the signal-to-noise ratio is increased by a factor of up to 1.6.     

[2-3H]ATP synthesis and 3H NMR spectroscopy of enzyme-nucleotide complexes: ADP and ADP.Vi bound to myosin subfragment 1

Summary The synthesis of [2-³H]ATP with specific activity high enough to use for ³H NMR spectroscopy at micromolar concentrations was accomplished by tritiodehalogenation of 2-Br-ATP. ATP with greater than 80% substitution at the 2-position and negligible tritium levels at other positions had a single ³H NMR peak at 8.20 ppm in 1D spectra obtained at 533 MHz. This result enables the application of tritium NMR spectroscopy to ATP utilizing enzymes.The proteolytic fragment of skeletal muscle myosin, called S1, consists of a heavy chain (95 kDa) and one alkali light chain (16 or 21 kDa) complex that retains myosin ATPase activity. In the presence of Mg²⁺, S1 converts [2-³H]ATP to [2-³H]ADP and the complex S1.Mg[2-³H]ADP has ADP bound in the active site. At 0°C, 1D ³H NMR spectra of S1.Mg[2-³H]ADP have two broadened peaks shifted 0.55 and 0.90 ppm upfield from the peak due to free [2-³H]ADP. Spectra with good signal-to-noise for 0.10 mM S1.Mg[2-³H]ADP were obtained in 180 min. The magnitude of the chemical shift caused by binding is consistent with the presence of an aromatic side chain being in the active site. Spectra were the same for S1 with either of the alkali light chains present, suggesting that the alkali light chains do not interact differently with the active site. The two broad peaks appear to be due to the two conformations of S1 that have been observed previously by other techniques. Raising the temperature to 20 °C causes small changes in the chemical shifts, narrows the peak widths from 150 to 80 Hz, and increases the relative area under the more upfield peak. Addition of orthovanadate (V_i) to produce S1.Mg[2-³H]ADP.V_i shifts both peaks slightly more upfield without chaning their widths or relative areas.     

Quantitative analysis of metabolite concentrations in human urine samples using 13C{1H} NMR spectroscopy

Targeted profiling is a library-based method of using mathematically modeled reference spectra for quantification of metabolite concentrations in NMR mixture analysis. Metabolomics studies of biofluids, such as urine, represent a highly complex problem in this area, and for this reason targeted profiling of ¹H NMR spectra can be hampered. A number of the issues relating to ¹H NMR spectroscopy can be overcome using ¹³C{¹H} NMR spectroscopy. In this work, a ¹³C{¹H} NMR database was created using Chenomx NMR Suite, incorporating 120 metabolites. The ¹³C{¹H} NMR database was standardized through the analysis of a series of metabolite solutions containing varying concentrations of 19 distinct metabolites, where the metabolite concentrations were varied across a range of values including biological ranges. Subsequently, the NMR spectra of urine samples were collected using ¹³C{¹H} NMR spectroscopy and profiled using the ¹³C{¹H} NMR library. In total, about 30 metabolites were conclusively identified and quantified in the urine samples using ¹³C{¹H} NMR targeted profiling. The proton decoupling and larger spectral window provided easier identification and more accurate quantification for specific classes of metabolites, such as sugars and amino acids with overlap in the aliphatic region of the ¹H NMR spectrum. We discuss potential application areas in which ¹³C{¹H} NMR targeted profiling may be superior to ¹H NMR targeted profiling.     

H NMR study of rehydration/dehydration and water mobility in β-cyclodextrin

The processes of dehydration and rehydration of β-cyclodextrin were studied by analysis of the ¹H NMR (nuclear magnetic resonance) line shape. Dehydration was carried in an open ampoule as a function of temperature and above 400 K total dehydration of β-cyclodextrin was observed. This result was confirmed by the thermogravimetry (TG) measurements. Rehydration was studied as a function of time at room temperature. After 40 days, β-cyclodextrin was found to absorb eight water molecules. The analysis of temperature changes in the shape of the ¹H NMR line of β-cyclodextrin kept in a closed ampoule and its dielectric measurements provided information on the mobility of water molecules. The water molecules were found to perform complex molecular motions, that is, reorientational jumps below 200 K and additionally, translational motion (diffusion) above 200 K.     

Micro-coil NMR to monitor optimization of the reconstitution conditions for the integral membrane protein OmpW in detergent micelles

Optimization of aqueous solutions of the integral membrane protein (IMP) OmpW for NMR structure determination has been monitored with micro-coil NMR, which enables the acquisition of NMR spectra using only micrograms of protein and detergent. The detergent 30-Fos (2-undecylphosphocholine) was found to yield the best 2D [¹⁵N, ¹H]-TROSY correlation NMR spectra of [²H, ¹⁵N]-labeled OmpW. For the OmpW structure determination we then optimized the 30-Fos concentration, the sample temperature and long-time stability, and the deuteration level of the protein. Some emerging guidelines for reconstitution of ??-barrel integral membrane proteins in structural biology are discussed.     

Adiabatic Heteronuclear Decoupling in Rotating Solids

In magic angle spinning solid state NMR experiments the potential of heteronuclear (1)H decoupling employing a continuous train of adiabatic inversion pulses has been assessed via numerical simulations and experimental measurements. It is shown that, with a (1)H RF field strength of approximately 100 kHz that is typically available in MAS NMR probes, it is possible to achieve efficient adiabatic (1)H decoupling at low magic angle spinning frequencies. It is pointed out that in the presence of H (1) inhomogeneities it will be advantageous to employ adiabatic decoupling in MAS solid state NMR experiments.     

Deuteron NMR spectroscopy of copper(II) complexes in solution. 1. (5,7,7,12,14,14,-hexamethyl-1,4,8,1 1-tetraazacyclotetradeca-4,11-diene)-copper(II) and its derivatives in nonaqueous solvent

¹H and ²H NMR spectra of the title copper(II) complexes and its derivatives have been measured. In contrast with their ¹H NMR spectra, ²H NMR spectra gave well resolved sharp signals, and demonstrated that two diastereomers attributable to two asymmetric ligand nitrogens are readily resolved. The remarkable linewidth-narrowing was found in the peripheral methyl groups, which make ²H NMR spectra very useful even for copper(II) complexes with a long electron spin relaxation time. By using ²H NMR spectra, meso-racemate equilibrium was pursued and examined in aqueous and acetonitrile solutions.     

Preparation of sulfated-chitins under homogeneous conditions

Sulfated-chitins of varying degrees of sulfation were prepared by the reaction of chitin with sulfur trioxide–pyridine complex under homogeneous conditions in 5% LiCl/DMAc solvent system. Sulfation at 8 °C or room temperature was regio-selective for the C6–OH position with the degree of sulfation (D.S.) ranging from 0.53 to 1.00 depending on the reaction time. When the reaction temperature was elevated, sulfation at the C3–OH position also occurred. The extent of sulfation at the C3 position was a function of the concentration of sulfating reagent, reaction time and temperature. The structure of sulfated-chitins was established by ¹H, ¹³C NMR and 2D HMQC. The degree of sulfation at the C6 position was estimated by ¹H NMR while that of the C3 position was by elemental analyses. The anticoagulant activity of the prepared sulfated-chitins correlated closely with D.S. The higher the D.S. yielded, the better the anticoagulant activity. In particular, a continuous sequence of 36S units was critical for obtaining high anticoagulation activity.     

Complete H and C NMR chemical shift assignments of mono-, di-, and trisaccharides as basis for NMR chemical shift predictions of polysaccharides using the computer program casper

The computer program casper uses ¹H and ¹³C NMR chemical shift data of mono- to trisaccharides for the prediction of chemical shifts of oligo- and polysaccharides. In order to improve the quality of these predictions the ¹H and ¹³C, as well as ³¹P when applicable, NMR chemical shifts of 30 mono-, di-, and trisaccharides were assigned. The reducing sugars gave two distinct sets of NMR resonances due to the α- and β-anomeric forms. In total 35 ¹H and ¹³C NMR chemical shift data sets were obtained from the oligosaccharides. One- and two-dimensional NMR experiments were used for the chemical shift assignments and special techniques were employed in some cases such as 2D ¹H,¹³C-HSQC Hadamard Transform methodology which was acquired approximately 45 times faster than a regular t₁ incremented ¹H,¹³C-HSQC experiment and a 1D ¹H,¹H-CSSF-TOCSY experiment which was able to distinguish spin-systems in which the target protons were only 3.3 Hz apart. The ¹H NMR chemical shifts were subsequently refined using total line-shape analysis with the PERCH NMR software. The acquired NMR data were then utilized in the casper program (http://www.casper.organ.su.se/casper/) for NMR chemical shift predictions of the O-antigen polysaccharides from Klebsiella O5, Shigella flexneri serotype X, and Salmonella arizonae O62. The data were compared to experimental data of the polysaccharides from the two former strains and the lipopolysaccharide of the latter strain showing excellent agreement between predicted and experimental ¹H and ¹³C NMR chemical shifts.     

Effective strategy to assign 1H-15N heteronuclear correlation NMR signals from lysine side-chain NH3 + groups of proteins at low temperature

Recent studies have shown that lysine side-chain NH₃ ⁺ groups are excellent probes for NMR investigations of dynamics involving hydrogen bonds and ion pairs relevant to protein function. However, due to rapid hydrogen exchange, observation of ¹H-¹⁵N NMR cross peaks from lysine NH₃ ⁺ groups often requires use of a relatively low temperature, which renders difficulty in resonance assignment. Here we present an effective strategy to assign ¹H and ¹⁵N resonances of NH₃ ⁺ groups at low temperatures. This strategy involves two new ¹H/¹³C/¹⁵N triple-resonance experiments for lysine side chains. Application to a protein-DNA complex is demonstrated.     

Review of NMR and μSR studies in the molecular nanomagnet Mn12-ac

We present a comprehensive review of the NMR and μSR studies performed in the molecular nanomagnet Mn12 a system characterized and widely studied by Prof. Gatteschi’s group in Florence. The proton (¹H, ²D) NMR, the ⁵⁵Mn NMR and the μSR investigations have yielded important information regarding both static and dynamic magnetic properties of the molecule. The magnetic and quadrupole hyperfine interactions have been extracted from NMR data. The spin dynamics at high and intermediate temperature associated with the zero dimensionality and with the spin–phonon coupling has been studied together with the spin dynamics in the quantum tunneling low temperature regime. The local spin configuration in the giant S = 10 total spin ground state has been determined via ⁵⁵Mn NMR in zero magnetic field and with fields parallel and perpendicular to the anisotropy axis. Finally a novel method is described to monitor the relaxation of the magnetization from the time evolution of the NMR spectrum.     

CHARACTERIZATION OF THE MAIN PHASE TRANSITION IN 1,2-DIPALMITOYL-PHOSPHATIDYLCHOLINE LUVS BY 1H NMR*

ABSTRACT

The main phase transition (Tm) of 100 nm large unilamellar vesicles (LUVs) of 1,2-dipalmitoylphosphatidylcholine (DPPC) was investigated using ¹H NMR (proton magnetic resonance) in deuterium oxide, and both DSC (differential scanning calorimetry) and IR (infrared) spectroscopy in water and deuterium oxide. The ability of ¹H NMR to determine Tm was demonstrated and the values obtained were in general agreement with those observed with DSC and IR. However, the temperature range of the transition observed by NMR was significantly broader than that observed with either DSC or IR. The effect of deuterium oxide on Tm was studied by comparing results obtained in water and deuterium oxide with DSC and IR. The results showed no significant difference in Tm or temperature range of transition determined in these solvents.