Response of lysozyme internal dynamics to hydration probed by13C and1H solid-state NMR relaxation

We have studied the hydration dependence of the internal protein dynamics of hen egg white lysozyme by naturally abundant¹³C and¹H nuclear magnetic resonance (NMR) relaxation. NMR relaxation timesT ₁, off-resonanceT _1p and proton-decoupled on-resonanceT _1p (only for carbon expriments) were measured in the temperature range from 0 to 50°C. The spectral resolution in carbon cross-polarization magic angle spinning spectrum allows to treat methine, methylene and methyl carbons separately, while proton experiments provide only one integral signal from all protons at a time. The relaxation times were quantitatively analyzed by the well-established correlation function formalism and model-free approach. The whole set of the data could be adequately described by a model assuming three types of motion having correlation times around 10^?4, 10^?9 and 10^?12 s. The slowest process originated from correlated conformational transitions between different energy minima, the intermediate process could be identified as librations within one energy minimum, and the fastest one is a fast rotation of methyl protons the symmetry axis of methyl groups. A comparison of the dynamic behavior of lysozyme and polylysine obtained from a previous study (A. Krushelnitsky, D. Faizullin, D. Reichert, Biopolymers 73, 1–15, 2004) reveals that in the dry state both biopolymers are rigid on both fast and slow time scales. Upon hydration, lysozyme and polylysine reveal a considerable enhancement of the internal mobility, however, in different ways. The side chains of polylysine are more mobile than those of lysozyme, whereas for the backbone a reversed picture is observed. This difference correlates with structural features of lysozyme and polylysine discussed in detail. Due to the presence of a fast spin diffusion, the analysis of proton relaxation data is a more difficult task. However, our data demonstrate that the correlation functions of motion obtained from carbon and proton experiments are substantially different. We explained this by the fact that these two types of NMR relaxation experiments probe the motion of different internuclear vectors. The comparison of the proton data with our previous results on proton relaxation timesT ₁ measured over a wide temperature range indicates that at low temperatures lysozyme undergoes structural rearrangements affecting the amplitudes and/or activation energies of motions.     

Mobility and structure of human serum albumin powders in water-acetonitrile mixtures by1H NMR relaxation and FTIR spectroscopy

The effect of acetonitrile on protein dynamics was investigated for solid human serum albumin samples at various hydration levels. Temperature dependences of¹H nonselective nuclear magnetic resonanceT ₁ andT ₂ relaxation times at 27 MHz have been measured and data were interpreted in terms of three kinds of internal motions in the protein. Microdynamic parameters of the motions were obtained within a “model-free” approach. It was found that acetonitrile hardly affects the fast motions but noticeably influences the slow motion of side chain groups, shortening the correlation time and increasing the amplitude of the motion. The acetonitrile effect on dynamics is likely based on the appearance of additional free volume as a result of the formation of rigid helical parts in the protein structure. Water, plasticizing the protein structure, promotes the action of organic solvent. A definite part of side chain groups, slowly moving in the same frequency window as the rest of protein side chain groups, performs less constrained “liquidlike” motion. The relative population of these highly movable protons is closely correlated with the increment of the helical structure induced by water and acetonitrile.     

Molecular dynamics in solid pregnenolone studied by 1H spin-lattice relaxation

Spin-lattice relaxation times T1 in solid pregnenolone have been studied over a wide range of temperatures, from 77 up to 417 K. The dynamic processes arising from C3 motion of the three methyl substituents are separated, and their activation parameters are determined.     

Molecular dynamics in solid pyridoxine as studied by 1H NMR

Spin-lattice relaxation times T1 and T1d as well as NMR second moment were employed to study molecular dynamics of pyridoxine (vitamin B6) in the temperature range 10-350 K. The T1 minimum observed at low temperatures at 200 MHz is attributed to a motion of methyl group. The motion is interpreted in terms of Haupt's theory, which takes into account the tunneling assisted relaxation. At low temperatures, where T1 is temperature independent, occupation of the ground state only is assumed. A motion of proton of the hydroxyl groups or CH2OH groups probably provides additional mechanism of relaxation, in the high-temperature region.     

Molecular dynamics in solid riboflavin as studied by 1H NMR

Spin–lattice relaxation times T₁ and T_1d as well as NMR second moment were employed to study the molecular dynamics of riboflavin (vitamin B₂) in the temperature range 55–350 K. The broad and flat T₁ minimum observed at low temperatures is attributed to the motion of two nonequivalent methyl groups. The motion of the methyl groups is interpreted in terms of Haupt's theory, which takes into account the tunneling assisted relaxation. An additional mechanism of relaxation in the high temperature region is provided by the motion of a proton in one of the hydroxyl groups. The Davidson–Cole distribution of correlation times for this motion is assumed.     

Molecular dynamics in solid anhydrous beta-estradiol studied by 1H NMR.

Proton second moment and spin-lattice relaxation times T1 and T1p in solid anhydrous beta-estradiol are measured as a function of temperature. The results show that the C3 reorientation of the single methyl group provides the mechanism dominating relaxation at low temperatures and reveal the existence of a conformational motion of the carbon skeleton dominating relaxation at high temperatures. The activation energies of the respective motions are found to be 9.3 and 37.3 kJ/mol.     

13C solid-state NMR chromatography by magic angle spinning 1H T1 relaxation ordered spectroscopy

An efficient method to separate the ¹³C NMR spectra of solid mixtures is introduced. The ¹H longitudinal (T₁) relaxation time is used to separate the overlapping ¹³C chemical shift spectra of solid mixtures via an inverse Laplace transform (ILT) of the relaxation dimension. The resulting 2D spectrum of the mixture contains separate ¹³C spectra for each component of the mixture that are identical to ¹³C spectra of the isolated materials. The separation is based on the equalization of ¹H T₁ values in a single domain by rapid ¹H spin diffusion and on the ¹H T₁ value differences between different domains. The introduction of a general ILT scheme enables efficient and reduced data acquisition time. The method is demonstrated on a mixture of two disaccharides and on a commercial drug containing several compounds.     

Molecular dynamics of poly(L-lactide) biopolymer studied by wide-line solid-state 1H and 2H NMR spectroscopy

The molecular dynamics of poly(L-lactide) (PLLA) biopolymer was characterized through analyses of 1H and 2H NMR line-shapes and spin-lattice relaxation times at different temperatures. At low temperatures (e.g. 90 K), the methyl group rotation is dominant leading to a significant reduction in the proton second moment. Fast methyl group reorientation occurs at ca. 130 K. In additional to the fast methyl group rotation, hydroxyl groups start to reorient as the temperature increases further, eventually leading to the breakdown of the segments of the biopolymer chains above its glass transition temperature Tg of 323 K. The analyses of the 2H NMR line-shapes indicate that both the methyl and hydroxyl reorientations can be described by the so-called cone model, in which the former has three equilibrium positions with theta(C-D) = 70.5 degrees and phi = 120 degrees while the latter one exhibits two equilibrium positions with theta(O-D) = 78 degrees and phi = 180 degrees .     

原油降粘剂组分的红外光谱及核磁共振氢谱的分析研究

为了明确原油降粘剂的主要组分,采取蒸馏、溶解一沉淀法及柱色谱法对原油降粘剂进行分离与纯化,用红外光谱法对其主要组分官能团的结构进行了鉴定,推断其主要组分是丙烯酸乙酯/甲基丙烯酸甲酯/丙烯酸三元共聚物,用核磁共振氢谱及质谱进行了结构确认及定量分析,共聚物中3种单体:丙烯酸乙酯、甲基丙烯酸甲酯、丙烯酸的摩尔数比为37.1:25.8:37.1;重量百分比为41.1:28.8:29.8.用红外光谱对甲醇溶液部分进行了结构分析,结果表明,非离子表面活性剂为聚氧乙烯醚,分子量范围是800～1 600,阴离子表面活性剂为烷基苯磺酸钠,其余为助剂和水.     

Pore-size determination of mesoporous materials by1H NMR spectroscopy

The pore-size distributions of a series of mesoporous silica materials were determined by measuring the¹H nuclear magnetic resonance (NMR) signal from the nonfrozen fraction of organic probe molecules as a function of temperature. The melting point distribution curves of confined benzene reveal 2–3 transition points. The high-temperature transition point, corresponding to the temperature at the first maximum of the melting point distribution curve, is interpreted as the average depressed melting point of the confined substance. However, the intensity data reveal that a measurable portion of the confined benzene apparently remains nonfrozen even 120 K below the bulk melting point in the 4–10 nm pore systems. The component at lowest temperature is largely attributed to the liquidlike molecules at the pore wall, while the component at the intermediate temperature might result from pockets in the solid matrix or even a bimodal pore-size distribution. The average pore-size distributions obtained by NMR agree fairly well with those obtained by N₂ sorption. However, NMR gives a more detailed picture of the distribution, revealing two or three well-defined peaks. The peak at the smallest pore size, however, reflects the surface layer rather than a pore-size distribution.     

Molecular dynamics in solid pregnenolone studied by H spin–lattice relaxation

Spin–lattice relaxation times T₁ in solid pregnenolone have been studied over a wide range of temperatures, from 77 up to 417 K. The dynamic processes arising from C₃ motion of the three methyl substituents are separated, and their activation parameters are determined.     

Picoliter (1)H NMR spectroscopy

In this study, a 267-microm-diameter solenoid transceiver is used to acquire localized (1)H NMR spectra and the measured signal-to-noise ratio (SNR) at 500 MHz is shown to be within 20--30% of theoretical limits formulated by considering only its resistive losses. This is illustrated using a 100-microm-diameter globule of triacylglycerols (approximately 900mM) that may be an oocyte precursor in young Xenopus laevis frogs and a water sample containing choline at a concentration often found in live mammalian cells (approximately 33 mM). In chemical shift imaging (CSI) experiments performed using a few thousand total scans, the choline methyl line is shown to have an acceptable SNR in resolved volume elements containing only 50 pL of sample, and localized spectra are resolved from just 5 pL in the Xenopus globule. These findings demonstrate the feasibility of performing (1)H NMR on picoliter-scale sample volumes in biological cells and tissues and illustrate how the achieved SNR in spectroscopic images can be predicted with reasonable accuracy at microscopic spatial resolutions.     

2H T2rho relaxation dynamics and double-quantum filtered NMR studies

In this study 2H T2rho DQF NMR spectra of water in MCM-41 were measured. The T2rho double-quantum filtered (DQF) NMR signal is generated by applying a radio frequency (RF) field for various durations and then observed after a monitor RF pulse. It was found that the transfer between different quantum coherences by the couplings during long-duration RF fields (i.e., soft pulses) and that residual quadrupolar interaction dominates the signal decay. Knowledge of coherence transfer during long-RF pulses has special significance for the development of sophisticated multi-quantum NMR experiments especially multi-quantum MRI applications.     

Molecular dynamics of n-dodecylammonium chloride in aqueous solutions investigated by 2H NMR and 1H NMR relaxometry

Molecular dynamics in n-dodecylammonium chloride/water solutions for concentrations of 34 and 45 wt% was studied by 2H NMR and by 1H NMR dispersion of spin-lattice relaxation in the 2 kHz-90 MHz frequency range. The system exhibits a number of lyotropic liquid crystalline phases, which differ in symmetry and involve motions characterized by a wide frequency scale. The analysis of 2H NMR lineshapes of selectively deuterated DDACl molecules gave us an evidence for local trans-gauche conformational changes in the chains, whereas the dispersion of spin-lattice relaxation times T1 explored by fast field cycling method revealed fast local motions, translational diffusion and collective molecular dynamics of the chains. In particular, we have found that the order director fluctuation mechanism in smectic and nematic phases dominates spin-lattice relaxation below 1 MHz and that local motions and translational diffusion are responsible for the spin-lattice relaxation in the higher Larmor frequency range.     

Quantitative 2H NMR spectroscopy with 1H lock extender

An inexpensive external unit that allows the use of the commercial high-resolution NMR spectrometer for (2)H observation with an (1)H lock system is described. The external unit does not require any tuning, is extremely easy to use, and could be a cheaper and more straightforward alternative to the more expensive (19)F lock configuration. An application for the quantitative determination of the natural isotopic ratio (2)H/(1)H of ethanol and acetic acid is reported.     

Measurement of longitudinal relaxation times in crowded 1H NMR spectra using one- and two-dimensional maximum quantum (MAXY) NMR spectroscopy

Methods for measuring longitudinal relaxation times of protons in heavily overlapped ¹H NMR spectra are introduced and exemplified using a solution of cholesteryl acetate. The methods are based on 1-dimensional and 2-dimensional maximum quantum NMR spectroscopy (MAXY), which makes possible the selective detection of CH, CH₂ and CH₃ ¹H NMR resonances. A modification of the BIRD pulse sequence to achieve selective inversion of protons bonded to either ¹²C or ¹³C is given. The approach should find application in studies of molecular dynamics where isotopic enrichment is not possible and the level of available sample dictates the use of ¹H NMR spectroscopy.     

Proton relaxation studies of 17α hydroxy- and 21 hydroxy-progesterones by1H NMR

Proton spin-lattice relaxation timesT ₁ in 17α hydroxy- and 21 hydroxy-progesterones have been performed in the temperature range from 100 to 400 K at the frequencies of 30 and 90 MHz. The dynamic processes involving the methyl group reorientation about the threefold symmetry axis of the C−C bond are separated, and their activation parameters are determined.     

Characterisation of crosslinked elastomeric materials by 1H NMR relaxation time distributions.

One of the most critical structural parameters in elastomeric materials is the density of cross-linking between the polymeric chains. This chemical feature greatly affects chain motions and is determinant in controlling mechanical properties of the final product. NMR techniques are widely and efficiently applied to investigation of such materials. In this study we have measured both transverse and longitudinal 1H relaxation times of a series of polybutadiene rubber samples with increasing crosslink density induced by chemical treatment. This approach allowed the observation of T(1) and T(2) decrease with the increase of crosslink density in the samples examined. The data obtained have been analyzed and compared to theoretical models.