Relaxation Probes Research Articles

Site-specific protein methyl deuterium quadrupolar patterns by proton-detected 3D 2H-13C-1H MAS NMR spectroscopy.

Determination of protein structure and dynamics is key to understand the mechanism of protein action. Perdeuterated proteins have been used to obtain high resolution/sensitivty NMR experiments via proton-detection. These methods utilizes 1H, 13C and 15N nuclei for chemical shift dispersion or relaxation probes, despite the existing abundant deuterons. However, a high-sensitivity NMR method to utilize deuterons and e.g. determine site-specific deuterium quadrupolar pattern information has been lacking due to technical difficulties associated with deuterium's large quadrupolar couplings. Here, we present a novel deuterium-excited and proton-detected three-dimensional 2H-13C-1H MAS NMR experiment to utilize deuterons and to obtain site-specific methyl 2H quadrupolar patterns on detuterated proteins for the first time. A high-resolution fingerprint 1H-15N HSQC-spectrum is correlated with the anisotropic deuterium quadrupolar tensor in the third dimension. Results from a model perdeuterated protein has been shown.

The SAMI Galaxy Survey: Kinematics of Stars and Gas in Brightest Group Galaxies—The Role of Group Dynamics

Abstract We study the stellar and gas kinematics of the brightest group galaxies (BGGs) in dynamically relaxed and unrelaxed galaxy groups for a sample of 154 galaxies in the SAMI galaxy survey. We characterize the dynamical state of the groups using the luminosity gap between the two most luminous galaxies and the BGG offset from the luminosity centroid of the group. We find that the misalignment between the rotation axis of gas and stellar components is more frequent in the BGGs in unrelaxed groups, although with quite low statistical significance. Meanwhile, galaxies whose stellar dynamics would be classified as “regular rotators” based on their kinemetry are more common in relaxed groups. We confirm that this dependency on group dynamical state remains valid at fixed stellar mass and Sérsic index. The observed trend could potentially originate from a differing BGG accretion history in virialized and evolving groups. Among the halo relaxation probes, the group BGG offset appears to play a stronger role than the luminosity gap on the stellar kinematic differences of the BGGs. However, both the group BGG offset and luminosity gap appear to roughly equally drive the misalignment between the gas and stellar component of the BGGs in one direction. This study offers the first evidence that the dynamical state of galaxy groups may influence the BGG's stellar and gas kinematics, and we call for further studies using a larger sample with higher signal-to-noise.

Conformational Entropy from Restricted Bond-Vector Motion in Proteins: The Symmetry of the Local Restrictions and Relation to NMR Relaxation.

Locally mobile bond-vectors contribute to the conformational entropy of the protein, given by Sk ≡ S/k = −∫(Peq ln Peq)dΩ – ln∫dΩ. The quantity Peq = exp(−u)/Z is the orientational probability density, where Z is the partition function and u is the spatially restricting potential exerted by the immediate internal protein surroundings at the site of the motion of the bond-vector. It is appropriate to expand the potential, u, which restricts local rotational reorientation, in the basis set of the real combinations of the Wigner rotation matrix elements, D0KL. For small molecules dissolved in anisotropic media, one typically keeps the lowest even L, L = 2, nonpolar potential in axial or rhombic form. For bond-vectors anchored at the protein, the lowest odd L, L = 1, polar potential is to be used in axial or rhombic form. Here, we investigate the effect of the symmetry and polarity of these potentials onSk. For L = 1 (L = 2), Sk is the same (differs) for parallel and perpendicular ordering. The plots of Sk as a function of the coefficients of the rhombic L = 1 (L = 2) potential exhibit high-symmetry (specific low-symmetry) patterns with parameter-range-dependent sensitivity. Similar statements apply to analogous plots of the potential minima. Sk is also examined as a function of the order parameters defined in terms of u. Graphs displaying these correlations, and applications illustrating their usage, are provided. The features delineated above are generally useful for devising orienting potentials that best suit given physical circumstances. They are particularly useful for bond-vectors acting as NMR relaxation probes in proteins, when their restricted local motion is analyzed with stochastic models featuring Wigner-function-made potentials. The relaxation probes could also be molecules adsorbed at surfaces, inserted into membranes, or interlocked within metal–organic frameworks.

NMR contributions to structural dynamics studies of intrinsically disordered proteins

Intrinsically disordered proteins (IDPs) are characterized by substantial conformational plasticity. Given their inherent structural flexibility X-ray crystallography is not applicable to study these proteins. In contrast, NMR spectroscopy offers unique opportunities for structural and dynamic studies of IDPs. The past two decades have witnessed significant development of NMR spectroscopy that couples advances in spin physics and chemistry with a broad range of applications. This article will summarize key advances in basic physical-chemistry and NMR methodology, outline their limitations and envision future R&D directions.

Modeling helical proteins using residual dipolar couplings, sparse long-range distance constraints and a simple residue-based force field

We present a fast and simple protocol to obtain moderate-resolution backbone structures of helical proteins. This approach utilizes a combination of sparse backbone NMR data (residual dipolar couplings and paramagnetic relaxation enhancements) or EPR data with a residue-based force field and Monte Carlo/simulated annealing protocol to explore the folding energy landscape of helical proteins. By using only backbone NMR data, which are relatively easy to collect and analyze, and strategically placed spin relaxation probes, we show that it is possible to obtain protein structures with correct helical topology and backbone RMS deviations well below 4 Å. This approach offers promising alternatives for the structural determination of proteins in which nuclear Overha-user effect data are difficult or impossible to assign and produces initial models that will speed up the high-resolution structure determination by NMR spectroscopy.

Experimental Measurement and Theoretical Assessment of Fast Lanthanide Electronic Relaxation in Solution with Four Series of Isostructural Complexes

The rates of longitudinal relaxation for ligand nuclei in four isostructural series of lanthanide(III) complexes have been measured by solution state NMR at 295 K at five magnetic fields in the range 4.7-16.5 T. The electronic relaxation time T(le) is a function of both the lanthanide ion and the local ligand field. It needs to be considered when relaxation probes for magnetic resonance applications are devised because it affects the nuclear relaxation, especially over the field range 0.5 to 4.7 T. Analysis of the data, based on Bloch-Redfield-Wangsness theory describing the paramagnetic enhancement of the nuclear relaxation rate has allowed reliable estimates of electronic relaxation times, T(1e), to be obtained using global minimization methods. Values were found in the range 0.10-0.63 ps, consistent with fluctuations in the transient ligand field induced by solvent collision. A refined theoretical model for lanthanide electronic relaxation beyond the Redfield approximation is introduced, which accounts for the magnitude of the ligand field coefficients of order 2, 4, and 6 and their relative contributions to the rate 1/T(le). Despite the considerable variation of these contributions with the nature of the lanthanide ion and its fluctuating ligand field, the theory explains the modest change of measured T(le) values and their remarkable statistical ordering across the lanthanide series. Both experiment and theory indicate that complexes of terbium and dysprosium should most efficiently promote paramagnetic enhancement of the rate of nuclear relaxation.

Single-walled carbon nanotubes as anisotropic relaxation probes for magnetic resonance imaging

We report on the preparation and characterization of magnetically oriented single-walled carbon nanotube arrangements as novel nanoprobes to enhance anisotropically water relaxation as detected by magnetic resonance imaging methods. SWCNT suspensions immobilized in agarose gels showed evident magnetic anisotropy with significantly longer T2 in the parallel than in the perpendicular orientations.

Sorption Selectivity in Natural Organic Matter Studied with Nitroxyl Paramagnetic Relaxation Probes

Sorption site selectivity and mechanism in natural organic matter (NOM) were addressed spectroscopically by the sorption of paramagnetic nitroxyl compounds (spin probes) of different polarity, TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl) and HTEMPO (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl). The sorbents were Pahokee peat, Beulah-Zap lignite, and a polystyrene-poly(vinyl methyl ether) (PS-PVME) polymer blend representing the mixed aliphatic-aromatic, polar-nonpolar character of NOM. Nuclear-electron spin interaction serves as an efficient relaxation pathway, resulting in attenuation of the (13)C-CP/TOSS NMR signal for (13)C nuclei in proximity to the N-O· group (r(-6) dependence). In the natural solids the spin probes sorbed more specifically (greater isotherm nonlinearity) and had lower rotational mobility (broader electron paramagnetic resonance signals) than in PS-PVME. Titration with spin probe indicated almost no selectivity for the different carbon functional groups of PS-PVME, and little to no selectivity for the different carbon moieties of Pahokee and Beulah, including aromatic, alkyl, O-alkyl, di-O-alkyl, and O-methyl. In any case, sorption site selectivity of spin probes to NOM was always weaker than partition selectivity found in model solvent-water (toluene, hexadecane, anisole, octanol) and cellulose-water systems. The results indicate little or no preferential sorption in NOM based on functional group chemistry or putative microdomain character, but rather are consistent with the filling of pores whose walls have an average chemical environment reflecting the bulk chemical composition of the solid. This work demonstrates for the first time the use of paramagnetic probes to study sorption specificity.

Influence of aging time of oleate precursor on the magnetic relaxation of cobalt ferrite nanoparticles synthesized by the thermal decomposition method

Cobalt ferrite nanoparticles are of interest because of their room temperature coercivity and high magnetic anisotropy constant, which make them attractive in applications such as sensors based on the Brownian relaxation mechanism and probes to determine the mechanical properties of complex fluids at the nanoscale. These nanoparticles can be synthesized with a narrow size distribution by the thermal decomposition of an iron–cobalt oleate precursor in a high boiling point solvent. We studied the influence of aging time of the iron–cobalt oleate precursor on the structure, chemical composition, size, and magnetic relaxation of cobalt ferrite nanoparticles synthesized by the thermal decomposition method. The structure and thermal behavior of the iron–cobalt oleate was studied during the aging process. Infrared spectra indicated a shift in the coordination state of the oleate and iron/cobalt ions from bidentate to bridging coordination. Aging seemed to influence the thermal decomposition of the iron–cobalt oleate as determined from thermogravimmetric analysis and differential scanning calorimetry, where shifts in the temperatures corresponding to decomposition events and a narrowing of the endotherms associated with these events were observed. Aging promoted formation of the spinel crystal structure, as determined from X-ray diffraction, and influenced the nanoparticle magnetic properties, resulting in an increase in blocking temperature and magnetocrystalline anisotropy. Mossbauer spectra also indicated changes in the magnetic properties resulting from aging of the precursor oleate. Although all samples exhibited some degree of Brownian relaxation, as determined from complex susceptibility measurements in a liquid medium, aging of the iron–cobalt oleate precursor resulted in crossing of the in-phase χ′and out-of-phase χ″ components of the complex susceptibility at the frequency of the Brownian magnetic relaxation peak, as expected for nanoparticles that relax through a single relaxation mechanism. The resulting nanoparticles would be suitable for sensors based on the Brownian relaxation mechanism and in determining mechanical properties of complex fluids at the size scale of the nanoparticles.

Spatially homogeneous ferromagnetism of (Ga, Mn)As

Mn-doped GaAs is a ferromagnetic semiconductor, widely studied because of its possible application for spin-sensitive 'spintronics' devices. The material also attracts great interest in fundamental research regarding its evolution from a paramagnetic insulator to a ferromagnetic metal. The high sensitivity of its physical properties to preparation conditions and heat treatments and the strong doping and temperature dependencies of the magnetic anisotropy have generated a view in the research community that ferromagnetism in (Ga, Mn)As may be associated with unavoidable and intrinsic strong spatial inhomogeneity. Muon spin relaxation (muSR) probes magnetism, yielding unique information about the volume fraction of regions having static magnetic order, as well as the size and distribution of the ordered moments. By combining low-energy muSR, conductivity and a.c. and d.c. magnetization results obtained on high-quality thin-film specimens, we demonstrate here that (Ga, Mn)As shows a sharp onset of ferromagnetic order, developing homogeneously in the full volume fraction, in both insulating and metallic films. Smooth evolution of the ordered moment size across the insulator-metal phase boundary indicates strong ferromagnetic coupling between Mn moments that exists before the emergence of fully itinerant hole carriers.

Dynamics in Organic Ionic Liquids in Distinct Regions Using Charged and Uncharged Orientational Relaxation Probes

The temperature-dependent fluorescence anisotropy decay (orientational relaxation) of perylene and sodium 8-methoxypyrene-1,3,6-sulfonate (MPTS) were measured in a series of 1-alkyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide (alkyl = ethyl, butyl, hexyl, octyl) organic room temperature ionic liquids (RTIL). The two fluorescent probe molecules display markedly different rotational dynamics when analyzed using Stokes-Einstein-Debye theory, demonstrating that they are located in distinct environments within the RTILs and have very different interactions with their surroundings. Perylene rotates with subslip behavior, becoming increasingly subslip as the length of ionic liquid alkyl chain is increased. The dynamics approach those of perylene in an organic oil. In contrast, MPTS shows superstick behavior, likely reflecting very strong coordination with the RTIL cations. These results are consistent with different elements of rotational friction within the ionic liquid structure, which are available to solutes depending on their chemical functionality.

Despite a Conserved Cystine Knot Motif, Different Cyclotides Have Different Membrane Binding Modes

Cyclotides are cyclic proteins produced by plants for defense against pests. Because of their remarkable stability and diverse bioactivities, they have a range of potential therapeutic applications. The bioactivities of cyclotides are believed to be mediated through membrane interactions. To determine the structural basis for the biological activity of the two major subfamilies of cyclotides, we determined the conformation and orientation of kalata B2 (kB2), a Möbius cyclotide, and cycloviolacin O2 (cO2), a bracelet cyclotide, bound to dodecylphosphocholine micelles, using NMR spectroscopy in the presence and absence of 5- and 16-doxylstearate relaxation probes. Analysis of binding curves using the Langmuir isotherm indicated that cO2 and kB2 have association constants of 7.0 × 10 3 M −1 and 6.0 × 10 3 M −1, respectively, consistent with the notion that they are bound near the surface, rather than buried deeply within the micelle. This suggestion is supported by the selective broadening of micelle-bound cyclotide NMR signals upon addition of paramagnetic Mn ions. The cyclotides from the different subfamilies exhibited clearly different binding orientations at the micelle surface. Structural analysis of cO2 confirmed that the main element of the secondary structure is a β-hairpin centered in loop 5. A small helical turn is present in loop 3. Analysis of the surface profile of cO2 shows that a hydrophobic patch stretches over loops 2 and 3, in contrast to the hydrophobic patch of kB2, which predominantly involves loops 2 and 5. The different location of the hydrophobic patches in the two cyclotides explains their different binding orientations and provides an insight into the biological activities of cyclotides.

Electrical properties of ions in the atmosphere of Titan

The Permittivity, Wave and Altimetry (PWA) package on the Huygens probe measured atmospheric electrical activity during its 14 January 2005 descent into Titan's atmosphere. Electrical mobilities of positive ions have been estimated using a combination of predictions of mass from the available chemical models, and the atmospheric properties measured by Huygens. Disc-shaped relaxation probes were used on the PWA to measure air conductivity. In this paper we present the extension of an inversion technique, originally developed to extract ion mobility information from cylindrical relaxation probe data, to the disc geometry. The maximum distance from which typical positive ion species are influenced by the electric field of the relaxation probe in Titan's troposphere and stratosphere is calculated, and found to be ∼10μm. However, loss of positive ion data from the Huygens probe prevents the application of the new technique. Based on the Huygens data, evidence for, and possible characteristics of, a global electric circuit on Titan are also discussed.

Divalent cation coordination and mode of membrane interaction in cyclotides: NMR spatial structure of ternary complex Kalata B7/Mn2+/DPC micelle

The cyclotides are the family of hydrophobic bioactive plant peptides, characterized by a circular protein backbone and three knot forming disulfide bonds. It is believed that membrane activity of the cyclotides underlines their antimicrobial, cytotoxic and hemolytic properties, but the specific interactions with divalent cations can be also involved. To assess the mode of membrane interaction and divalent cation coordination in cyclotides, the spatial structure of the Möbius cyclotide Kalata B7 from the African perennial plant Oldenlandia affinis was determined in the presence of anisotropic membrane mimetic (dodecylphosphocholine micelles). The model of peptide/cation/micelle complex was built using 5-doxylstearate and Mn2+ relaxation probes. Results show that the peptide binds to the micelle surface with relatively high affinity by two hydrophobic loops (loop 2 - Thr6-Leu7 and loop 5 - Trp19-Ile21). The partially hydrated divalent cation is coordinated by charged side-chain of Glu3, aromatic side chain of Tyr11 and free carbonyls of Thr4 and Thr9, and is located in direct contact with the polar head-groups of detergent. The comparison with data about other cyclotides indicates that divalent cation coordination is the invariant property of all cyclotides, but the mode of peptide/membrane interactions is varied. Probably, the specific cation/peptide interactions play a major, but yet not known, role in the biological activity of the cyclotides.

The use of solvent relaxation technique to investigate headgroup hydration and protein binding of simple and mixed phosphatidylcholine/surfactant bilayer membranes

The subject of this report was to investigate headgroup hydration and mobility of two types of mixed lipid vesicles, containing nonionic surfactants; straight chain Brij 98, and polysorbat Tween 80, with the same number of oxyethylene units as Brij, but attached via a sorbitan ring to oleic acid. We used the fluorescence solvent relaxation (SR) approach for the purpose and revealed differences between the two systems. Fluorescent solvent relaxation probes (Prodan, Laurdan, Patman) were found to be localized in mixed lipid vesicles similarly as in pure phospholipid bilayers. The SR parameters (i.e. dynamic Stokes shift, Δν, and the time course of the correlation function, C( t)) of such labels are in the same range in both kinds of systems. Each type of the tested surfactants has its own impact on water organization in the bilayer headgroup region probed by Patman. Brij 98 does not modify the solvation characteristics of the dye. In contrast, Tween 80 apparently dehydrates the headgroup and decreases its mobility. The SR data measured in lipid bilayers in presence of Interferon alfa-2b reveal that this protein, a candidate for non-invasive delivery, affects the bilayer in a different way than the peptide melittin. Interferon alfa-2b binds to mixed lipid bilayers peripherally, whereas melittin is deeply inserted into lipid membranes and affects their headgroup hydration and mobility measurably.

NMR Studies of Lysozyme Surface Accessibility by Using Different Paramagnetic Relaxation Probes

Paramagnetic probes, whose approach to proteins can be monitored by nuclear magnetic resonance (NMR) studies, have been found to be of primary relevance for investigating protein surfaces' accessibility. Here, a Gd(III) neutral complex which contains two metal ions, [Gd2(L7)(H2O)2], is suggested as a paramagnetic probe particularly suited for systematic NMR investigation of protein surface accessibility, due to an expected high relaxivity and to the lack of electric charge which could favor specific interactions. Hen egg white lysozyme has been used as a model system to verify the absence of preferential approaches of this paramagnetic probe to specific protein moieties by comparing paramagnetic perturbation profiles of 1H-13C HSQC signals obtained in the presence of TEMPOL and [Gd2(L7)(H2O)2]. From the similarity of the measured paramagnetic perturbation profiles induced by the two different probes, specific interactions of [Gd2(L7)(H2O)2] with the enzyme could be ruled out. The large size of the latter probe is suggested to be responsible for the strong paramagnetic perturbations observed for CalphaH groups which are located in convex surface-exposed regions. The combined use of the two probes reveals fine details of the dynamics controlling their approach toward the protein surface.

Conformation and mode of membrane interaction in cyclotides

Cyclotides are a family of bioactive plant peptides that are characterized by a circular protein backbone and three conserved tightly packed disulfide bonds. The antimicrobial and hemolytic properties of cyclotides, along with the relative hydrophobicity of the peptides, point to the biological membrane as a target for cyclotides. To assess the membrane-induced conformation and orientation of cyclotides, the interaction of the Möbius cyclotide, kalata B1, from the African perennial plant Oldenlandia affinis, with dodecylphosphocholine micelles was studied using NMR spectroscopy. Under conditions where the cyclotide formed a well-defined complex with micelles, the spatial structure of kalata B1 was calculated from NOE and J couplings data, and the model for the peptide-micelle complex was built using 5- and 16-doxylstearate relaxation probes. The binding of divalent cations to the peptide-micelle complex was quantified by Mn2+ titration. The results show that the peptide binds to the micelle surface, with relatively high affinity, via two hydrophobic loops (loop 5, Trp19-Val21; and loop6, Leu27-Val29). The charged residues (Glu3 and Arg24), along with the cation-binding site (near Glu3) are segregated on the other side of the molecule and in contact with polar head groups of detergent. The spatial structure of kalata B1 is only slightly changed during incorporation into micelles and represents a distorted triple-stranded beta-sheet cross-linked by a cystine knot. Detailed structural analysis and comparison with other knottins revealed structural conservation of the two-disulfide motif in cyclic and acyclic peptides. The results thus obtained provide the first model for interaction of cyclotides with membranes and permit consideration of the cyclotides as membrane-active cationic antimicrobial peptides.

Accurate relaxation parameters for large proteins

The practical applicability, performance, and robustness of three-way decomposition (TWD) for the extraction of relaxation parameters are demonstrated for a large protein with 370 residues, the maltose binding protein. An ordinary set of seven relaxation-modulated 15N HSQC spectra, recorded at another site, is systematically analyzed. For all 341 assigned backbone amide groups, including 21 pairs and one group of three overlapped peaks, T 1 decay values were determined. On isolated peaks, TWD extracts T 1 values with systematically lower error bounds compared to conventional tools, although for these simple cases the improvements remain limited. However, in the presence of spectral artifacts, the decrease in errors can become significant, demonstrating the higher robustness of TWD. For about half of the peaks in overlapped regions, the decomposition allowed separation of the signals, yielding significantly different T 1 values between overlapping signals. For the rest, similarity of the decay times for the two or three overlapping signals could be confirmed within usually low error bounds. The use of TWD thus leads to a significant increase in the number of accessible relaxation probes in large proteins. With a newly implemented graphical user interface, the application of TWD requires merely a peak list, and thus no additional effort compared to conventional approaches is needed.

Spatial Structure of Zervamicin IIB Bound to DPC Micelles: Implications for Voltage-Gating

Zervamicin IIB is a 16-amino acid peptaibol that forms voltage-dependent ion channels with multilevel conductance states in planar lipid bilayers and vesicular systems. The spatial structure of zervamicin IIB bound to dodecylphosphocholine micelles was studied by nuclear magnetic resonance spectroscopy. The set of 20 structures obtained has a bent helical conformation with a mean backbone root mean square deviation value of ∼0.2 Å and resembles the structure in isotropic solvents (Balashova et al., 2000. NMR structure of the channel-former zervamicin IIB in isotropic solvents. FEBS Lett 466:333–336). The N-terminus represents an α-helix, whereas the C-terminal part has a mixed 3 10/ α R hydrogen-bond pattern. In the anisotropic micelle environment, the bending angle on Hyp10 (23°) is smaller than that (47°) in isotropic solvents. In the NOESY (Nuclear Overhauser Effect Spectroscopy) spectra, the characteristic attenuation of the peptide signals by 5- and 16-doxylstearate relaxation probes indicates a peripheral mode of the peptaibol binding to the micelle with the N-terminus immersed slightly deeper into micelle interior. Analysis of the surface hydrophobicity reveals that the zervamicin IIB helix is amphiphilic and well suited to formation of a tetrameric transmembrane bundle, according to the barrel-stave mechanism. The results are discussed in a context of voltage-driven peptaibol insertion into membrane.

A Calix[4]arene Gd(III) Complex Endowed with High Stability, Relaxivity, and Binding Affinity to Serum Albumin This work was supported by CNR (Programma M.U.R.S.T. - Chimica Legge 95/95) "Agenti di contrasto, di shift e sonde luminescenti". We thank C.I.M. (Centro Interdipartimentale Misure) dell'Università di Parma for the NMR and mass spectroscopy facilities.

Contrasting calixarenes: Thermodynamically stable and water-soluble calix[4]arene–LnIII (see picture) complexes may represent a new class of relaxation (Ln=Gd) or luminescent (Ln=Eu, Tb) probes for biomedical studies. The first complete characterization of one of these complexes in water is reported. The GdIII chelate shows a strong binding affinity with human serum albumin as a result of the cooperative effects of hydrophobic interaction and coordination by donors groups on the protein.