Motional Correlation Times Research Articles

Time-Resolved CIDNP Study of Native-State Bovine and Human α-Lactalbumins

The reaction mechanism and details of the formation of CIDNP (chemically induced dynamic nuclear polarization) in the photoreactions of two aromatic dyes (2,2‘-dipyridyl, DP, and flavin mononucleotide, FMN) with bovine and human α-lactalbumins (BLA and HLA) in aqueous solution have been studied using the time-resolved CIDNP technique. With DP as the photosensitizer, polarization in BLA is observed for the protons of Trp118, His68, Tyr18, and Tyr103. The latter is not manifested in the CIDNP spectra obtained with FMN. This threshold effect on CIDNP formation indicates that accessibility is a combined property of both the residue and the dye. The nuclear spin−lattice relaxation times in the radicals formed in these reactions have been determined from the CIDNP kinetics: T1 = 60 μs for H2,4,7 of Trp118, T1 = 53 μs for H3,5 of Tyr103, T1 = 16 μs for H3,5 of Tyr18, and T1 = 10 μs for the H2 and H4 of His68. The correlation times for the side chain motion as determined from the T1 of the radicals are correlated with the accessibility of the side chains in the intact protein.

Influence of confined geometry on nuclear spin relaxation and self-diffusion in liquid indium

The spin-lattice relaxation times for the $^{115}\mathrm{In}$ isotope in liquid indium embedded into artificial opals and porous glass were found to be shortened remarkably compared to the bulk indium melt. The spin relaxation acceleration was ascribed mainly to the enhancement of the quadrupole contribution caused by the translational diffusion in liquid indium; the magnetic contribution was implied unchanged on the base of the Knight shift measurements and the Korringa relation. Calculations made for quadrupole relaxation showed that it dominated the spin relaxation process for indium in nanopores and that the correlation times of atomic motion increased by more than a factor of 6 depending on pore sizes. The increase in the correlation time evidenced drastic slowing down the atomic diffusion for confined melted indium.

Backbone dynamics of oxidised and reduced forms of human atrial natriuretic peptide

The backbone dynamics of the 28 residue 15N-labelled human atrial natriuretic peptide have been examined by 15N NMR methods. 15N R 1, R 2 and { 1H}– 15N NOE values were determined for the oxidised and reduced forms of the peptide (ANP ox and ANP rd, respectively), and analysed using reduced spectral density mapping and an extended model-free approach. The two forms possessed correlation times for overall molecular motion of 4.7 ns and were highly flexible, with substantial contributions to relaxation processes from internal motions on picosecond to nanosecond time scales. Reduction of the Cys7–Cys23 disulphide bond to form ANP rd produced a very dynamic linear peptide with a mean overall order parameter of 0.2; the intramolecular cross-link in ANP ox increased this to a mean value of 0.4. A simple model for segmental backbone motion accounted for the R 2 values of both species using only two variable parameters, indicating that relaxation is dominated by interactions with sites <7 residues distant in the covalent network and that changes in the conformation of the disulphide bond lead to significant chemical exchange broadening in ANP ox. The contributions of backbone dynamics to configurational entropy were determined and accounted for the different receptor binding affinities of cyclised and linear natriuretic peptides.

Dynamic Heterogeneity in Interfacial Region of Microphase-Separated Polystyrene-block-poly(methyl acrylate) Studied by the ESR Spin-Label Technique

Molecular motion in an interfacial region of microdomains of polystyrene-block-poly(methyl acrylate) (PS-block-PMA) was studied by electron spin resonance (ESR) technique. The junction points between the blocks, which are located in the interfacial region, were labeled with stable nitroxide radicals. Mobility of the spin-labels reflected the dynamic environments in the interfacial region. The transition temperature of the motion of the spin-labels, T5.0mT, at which the extreme separation width due to 14N anisotropic hyperfine splitting is 5.0 mT, was estimated, and it reflects a glass transition of the region around the labels. The T5.0mT of the PS-block-PMA labeled at the junction point was almost the mean value of those of the spin-labeled PS and PMA homopolymers, and the distribution of the motional correlation times (τc) in the interfacial region was much broader than that in the homopolymers. These results are considered to be caused by the heterogeneous mixture of the each segment in the interfacial region at a certain length scale. The molecular weight strongly influenced the interfacial thickness and the segmental mobility and the width of the distribution of the τc in the interfacial region as well as on the glass transition temperatures (Tg's) of the microdomains. It was revealed that the width of the distribution of the τc depended on not the interfacial thickness so much as the difference between the mobilities of the block chains in the microdomains. On the other hand, extremely small effects of the overall composition and the morphology of the PS-block-PMA on the segmental mobility in the interfacial region were observed. From these results, it was considered that the dynamic environment in the interfacial region was strongly affected by the gradient of the segmental concentration in the interfacial region and the mobility of the block chains in the microdomains.

Dynamics of the Escherichia coli O91 O-antigen polysaccharide in solution as studied by carbon-13 NMR relaxation.

The dynamics of the O-antigen part of the lipopolysaccharide from the enterohemorrhagic Escherichia coli O91 has been determined in solution using (13)C NMR relaxation measurements at two magnetic field strengths, 9.4 and 14.1 T, thereby facilitating the testing of several dynamical models. The biological repeating unit, consisting of five sugar residues and substituents, could be determined by spectral analysis of different (1)H,(13)C correlations and corroborated by the relaxation data. The site specifically (13)C-labeled material was shown to have approximately 10 repeating units with a narrow distribution. A model-free analysis of the relaxation data revealed a complex dynamical behavior where the sugar residues could be described by a global correlation time (tau(m) = 5.4 ns), generalized order parameters (S(2) approximately 0.63), and different correlation times for internal motions related to their position in the repeating unit along the polymer (tau(e) approximately 360-520 ps). One of the sugar residues showed, in addition, a chemical exchange contribution. Furthermore, a substituent on another sugar residue was described by two order parameters (S(f)(2) = 0.51 and S(s)(2) = 0.21). The solution dynamics of the polysaccharide are thus described by highly intricate motions, both in amplitude and time scales. These results are of significance in the general description of polysaccharides surrounding bacterial cell surfaces and in the presentation of antigenic epitopes to the immune system of an invaded host.

Nuclear Magnetic Resonance Investigation of the Micellar Properties of Two-Headed Surfactant Systems: The Disodium 4-Alkyl-3-sulfonatosuccinates. 2. The Dynamics of the Chains Comprising the Interior of Two-Headed Surfactant Micelles

The dynamics of the carbons comprising the micelles of two members of the family of two-headed surfactants, the disodium 4-alkyl-3-sulfonatosuccinates, has been determined via the application of the two-step model to the 13C relaxation rates and the nuclear Overhauser enhancements (nOe's) at 200 MHz. The NMR relaxation times, determined from the inversion recovery method, increase steadily as we descend the chain from the headgroup region. The relaxation rate profiles and the order parameters have been calculated from the two-step model for the micellar sulfosuccinate aggregates. We note that the order parameter profile and the fast motion correlation time profile for these two-headed surfactants are distinctly different from those of a typical single-headed, single-tailed surfactant such as dodecyltrimethylammonium bromide, particularly in the headgroup region of the micelle. All these results are interpreted in terms of the effect of adding a second headgroup to a single-headed, single-tailed surfactant.

1H NMR relaxation investigation of acetylcholinesterase inhibitors from huperzine A and derivative

The binding properties of huperzine A ( 1) with Torpediniforms Nacline acetylcholinesterase (TnAChE) were investigated by 1H NMR methods. The noselective, selective and double-selective spin-lattice relaxation rates were acquired in absent and present of TnAChE at a ratio [ligand]/[protein]=1:0.005. The selective relaxation rates shown protons of 1 had dipole–dipole interaction with protein active site protons. The motional correlation time of bound ligand was calculated by double-selective relaxation rate at 1 τ 2,3=40.5 ns at 298 K, which showed 1 had high affinity with TnAChE. The experiments give a possible method to use TnAChE to locate the new huperzine A derivatives as AChE inhibitors.

Backbone dynamic properties of the central linker region of calcium-calmodulin in 35% trifluoroethanol

The backbone dynamic properties of uniformly 15N-labeled calcium-saturated calmodulin (Ca 2+-CaM) in 35% 2,2,2-trifluoroethanol (TFE) have been examined by 15N NMR relaxation methods. This particular solvent was chosen in order to mimic the conditions in which CaM was crystallized, which included the presence of alcohols. Special attention was paid to the central linker region of Ca 2+-CaM, which is a long, solvent-exposed α-helix in the crystal structure but is known to be partially unwound and flexible in solution. 15N T 1, T 2, and 15N–{ 1H} NOE values were determined for both Ca 2+-CaM in H 2O and Ca 2+-CaM in 35% TFE, and the results indicated that the presence of 35% TFE did indeed induce a more ordered conformation in the central linker, with order parameters for Asp78–Glu80 of 0.29, 0.17, and 0.27 in H 2O and 0.82, 0.66, and 0.64 in 35% TFE. However, 15N–{ 1H} NOE values showed that these residues were still slightly more flexible than the rest of the molecule in 35% TFE (Asp78–Glu80 15N–{ 1H} NOE=0.46, 0.46, and 0.51). Furthermore, there is still independent motion of the two lobes of Ca 2+-CaM in 35% TFE, with motional correlation times of ∼10 and ∼9 ns for the N- and C-lobes, respectively, indicating that 35% TFE was not sufficient to force Ca 2+-CaM into a rigid dumbbell-shaped molecule as seen in the crystal structure. Additional factors that could further stabilize the structure of CaM in the crystal include pH, temperature, and crystal packing.

Size effect in nuclear spin-lattice relaxation and atomic mobility for molten gallium particles

The spin-lattice relaxation rate of gallium isotopes in a melt was shown by NMR to increase considerably with a decrease in gallium particle size. The dominant relaxation mechanism of gallium embedded in an synthetic opal matrix changes from magnetic dipole to electrical quadrupole. The increase in the correlation time of atomic motion in gallium particles with a decrease in their size was estimated. For gallium in opal, the correlation time was found to increase by more than an order of magnitude. It was shown that a variation in atomic mobility becomes noticeable already in gallium particles about 5 µm in size.

Hydration dependence of backbone and side chain polylysine dynamics: a 13C solid-state NMR and IR spectroscopy study.

The molecular dynamics of solid poly-L-lysine has been studied by the following natural abundance (13)C-NMR relaxation methods: measurements of the relaxation times T(1) at two resonance frequencies, off-resonance T(1rho) at two spin-lock frequencies, and proton-decoupled T(1rho). Experiments were performed at different temperatures and hydration levels (up to 17% H(2)O by weight). The natural abundance (13)C-CPMAS spectrum of polylysine provides spectral resolution of all types of backbone and side chain carbons and thus, dynamic parameters could be determined separately for each of them. At the same time, the conformational properties of polylysine were investigated by Fourier transform infrared spectroscopy. The data obtained from the different NMR experiments were simultaneously analyzed using the correlation function formalism and model-free approach. The results indicate that in dry polylysine both backbone and side chains take part in two low amplitude motions with correlation times of the order of 10(-4) s and 10(-9) s. Upon hydration, the dynamic parameters of the backbone remain almost constant except for the amplitude of the slower process that increases moderately. The side chain dynamics reveals a much stronger hydration response: the amplitudes of both slow and fast motions increase significantly and the correlation time of the slow motion shortens by about five orders of magnitude, and at hydration levels of more than 10% H(2)O fast and slow side chain motions are experimentally indistinguishable. These changes in the molecular dynamics cannot be ascribed to any hydration-dependent conformational transitions of polylysine because IR spectra reveal almost no hydration dependence in either backbone or side chain absorption domains. The physical nature of the fast and slow motions, their correlation time distributions, and hydration dependence of microdynamic parameters are discussed.

Dynamics and phase behaviour of materials in confined geometry

Phase transitions and dynamics of the liquid crystal MBBA [N-( p-methoxybenzylidene)-p-n-butylaniline] microconfined within porous glasses of average pore diameters: 82 and 337 Å were investigated by 1H Nuclear Magnetic Resonance (NMR). Confinement is found to favour the depression of phase transition temperatures below the corresponding transitions of bulk MBBA. Confinement induces further effects for the molecular dynamics related to the phase investigated. The correlation times of the molecular motions were obtained and the results are discussed by making comparison with the bulk analysis.

An NMR Study on the Dynamic Behavior of Triethylamine Included in AFI Crystals — Influence of Acid Sites on the Motional State of Triethylamine Molecules —

Abstract The 1H NMR spin-lattice relaxation times (T1) of triethylamine (TEA) molecules included in five kinds of AFI crystals with different concentrations of Si atoms in the wall were measured. The temperature and Larmor frequency dependences of T1 were well explained by introducing a distributed motional correlation time (τ) for the pseudo-C3 TEA reorientation. An obtained linear decrease in the τ distribution width with an increase of the Si concentration, i.e., the number of Brønsted acid sites, was shown to be related to the orientations of TEA molecules in channels.

Comparison of (13)C(alpha)H and (15)NH backbone dynamics in protein GB1.

This study presents a site-resolved experimental view of backbone C(alpha)H and NH internal motions in the 56-residue immunoglobulin-binding domain of streptococcal protein G, GB1. Using (13)C(alpha)H and (15)NH NMR relaxation data [T(1), T(2), and NOE] acquired at three resonance frequencies ((1)H frequencies of 500, 600, and 800 MHz), spectral density functions were calculated as F(omega) = 2omegaJ(omega) to provide a model-independent way to visualize and analyze internal motional correlation time distributions for backbone groups in GB1. Line broadening in F(omega) curves indicates the presence of nanosecond time scale internal motions (0.8 to 5 nsec) for all C(alpha)H and NH groups. Deconvolution of F(omega) curves effectively separates overall tumbling and internal motional correlation time distributions to yield more accurate order parameters than determined by using standard model free approaches. Compared to NH groups, C(alpha)H internal motions are more broadly distributed on the nanosecond time scale, and larger C(alpha)H order parameters are related to correlated bond rotations for C(alpha)H fluctuations. Motional parameters for NH groups are more structurally correlated, with NH order parameters, for example, being larger for residues in more structured regions of beta-sheet and helix and generally smaller for residues in the loop and turns. This is most likely related to the observation that NH order parameters are correlated to hydrogen bonding. This study contributes to the general understanding of protein dynamics and exemplifies an alternative and easier way to analyze NMR relaxation data.

Influence of Chain End and Molecular Weight on Molecular Motion of Polystyrene, Revealed by the ESR Selective Spin-Label Method

A local segmental mobility was determined by electron spin resonance (ESR) spin-label method for a series of polystyrene (PS) with various molecular weights. Each PS specimen was selectively spin-labeled with stable nitroxide radicals at a chain end or inside sites. Molecular motion at the inside of the chain was compared with that at the chain end from the temperature dependence of ESR spectra of the nitroxide radicals. The transition temperature of molecular motion, T5.0mT, at which the extreme separation width due to 14N anisotropic hyperfine splitting is 5.0 mT, increased with an increase in molecular weight. The WLF equation confirmed that the T5.0mT correlated with a glass transition temperature, Tg, of PS. The T5.0mT for the spin-labeled PS at the chain end was ca. 5 K lower than that for the spin-labeled PS at the inside sites due to the enrichment of the specific free volume around the chain end. The transition temperature, T5.0mT, for both labeled PS depended on the molecular weight in accordance with the Unberreiter−Kanig equation for a glass transition. The T5.0mT for the spin-labeled PS at the chain end had a strong dependence on the molecular weight as compared with that at the inside sites because the molecular motion of the chain end was accelerated by an encounter of more than two chain ends. From the molecular weight dependence, we determined the short correlation time for segmental motion of the chain end, ca. 40 s, and the segment size undergoing the segmental motion at the Tg. The obtained segment size agreed well with the general segment size reported by others, 5−10 monomeric unit size.

Motions of IsolatedG‐Band Bright Points in the Solar Photosphere

Magnetic elements on the quiet Sun are buffeted by convective flows that cause lateral motions on timescales of minutes. The magnetic elements can be observed as bright points (BPs) in the G band at 4305 A ˚ .W e present observations of BPs based on a long sequence of G-band images recorded with the Dutch Open Telescope and postprocessed using speckle-masking techniques. From these images we measured the proper motions of isolated BPs and derived the autocorrelation function of their velocity relative to the solar granulation pattern. The accuracy of BP position measurements is estimated to be less than 23 km on the Sun. The rms velocity of BPs (corrected for measurement errors) is about 0.89 km s � 1 , and the correlation time of BP motions is about 60 s. This rms velocity is about 3 times the velocity measured using cork tracking, almost certainly due to the fact that isolated BPs move more rapidly than clusters of BPs. We also searched for evidence of vorticity in the motions of G-band BPs. Subject headings: Sun: atmospheric motions — Sun: faculae, plages — Sun: magnetic fields — techniques: high angular resolution

15NH Backbone Dynamics of Protein GB1: Comparison of Order Parameters and Correlation Times Derived Using Various “Model-Free” Approaches

A new approach that allows visualization of the distribution of motional correlation times is presented and is used to analyze 1 5 N NMR relaxation data [T 1 , T 2 , and { 1 H}- 1 5 N NOE], acquired at 500. 600, and 800 MHz, on the uniformly 1 5 N-enriched, 56 residue B1 domain from immunoglubulin protein G (GB I ). Nanosecond time scale internal motions are found for all NHs of residues in protein GB1, a finding which is consistent with the concept of hierarchical internal motions in proteins first forwarded by Frauenfelder. Order parameters and overall tumbling correlation times derived using this approach are not influenced by the number of internal motional modes. Comparatively, use of the Lipari-Szabo, Clore et al., or Lemaster model-free methods yields underestimated order parameters and overestimated overall tumbling correlation times due to the presence of nanosecond time scale internal motions. This new approach provides a straightforward way to derive more accurate order parameters and correlation times for overall tumbling and internal motions.

Heat Capacities and a Snapshot of the Energy Landscape in Protein GB1 from the Pre-denaturation Temperature Dependence of Backbone NH Nanosecond Fluctuations

Protein stability is usually characterized calorimetrically by a melting temperature and related thermodynamic parameters. Despite its importance, the microscopic origin of the melting transition and the relationship between thermodynamic stability and dynamics remains a mystery. Here, NMR relaxation parameters were acquired for backbone 15NH groups of the 56 residue immunoglobulin-binding domain of streptococcal protein G over a pre-denaturation temperature range of 5–50 °C. Relaxation data were analyzed using three methods: the standard three-Lorentzian model free approach; the F(ω)=2ω J(ω) spectral density approach that yields motional correlation time distributions, and a new approach that determines frequency-dependent order parameters. Regardless of the method of analysis, the temperature dependence of internal motional correlation times and order parameters is essentially the same. Nanosecond time-scale internal motions are found for all NHs in the protein, and their temperature dependence yields activation energies ranging up to about 33 kJ/mol residue. NH motional barrier heights are structurally correlated, with the largest energy barriers being found for residues in the most “rigid” segments of the fold: β-strands 1 and 4 and the α-helix. Trends in this landscape also parallel the free energy of folding–unfolding derived from hydrogen–deuterium (H–D) exchange measurements, indicating that the energetics for internal motions occurring on the nanosecond time-scale mirror those occurring on the much slower time-scale of H–D exchange. Residual heat capacities, derived from the temperature dependence of order parameters, range from near zero to near 100 J/mol K residue and correlate with this energy landscape. These results provide a unique picture of this protein's energy landscape and a relationship between thermodynamic stability and dynamics that suggests thermosensitive regions in the fold that could initiate the melting process.

Large-scale molecular dynamics simulations of general anesthetic effects on the ion channel in the fully hydrated membrane: the implication of molecular mechanisms of general anesthesia.

Interactions of volatile anesthetics with the central nervous system are characterized by low yet specific binding affinities. Although neurotransmitter-gated ion channels are considered the primary anesthetic targets, the mechanism of action at the molecular level remains elusive. We consider here the theoretical implications of channel dynamics on anesthetic action in a simplified membrane-channel system. Large-scale 2.2-ns all-atom molecular dynamics simulations were performed to study the effects of halothane, a clinical anesthetic, on a gramicidin A (gA) channel in a fully hydrated dimyristoyl phosphatidylcholine membrane. In agreement with experimental results, anesthetics preferentially target the anchoring residues at the channel-lipid-water interface. Although the anesthetic effect on channel structure is minimal, the presence of halothane profoundly affects channel dynamics. For 2.2-ns simulation, the rms fluctuation of gA backbone in the lipid core increases from approximately equal 1 A in the absence of anesthetics to approximately equal 1.5 A in the presence of halothane. Autocorrelation analysis reveals that halothane (i) has no effect on the subpicosecond librational motion, (ii) prolongs the backbone autocorrelation time in the 10- to 100-ps time scale, and (iii) significantly decreases the asymptotic values of generalized order parameter and correlation time of nanosecond motions for the inner but not the outer residues. The simulation results discount the viewpoint of a structure-function paradigm that overrates the importance of structural fitting between general anesthetics and yet-unidentified hydrophobic protein pockets. Instead, the results underscore the global, as opposed to local, effects of anesthetics on protein dynamics as the underlying mechanisms for the action of general anesthetics and possibly of other low-affinity drugs.

Production and characterization of recombinant tachycitin, the Cys-rich chitin-binding protein.

Tachycitin is an invertebrate chitin-binding protein with an amidated C-terminus, and possesses antimicrobial activity against both fungi and bacteria. The (1)H-NMR-based tertiary structure of tachycitin was recently determined [Suetake et al. (2000) J. Biol. Chem., 275, 17929-17932]. In order to examine the structural and functional features of tachycitin more closely, we performed for the first time, gene expression, refolding, (15)N-NMR-based characterizations, and antimicrobial activity measurements of a recombinant tachycitin (rTcn) that does not have the amide group at the C-terminus. The NMR analysis indicated that rTcn possesses the same structural construction as the native tachycitin. The backbone (15)N relaxation measurements showed that the molecular motional correlation time of rTcn increases as its concentration increases, indicating that tachycitins have a tendency to aggregate with each other. rTcn exhibits antimicrobial activity against fungi but not against bacteria. The cell surface of fungi contains chitin as an essential constituent, but that of bacteria does not. These results suggest that not only the chitin-binding region but also the C-terminal amide group of tachycitin plays a significant role in its antimicrobial properties.

Probing oxygen motion in disordered anionic conductors with 17O and 51V MAS NMR spectroscopy.

Identification of the local environments of the ions in a solid-state electrolyte that contribute to the ionic conductivity or remain trapped in the lattice represents a challenge for many experimental probes of structure. We show that high-resolution 17O magic angle spinning nuclear magnetic resonance (MAS NMR) spectra may be obtained even from the highly disordered, layered materials alpha-Bi4V2O11 and gamma-Bi4V1.7Ti0.3O10.85, in which the different oxide sites in the lattice may be distinguished. The sites responsible for anionic conduction were determined directly from the variable-temperature 17O NMR spectra, and correlation times for motion were estimated. Double-resonance 17O/51V NMR methods were used as confirmation of the assignments of the resonances and as a second experimental probe of motion that is sensitive to mobility involving oxide ion hops between the same crystallographic sites.