Local Correlation Times Research Articles

Investigation of the Influence of Various Functional Groups on the Dynamics of Glucocorticoids.

The basic configuration of glucocorticoid consists of four-fused rings associated with one cyclohexadienone ring, two cyclohexane rings, and one cyclopentane ring. The ways the structure and dynamics of five glucocorticoids (prednisone, prednisolone, prednisolone acetate, methylprednisolone, and methylprednisolone acetate) are altered because of the substitution of various functional groups with these four-fused rings are studied thoroughly by applying sophisticated solid-state nuclear magnetic resonance (NMR) methodologies. The biological activities of these glucocorticoids are also changed because of the attachment of various functional groups with these four-fused rings. The substitution of the hydroxyl group (with the C11 atom of the cyclohexane ring) in place of the keto group enhances the potential of the glucocorticoid to cross the cellular membrane. As a result, the bioavailability of prednisolone (the hydroxyl group is attached with the C11 atom of the cyclohexane ring) is increased compared to prednisone (the keto group is attached with the C11 atom of cyclohexane rings). Another notable point is that the spin-lattice relaxation rate at crystallographically distinct carbon nuclei sites of prednisolone is increased compared to that of the prednisone, which implies that the motional degrees of freedom of glucocorticoid is increased because of the substitution of the hydroxyl group in place of the keto group of the cyclohexane ring. The attachment of the methyl group with the C6 atom of cyclohexane rings further reduces the spin-lattice relaxation time at crystallographically distinct carbon nuclei sites of glucocorticoid and its bioactivity is also increased. By comparing the spin-lattice relaxation time and the local correlation time at crystallographically different carbon nuclei sites of three steroids prednisone, prednisolone, and methylprednisolone, it is observed that both the spin-lattice relaxation time and the local correlation time gradually decrease at each crystallographically distinct carbon nuclei sites when we move from prednisone to prednisolone to methyl-prednisolone. On the other hand, if we compare the same for prednisolone, prednisolone acetate, and methylprednisolone acetate, then we also observe that both the spin-lattice relaxation time and the local-correlation time gradually decrease from prednisolone to prednisolone acetate to methylprednisolone acetate for all chemically different carbon nuclei. It is also noticeable that both the spin-lattice relaxation time and the local-correlation time gradually decrease from prednisone to prednisolone to prednisolone acetate to methylprednisolone to methylprednisolone acetate for most of the carbon nuclei sites. From in silico analysis, it is also revealed that the bioavailability and efficacy of the glucocorticoid increase from prednisone to prednisolone to prednisolone acetate to methylprednisolone to methylprednisolone acetate. Hence, it can be concluded that the biological activity and the motional degrees of freedom of the glucocorticoids are highly correlated. These types of studies provide a clear picture of the structure-activity relationship of the drug molecules, which will enlighten the path of developing highly potent glucocorticoids with minimum side effects. Another important aspect of these types of studies is to provide information about the electronics configuration and nuclear spin dynamics at crystallographically different carbon nuclei sites of five glucocorticoids, which will enrich the field of "NMR crystallography".

A Description of the Local Structure and Dynamics of Ketoconazole Molecule by Solid‐State NMR Measurements and DFT Calculations: Proposition for NMR Crystallography

AbstractAn azole class antifungal agent, ketoconazole, is widely used in the treatment of mucosal fungal infections that arise due to AIDS immunosuppression, organ transplantation, and cancer chemotherapy. The structure and dynamics of various molecular moieties of ketoconazole are thoroughly studied by measuring chemical shift anisotropy tensor and site‐specific spin‐lattice relaxation time. The local correlation time at crystallographically different carbon nuclei sites is also calculated. The chemical shift anisotropy (CSA) parameters at the crystallographically distinct sites of ketoconazole are determined by the two‐dimensional phase‐adjusted spinning sideband (2D PASS) cross‐polarization magic angle spinning (CP‐MAS) solid‐state NMR experiment. The site‐specific spin‐lattice relaxation time is measured by the method outlined by Torchia (T1CP). The values of the principal components of CSA parameters extracted by the 2DPASS CP‐MAS ssNMR experiment are supported by density functional theory (DFT) calculations. The CSA parameters are high for those carbon nuclei, whose spin‐lattice relaxation rate is slow, and it is low for those carbon nuclei whose spin‐lattice relaxation rate is fast. It suggests that the spin‐lattice relaxation mechanism is mainly governed by chemical‐shift anisotropy interaction for these carbon nuclei. A huge variation of the spin‐lattice relaxation time and the local correlation time are observed for numerous carbon nuclei that reside on the side‐chain of the molecule. The spin‐lattice relaxation time varies from 500 s to 8 s, and the order of the local correlation time varies from s to s. These types of studies in which the chemical shift anisotropy (CSA) parameters and spin‐lattice relaxation time provide information about the electronic configuration and the spin dynamics at the various crystallographic locations of the drug molecule will enrich the field “NMR crystallography.” It will also help to develop the strategies for the administration of antifungal drugs by providing information about the structure and dynamics of various parts of the drug molecule.

Study of the Variation of the Electronic Distribution and Motional Dynamics of Two Independent Molecules of an Asymmetric Unit of Atorvastatin Calcium by Solid-State NMR Measurements.

Significant changes in the spin-lattice time and chemical shift anisotropy (CSA) parameters are observed in two independent molecules of an asymmetric unit of atorvastatin calcium (ATC-I) (which is referred to as “a”- and “b”-type molecules by following Wang et al.). The longitudinal magnetization decay curve is fitted by two exponentials—one with longer relaxation time and another with shorter relaxation time for most of the carbon nuclei sites. The local correlation time also varies significantly. This is the experimental evidence of the coexistence of two different kinds of motional degrees of freedom within ATC-I molecule. The solubility and bioavailability of the drug molecule are enhanced due to the existence of two different kinds of dynamics. Hence, the macroscopic properties like solubility and bioavailability of a drug molecule are highly correlated with its microscopic properties. The motional degrees of freedom of “a”- and “b”-type molecules are also varied remarkably at certain carbon nuclei sites. This is the first time the change in the molecular dynamics of two independent molecules of an asymmetric unit of atorvastatin calcium is quantified using solid-state NMR methodology. These types of studies, in which the chemical shift anisotropy (CSA) parameters and spin-lattice relaxation time provide information about the change in electronic distribution and the spin dynamics at the various crystallographic location of the drug molecule, will enrich the field “NMR crystallography”. It will also help us to understand the electronic distribution around a nucleus and the nuclear spin dynamics at various parts of the molecule, which is essential to develop the strategies for the administration of the drug.

Study of the structure and dynamics at various parts of the antibacterial drug molecule cefpodoxime proxetil

The structure and dynamics of cefpodoxime proxetil are elucidated by measuring chemical shift anisotropy (CSA) tensor, spin-lattice relaxation time, and local correlation time at twenty-one crystallographically different 13C nuclei sites. The principal components of CSA tensor of cefpodoxime proxetil are extracted by the two-dimensional phase adjusted sinning sideband (2DPASS) cross-polarization magic angle spinning (CP-MAS) solid-state NMR experiment, and the spin-lattice relaxation time is measured by the method outlined by Torchia(T1CP). The local correlation time is calculated by bearing in mind that the spin-lattice relaxation mechanism of 13C nuclei is mainly governed by the CSA interaction and the heteronuclear dipole-dipole interaction. The aminothiazole ring, β-lactam ring, and dihydrothiazine ring provide stability to the drug molecule and increase the affinity of the drug to penicillin-binding proteins (PBPs) receptors. The principal components of CSA parameters, spin-lattice relaxation time, and local correlation time vary substantially for carbon nuclei residing on these three rings. These signify that not only the electronic environment, but the molecular conformation, and the local dynamics are also altered within the ring. The substitution of the acyl side chain, oxime group, and the aminothiazole ring at the C7 position of the β-lactam ring enhances the antibacterial activity and the binding affinity of the drug. A huge variation of the spin-lattice relaxation time and local correlation time is observed in those regions. The change in the electron charge distribution and nuclear spin dynamics at different parts of the drug molecule is clear by CSA and spin-lattice relaxation measurements, which will enrich the field “NMR crystallography”.

Air–water flow characteristics in high-velocity free-surface flows with 50% void fraction

High-velocity free-surface flows are complex two-phase flows and limited information is available about the interactions between air and water for void fractions of about 50%. Herein a detailed experimental study was conducted in the intermediate flow region (C ∼ 50%) on a stepped spillway and the microscopic air–water flow characteristics were investigated. The results showed differences in water and droplet chord times with comparatively larger number of air chord times (0–2ms), and larger number of water chord times (2–6ms). A monotonic decrease of particle chord modes was observed with increasing bubble count rates. Several characteristic time scales were identified based upon inter-particle arrival time analyses of characteristic chord time classes as well as spectral analyses of the instantaneous void fraction signal. Chord times of 3–5ms appeared to be characteristic time scales of the intermediate flow region having similar time scales compared to the local correlation and integral turbulent time scales and to time scales associated with bubble break-up and turbulent velocity fluctuations. A further characteristic time scale of 100ms was identified in a frequency analysis of instantaneous void fraction. This time scale was of the same order of magnitude as free-surface auto-correlation time scales suggesting that the air–water flow structure was affected by the free-surface fluctuations.

Dimensions and Dynamics of Highly Cooperative Sic1-WD40 Binding: smFRET through a Polymer Physics Lens

Sic1 is a cyclin-dependent kinase inhibitor which must be phosphorylated on at least six sites (termed Cdc4 phosphodegrons, CPDs) to allow its recognition by the WD40 binding domain of Cdc4. The highly-cooperative switch-like dependence on the number of phosphorylated sites on Sic1 cannot be accounted for by traditional thermodynamic models of cooperativity. Further experimental attention is necessary to determine the physicochemical/mechanistic basis of its highly cooperative binding.We used single molecule fluorescence techniques to study the dimensions and dynamics of Sic1's N-terminal targeting region (residues 1-90, henceforth Sic1), phosphorylated Sic1 (pSic1), and the pSic1-WD40 dynamic complex. Using time-resolved fluorescence anisotropy, we find local segment specific rotational correlation times which are complexly modulated by chain compactness and electrostatics (charge screening and phosphorylation).Previous single molecule Fӧrster Resonance Energy Transfer (smFRET) measurements [1] observed end-to-end reconfiguration on timescales larger than ∼1ms; resulting in FRET histograms with multiple conformational sub-ensembles. These sub-ensembles and their dynamics are further explored by trapping single Sic1, pSic1 and WD40 in nanometer sized surface-tethered lipid vesicles and modulating their electrostatics and compactness. In a refinement to the conventional approaches for inferring dimensions from smFRET experiments, we use distance distributions from Monte Carlo simulations which extensively sample coarse-grained protein conformations. The application of polymer physics theory/simulation towards smFRET data interpretation, and towards IDP binding, contributes to the growing toolkit for understanding the diverse behaviours of IDPs.[1] Liu B. et al., J. Phys. Chem. B. 2014 118(15):4088-97.

Field-Cycling Relaxometry as a Molecular Rheology Technique: Common Analysis of NMR, Shear Modulus and Dielectric Loss Data of Polymers vs Dendrimers

Linear poly(propylene glycol) (PPG) as well as a poly(propyleneimine) (PPI) dendrimer with different molar masses (M) are investigated by field-cycling (FC) 1H NMR, shear rheology (G) and dielectric spectroscopy (DS). The results are compared in a reduced spectral density representation: the quantity R1(ωτα)/R1α(0), where R1(ωτα) is the master curve of the frequency dependent spin–lattice relaxation rate with τα denoting the local correlation time, is compared to the rescaled dynamic viscosity η′(ωτα)/η′α(0). The quantities R1α(0) and η′α(0), respectively, are the zero-frequency limits of a simple liquid reference system. Analogously, the dielectric loss data can be included in the methodological comparison. This representation allows quantifying the sensitivity of each method with respect to the polymer-specific relaxation contribution. Introducing a “cumulative mode ratio” Fi(M) for each technique i, which measures the zero-frequency plateau of the rescaled spectral density, characteristic power-law beh...

Heteronuclear transverse and longitudinal relaxation in AX4 spin systems: Application to 15N relaxations in 15NH4+

The equations that describe the time-evolution of transverse and longitudinal 15N magnetisations in tetrahedral ammonium ions, 15NH4+, are derived from the Bloch-Wangsness-Redfield density operator relaxation theory. It is assumed that the relaxation of the spin-states is dominated by (1) the intra-molecular 15N–1H and 1H–1H dipole–dipole interactions and (2) interactions of the ammonium protons with remote spins, which also include the contribution to the relaxations that arise from the exchange of the ammonium protons with the bulk solvent. The dipole–dipole cross-correlated relaxation mechanisms between each of the 15N–1H and 1H–1H interactions are explicitly taken into account in the derivations. An application to 15N-ammonium bound to a 41kDa domain of the protein DnaK is presented, where a comparison between experiments and simulations show that the ammonium ion rotates rapidly within its binding site with a local correlation time shorter than approximately 1ns. The theoretical framework provided here forms the basis for further investigations of dynamics of AX4 spin systems, with ammonium ions in solution and bound to proteins of particular interest.

Magnetic field emergence in mesogranular-sized exploding granules observed with sunrise/IMaX data

We report on magnetic field emergences covering significant areas of exploding granules. The balloon-borne mission SUNRISE provided high spatial and temporal resolution images of the solar photosphere. Continuum images, longitudinal and transverse magnetic field maps and Dopplergrams obtained by IMaX onboard SUNRISE are analyzed by Local Correlation Traking (LCT), divergence calculation and time slices, Stokes inversions and numerical simulations are also employed. We characterize two mesogranular-scale exploding granules where $\sim$ 10$^{18}$ Mx of magnetic flux emerges. The emergence of weak unipolar longitudinal fields ($\sim$100 G) start with a single visible magnetic polarity, occupying their respective granules' top and following the granular splitting. After a while, mixed polarities start appearing, concentrated in downflow lanes. The events last around 20 min. LCT analyses confirm mesogranular scale expansion, displaying a similar pattern for all the physical properties, and divergence centers match between all of them. We found a similar behaviour with the emergence events in a numerical MHD simulation. Granule expansion velocities are around 1 \kms while magnetic patches expand at 0.65 \kms. One of the analyzed events evidences the emergence of a loop-like structure. Advection of the emerging magnetic flux features is dominated by convective motion resulting from the exploding granule due to the magnetic field frozen in the granular plasma. Intensification of the magnetic field occurs in the intergranular lanes, probably because of being directed by the downflowing plasma.

Methyl dynamics of a Ca2+-calmodulin-peptide complex from NMR/SRLS.

We developed the slowly relaxing local structure (SRLS) approach for analyzing NMR spin relaxation in proteins. SRLS accounts for dynamical coupling between the tumbling of the protein and the local motion of the probe and for general tensorial properties. It is the generalization of the traditional model-free (MF) method, which does not account for mode-coupling and treats only simple tensorial properties. SRLS is applied herein to ²H relaxation of ¹³CDH₂ groups in the complex of Ca(2+)-calmodulin with the peptide smMLCKp. Literature data comprising ²H T₁ and T₂ acquired at 14.1 and 17.6 T, and 288, 295, 308, and 320 K, are used. We find that mode-coupling is a small effect for methyl dynamics. On the other hand, general tensorial properties are important. In particular, it is important to allow for the asymmetry of the local spatial restrictions, which can be represented in SRLS by a rhombic local ordering tensor with components S(0)(2) and S(2)(2). The principal axes frame of this tensor is obviously different from the axial frames of the magnetic tensors. Here, we find that -0.2 ≤ S(0)(2) ≤ 0.5 and -0.4 ≤ S(2)(2) ≤ 0. MF features a single "generalized" order parameter, S, confined to the 0-0.316 range; the local geometry is inherently simple. The parameter S is inaccurate, having absorbed unaccounted for effects, notably S(2)(2) ≠ 0. We find that the methionine methyls (the other methyl types) reorient with rates of 8.6 × 10⁹ to 21.4 × 10⁹ (0.67 × 10⁹ to 6.5 × 10⁹) 1/s. The corresponding activation energies are 10 (10-27) kJ/mol. By contrast, MF yields inaccurate effective local motional correlation times, τ(e), with nonphysical temperature dependence. Thus, the problematic S- and τ(e)-based MF picture of methyl dynamics has been replaced with an insightful physical picture based on a local ordering tensor related to structural features, and a local diffusion tensor that yields accurate activation energies.

NMR Backbone Dynamics of VEK-30 Bound to the Human Plasminogen Kringle 2 Domain

To gain insights into the mechanisms for the tight and highly specific interaction of the kringle 2 domain of human plasminogen (K2 Pg) with a 30-residue internal peptide (VEK-30) from a group A streptococcal M-like protein, the dynamic properties of free and bound K2 Pg and VEK-30 were investigated using backbone amide 15N-NMR relaxation measurements. Dynamic parameters, namely the generalized order parameter, S 2, the local correlation time, τ e, and the conformational exchange contribution, R ex, were obtained for this complex by Lipari-Szabo model-free analysis. The results show that VEK-30 displays distinctly different dynamic behavior as a consequence of binding to K2 Pg, manifest by decreased backbone flexibility, particularly at the binding region of the peptide. In contrast, the backbone dynamics parameters of K2 Pg displayed similar patterns in the free and bound forms, but, nonetheless, showed interesting differences. Based on our previous structure-function studies of this interaction, we also made comparisons of the VEK-30/K2 Pg dynamics results from different kringle modules complexed with small lysine analogs. The differences in dynamics observed for kringles with different ligands provide what we believe to be new insights into the interactions responsible for protein-ligand recognition and a better understanding of the differences in binding affinity and binding specificity of kringle domains with various ligands.

Macrocyclic Gd3+ Chelates Attached to a Silsesquioxane Core as Potential Magnetic Resonance Imaging Contrast Agents: Synthesis, Physicochemical Characterization, and Stability Studies

Two macrocyclic ligands, 1,4,7,10-tetraazacyclododecane-1-glutaric-4,7,10-triacetic acid (H(5)DOTAGA) and the novel 1,4,7,10-tetraazacyclododecane-1-(4-(carboxymethyl)benzoic)-4,7,10-triacetic acid (H(5)DOTABA), were prepared and their lanthanide complexes (Ln = Gd(3+), Y(3+)) attached to an amino-functionalized T(8)-silsesquioxane. The novel compounds Gadoxane G (GG) and Gadoxane B (GB) possess eight monohydrated lanthanide complexes each, as evidenced by multinuclear ((1)H, (13)C, (29)Si) NMR spectroscopy and high resolution mass spectrometry (HR-MS). Pulsed-field gradient spin echo (PGSE) diffusion (1)H NMR measurements revealed hydrodynamic radii of 1.44 nm and global rotational correlation times of about 3.35 ns for both compounds. With regard to potential MRI contrast agent applications, a variable-temperature (17)O NMR and (1)H nuclear magnetic relaxation dispersion (NMRD) study was carried out on aqueous solutions of the gadolinium(III) complexes of the Gadoxanes and the corresponding monomeric ligands to yield relevant physicochemical properties. The water exchange rates of the inner-sphere water molecules are all very similar (k(ex)(298) between (5.3 +/- 0.5) x 10(6) s(-1) and (5.9 +/- 0.3) x 10(6) s(-1)) and only slightly higher than that reported for the gadolinium(III) complex of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (H(4)DOTA) (k(ex)(298) = 4.1 x 10(6) s(-1)). Despite their almost identical size and their similar water exchange rates, GB shows a significantly higher longitudinal relaxivity than GG over nearly the whole range of magnetic fields (e.g., 17.1 mM(-1) s(-1) for GB and 12.1 mM(-1) s(-1) for GG at 20 MHz and 25 degrees C). This difference arises from their different local rotational correlation times (tau(lR)(298) = 240 +/- 10 ps and 380 +/- 20 ps, respectively), because of the higher rigidity of the phenyl ring of GB as compared to the ethylene spacer of GG. A crucial feature of these novel compounds is the lability of the silsesquioxane core in aqueous media. The hydrolysis of the Si-O-Si moieties was investigated by (29)Si NMR and PGSE diffusion (1)H NMR spectroscopy, electrospray ionization mass spectrometry (ESI-MS), as well as by relaxivity measurements. Although frozen aqueous solutions (pH 7.0) of GG and GB can be stored at -28 degrees C for at least 10 months without any decomposition, with increasing temperature and pH the hydrolysis of the silsesquioxane core was observed (e.g., t(1/2) = 15 h at pH 7.4 and 55 min at pH 8.1 for GG at 37 degrees C). No change in relaxivity was detected within the first 3 h, since the hydrolysis of the initial Si-O-Si moieties has no influence on the rotational correlation time. However, the further hydrolysis of the silsesquioxane core leads to smaller fragments and therefore to a decrease in relaxivity.

Domain Mobility in Proteins from NMR/SRLS

Enhanced internal mobility in proteins is typically functional. Domain motion in enzymes, necessarily related to catalysis, is a prototype in this context. Experimental (15)N spin relaxation data from E. coli adenylate kinase report qualitatively on nanosecond motion experienced by the domains AMPbd and LID. Previous quantitative analysis based on the mode-coupling slowly relaxing local structure approach confirmed nanosecond mobility but yielded unduly small local ordering and local geometry not interpretable directly in terms of the local protein structure. Here, we show that these features ensue from having assumed axial local ordering and highly axial local diffusion. After eliminating these simplified second-rank tensor properties, a physically sound picture, with the local motion interpretable as domain motion, is obtained. Rhombic local ordering, with components given by <Sxx> = 0.471, <Syy> = -0.952 and <Szz> = 0.481, and main ordering axis, Y(M), lying along C(alpha)(i-1) - C(alpha)(i), has been determined. The associated rhombic potential is given by axial (rhombic) coefficients of <c(2)(0)> = -3.3 (<c(2)(2)> = 17.8). The average correlation time for domain motion is 10.4 (6.4) ns at 288 (302) K; the corresponding correlation time for global motion is 20.6 (14.9) ns. The rates for domain motion exhibit noteworthy Arrhenius-type temperature-dependence, yielding activation energies of 63.8 +/- 7.0 (53.0 +/- 9.1) kJ/mol for the AMPbd (LID) domain. The traditional model-free analysis ignores mode-coupling and simplifies tensor properties. Within its scope, the AKeco backbone emerges as largely rigid, <Szz> approximately = 0.94; the main ordering axis, Z(M), lies along N-H, <c(2)(0)> approximately = 16 (c(2)(2) = 0); and the slow local motional correlation time lies at the low end of the nanosecond time scale.

A benzene-core trinuclear GdIIIcomplex: towards the optimization of relaxivity for MRI contrast agent applications at high magnetic field

A novel ligand, H(12)L, based on a trimethylbenzene core bearing three methylenediethylenetriamine-N,N,N'',N''-tetraacetate moieties (-CH(2)DTTA(4-)) for Gd(3+) chelation has been synthesized, and its trinuclear Gd(3+) complex [Gd(3)L(H(2)O)(6)](3-) investigated with respect to MRI contrast agent applications. A multiple-field, variable-temperature (17)O NMR and proton relaxivity study on [Gd(3)L(H(2)O)(6)](3-) yielded the parameters characterizing water exchange and rotational dynamics. On the basis of the (17)O chemical shifts, bishydration of Gd(3+) could be evidenced. The water exchange rate, k(ex)(298)=9.0+/-3.0 s(-1) is around twice as high as k(ex)(298) of the commercial [Gd(DTPA)(H(2)O)](2-) and comparable to those on analogous Gd(3+)-DTTA chelates. Despite the relatively small size of the complex, the rotational dynamics had to be described with the Lipari-Szabo approach, by separating global and local motions. The difference between the local and global rotational correlation times, tau(lO)(298)=170+/-10 ps and tau(gO)(298)=540+/-100 ps respectively, shows that [Gd(3)L(H(2)O)(6)](3-) is not fully rigid; its flexibility originates from the CH(2) linker between the benzene core and the poly(amino carboxylate) moiety. As a consequence of the two inner-sphere water molecules per Gd(3+), their close to optimal exchange rate and the appropriate size and limited flexibility of the molecule, [Gd(3)L(H(2)O)(6)](3-) has remarkable proton relaxivities when compared with commercial contrast agents, particularly at high magnetic fields (r(1)=21.6, 17.0 and 10.7 mM(-1)s(-1) at 60, 200 and 400 MHz respectively, at 25 degrees C; r(1) is the paramagnetic enhancement of the longitudinal water proton relaxation rate, referred to 1 mM concentration of Gd(3+)).

Unique Dimeric Structure of BNip3 Transmembrane Domain Suggests Membrane Permeabilization as a Cell Death Trigger

BNip3 is a prominent representative of apoptotic Bcl-2 proteins with rather unique properties initiating an atypical programmed cell death pathway resembling both necrosis and apoptosis. Many Bcl-2 family proteins modulate the permeability state of the outer mitochondrial membrane by forming homo- and hetero-oligomers. The structure and dynamics of the homodimeric transmembrane domain of BNip3 were investigated with the aid of solution NMR in lipid bicelles and molecular dynamics energy relaxation in an explicit lipid bilayer. The right-handed parallel helix-helix structure of the domain with a hydrogen bond-rich His-Ser node in the middle of the membrane, accessibility of the node for water, and continuous hydrophilic track across the membrane suggest that the domain can provide an ion-conducting pathway through the membrane. Incorporation of the BNip3 transmembrane domain into an artificial lipid bilayer resulted in pH-dependent conductivity increase. A possible biological implication of the findings in relation to triggering necrosis-like cell death by BNip3 is discussed.

Quantifying Lipari–Szabo modelfree parameters from 13CO NMR relaxation experiments

It is proposed to obtain effective Lipari-Szabo order parameters and local correlation times for relaxation vectors of protein (13)CO nuclei by carrying out a (13)CO-R(1) auto relaxation experiment, a transverse (13)CO CSA/13CO-13Calpha CSA/dipolar cross correlation and a transverse (13)CO CSA/(13)CO-(15)N CSA/dipolar cross correlation experiment. Given the global rotational correlation time from (15)N relaxation experiments, a new program COMFORD (CO-Modelfree Fitting Of Relaxation Data) is presented to fit the (13)CO data to an effective order parameter S2CO, an effective local correlation time and the orientation of the CSA tensor with respect to the molecular frame. It is shown that the effective S2CO is least sensitive to rotational fluctuations about an imaginary Calpha-Calpha axis and most sensitive to rotational fluctuations about an imaginary axis parallel to the NH bond direction. As such, the Calpha-Calpha information is fully complementary to the (15)N relaxation order parameter, which is least sensitive to fluctuations about the NH axis and most sensitive to fluctuations about the Calpha-Calpha axis. The new paradigm is applied on data of Ca(2+) saturated Calmodulin, and on available literature data for Ubiquitin. Our data indicate that the S2CO order parameters rapport on slower, and sometimes different, motions than the (15)N relaxation order parameters. The CO local correlation times correlate well with the calmodulin's secondary structure.

Unexpected Aggregation of Neutral, Xylene‐Cored Dinuclear GdIII Chelates in Aqueous Solution

We have synthesized ditopic ligands L(1), L(2), and L(3) that contain two DO3A(3-) metal-chelating units with a xylene core as a noncoordinating linker (DO3A(3-) = 1,4,7,10-tetraazacyclododecane-1,4,7-triacetate; L(1) = 1,4-bis{[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl]methyl}benzene; L(2) = 1,3-bis{[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl]methyl}benzene; L(3) = 3,5-bis{[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl]methyl}benzoic acid). Aqueous solutions of the dinuclear Gd(III) complexes formed with the three ligands have been investigated in a variable-temperature, multiple-field (17)O NMR and (1)H relaxivity study. The (17)O longitudinal relaxation rates measured for the [Gd(2)L(1-3)(H2O)(2)] complexes show strong field dependence (2.35-9.4 T), which unambiguously proves the presence of slowly tumbling entities in solution. The proton relaxivities of the complexes, which are unexpectedly high for their molecular weight, and in particular the relaxivity peaks observed at 40-50 MHz also constitute experimental evidences of slow rotational motion. This was explained in terms of self-aggregation related to hydrophobic interactions, pi stacking between the aromatic linkers, or possible hydrogen bonding between the chelates. The longitudinal (17)O relaxation rates of the [Gd(2)L(1-3)(H2O)(2)] complexes have been analysed with the Lipari-Szabo approach, leading to local rotational correlation times tau(1)(298) of 150-250 ps and global rotational correlation times tau(g)(298) of 1.6-3.4 ns (c(Gd): 20-50 mM), where tau(1)(298) is attributed to local motions of the Gd segments, while tau(g)(298) describes the overall motion of the aggregates. The aggregates can be partially disrupted by phosphate addition; however, at high concentrations phosphate interferes in the first coordination sphere by replacing the coordinated water. In contrast to the parent [Gd(DO3A)(H2O)(1.9)], which presents a hydration equilibrium between mono- and dihydrated species, a hydration number of q = 1 was established for the [Ln(2)L(1-3)(H2O)(2)] chelates by (17)O chemical shift measurements on Ln = Gd and UV/Vis spectrophotometry for Ln = Eu. The exchange rate of the coordinated water is higher for [Gd(2)L(1-3)(H2O)(2)] complexes k(ex)(298) = 7.5-12.0 x 10(6) s(-1)) than for [Gd(DOTA)(H2O)](-). The proton relaxivity of the [Gd(2)L(1-3)(H2O)(2)] complexes strongly decreases with increasing pH. This is related to the deprotonation of the inner-sphere water, which has also been characterized by pH potentiometry. The protonation constants determined for this process are logK(OH) = 9.50 and 10.37 for [Gd(2)L(1)(H2O)(2)] and [Gd(2)L(3)(H2O)(2)], respectively.

A Starburst‐Shaped Heterometallic Compound Incorporating Six Densely Packed Gd3+ Ions

The heterotritopic ligand [bpy(DTTA)2]8- has two diethylenediamine-tetraacetate units for selective lanthanide(III) coordination and one bipyridine function for selective Fe(II) coordination. In aqueous solution and in the presence of these metals, the ligand is capable of self-assembly to form a rigid supramolecular metallostar structure, [Fe[Gd2bpy(DTTA)2(H2O)4]3]4-. We report here the physicochemical characterization of the dinuclear complex [Gd2bpy(DTTA)2(H2O)4]2- and the metallostar [Fe[Gd2bpy(DTTA)2(H2O)4]3]4- with regard to potential MRI contrast agent applications. A combination of pH potentiometry and 1H NMR spectroscopy has been used to determine protonation constants for the ligand [bpy(DTTA)2]8- and for the complexes [Fe[bpy(DTTA)2]3]22- and [Y2bpy(DTTA)2]2-. In addition, stability constants have been measured for the dinuclear chelates [M2bpy(DTTA)2]n- formed with M = Gd3+ and Zn2+ (log K(GdL) = 18.2; log K(ZnL) = 18.0; log K(ZnHL) = 3.4). A multiple field, variable-temperature 17O NMR and proton relaxivity study on [Gd2bpy(DTTA)2(H2O)4]2- and [Fe[Gd2bpy(DTTA)2(H2O)4]3](4-) yielded the parameters for water exchange and the rotational dynamics. The 17O chemical shifts are indicative of bishydration of the lanthanide ion. The exchange rates of the two inner-sphere water molecules are very similar in the dinuclear [Gd2bpy(DTTA)2(H2O)(4)]2- and in the metallostar (k(ex)298 = 8.1 +/- 0.3 x 10(6) and 7.4 +/- 0.2 x 10(6) s(-1), respectively), and are comparable to k(ex)298 for similar Gd(III) poly(amino carboxylates). The rotational dynamics of the metallostar has been described by means of the Lipari-Szabo approach, which involves separating global and local motions. The difference between the local and global rotational correlation times, tau(lO)298 = 190 +/- 15 ps and tau(gO)298 = 930 +/- 50 ps, respectively, shows that the metallostar is not completely rigid. However, the relatively high value of S2 = 0.60 +/- 0.04, describing the restriction of the local motions with regard to the global one, points to a limited flexibility compared with previously reported macromolecules such as dendrimers. As a result of the two inner-sphere water molecules, with their near-optimal exchange rate, and the limited flexibility, the metallostar has a remarkable molar proton relaxivity, particularly at high magnetic fields (r1 = 33.2 and 16.4 mM(-1) s(-1) at 60 and 200 MHz, respectively, at 25 degrees C). It packs six efficiently relaxing Gd(III) ions into a small molecular space, which leads, to the best of our knowledge, to the highest relaxivity per molecular mass ever reported for a Gd(III) complex. The [bpy(DTTA)2]8- ligand is also a prime candidate as a terminal ligand for constructing larger sized, Fe(II) (or Ru(II))-based metallostars or metallodendrimers loaded with Gd(III) on the surface.

Supramolecular Assembly of an Amphiphilic GdIII Chelate: Tuning the Reorientational Correlation Time and the Water Exchange Rate

We report the synthesis and characterization of the novel ligand H(5)EPTPA-C(16) ((hydroxymethylhexadecanoyl ester)ethylenepropylenetriaminepentaacetic acid). This ligand was designed to chelate the Gd(III) ion in a kinetically and thermodynamically stable way while ensuring an increased water exchange rate (kappa(ex)) on the Gd(III) complex owing to steric compression around the water-binding site. The attachment of a palmitic ester unit to the pendant hydroxymethyl group on the ethylenediamine bridge yields an amphiphilic conjugate that forms micelles with a long tumbling time (tau(R)) in aqueous solution. The critical micelle concentration (cmc = 0.34 mM) of the amphiphilic [Gd(eptpa-C(16))(H(2)O)](2-) chelate was determined by variable-concentration proton relaxivity measurements. A global analysis of the data obtained in variable-temperature and multiple-field (17)O NMR and (1)H NMRD measurements allowed for the determination of parameters governing relaxivity for [Gd(eptpa-C(16))(H(2)O)](2-); this is the first time that paramagnetic micelles with optimized water exchange have been investigated. The water exchange rate was found to be kappa(298)(ex) = 1.7 x 10(8) s(-1), very similar to that previously reported for the nitrobenzyl derivative [Gd(eptpa-bz-NO(2))(H(2)O)](2-) kappa(298)(ex) = 1.5 x 10(8) s(-1)). The rotational dynamics of the micelles were analysed by using the Lipari-Szabo approach. The micelles formed in aqueous solution show considerable flexibility, with a local rotational correlation time of tau(298)(l0) = 330 ps for the Gd(III) segments, which is much shorter than the global rotational correlation time of the supramolecular aggregates, tau(298)(g0) = 2100 ps. This internal flexibility of the micelles is responsible for the limited increase of the proton relaxivity observed on micelle formation (r(1) = 22.59 mM(-1) s(-1) for the micelles versus 9.11 mM(-1) s(-1) for the monomer chelate (20 MHz; 25 degrees C)).

The importance of including local correlation times in the calculation of inter-proton distances from NMR measurements: ignoring local correlation times leads to significant errors in the conformational analysis of the Glcα1–2Glcα linkage by NMR spectroscopy

The experimental determination of oligosaccharide conformations has traditionally used cross-linkage 1H-1H NOE/ROEs. As relatively few NOEs are observed, to provide sufficient conformational constraints this method relies on: accurate quantification of NOE intensities (positive constraints); analysis of absent NOEs (negative constraints); and hence calculation of inter-proton distances using the two-spin approximation. We have compared the results obtained by using 1H 2D NOESY, ROESY and T-ROESY experiments at 500 and 700 MHz to determine the conformation of the terminal Glc alpha1-2Glc alpha linkage in a dodecasaccharide and a related tetrasaccharide. For the tetrasaccharide, the NOESY and ROESY spectra produced the same qualitative pattern of linkage cross-peaks but the quantitative pattern, the relative peak intensities, was different. For the dodecasaccharide, the NOESY and ROESY spectra at 500 MHz produced a different qualitative pattern of linkage cross-peaks, with fewer peaks in the NOESY spectrum. At 700 MHz, the NOESY and ROESY spectra of the dodecasaccharide produced the same qualitative pattern of peaks, but again the relative peak intensities were different. These differences are due to very significant differences in the local correlation times for different proton pairs across this glycosidic linkage. The local correlation time for each proton pair was measured using the ratio of the NOESY and T-ROESY cross-relaxation rates, leaving the NOESY and ROESY as independent data sets for calculating the inter-proton distances. The inter-proton distances calculated including the effects of differences in local correlation times give much more consistent results.