Global Rotational Correlation Time Research Articles

Bromodomain Interactions with Acetylated Histone 4 Peptides in the BRD4 Tandem Domain: Effects on Domain Dynamics and Internal Flexibility.

The bromodomain and extra-terminal (BET) protein BRD4 regulates gene expression via recruitment of transcriptional regulatory complexes to acetylated chromatin. Like other BET proteins, BRD4 contains two bromodomains, BD1 and BD2, that can interact cooperatively with target proteins and designed ligands, with important implications for drug discovery. Here, we used nuclear magnetic resonance (NMR) spectroscopy to study the dynamics and interactions of the isolated bromodomains, as well as the tandem construct including both domains and the intervening linker, and investigated the effects of binding a tetra-acetylated peptide corresponding to the tail of histone 4. The peptide affinity is lower for both domains in the tandem construct than for the isolated domains. Using 15N spin relaxation, we determined the global rotational correlation times and residue-specific order parameters for BD1 and BD2. Isolated BD1 is monomeric in the apo state but apparently dimerizes upon binding the tetra-acetylated peptide. Isolated BD2 partially dimerizes in both the apo and peptide-bound states. The backbone order parameters reveal marked differences between BD1 and BD2, primarily in the acetyl-lysine binding site where the ZA loop is more flexible in BD2. Peptide binding reduces the order parameters of the ZA loop in BD1 and the ZA and BC loops in BD2. The AB loop, located distally from the binding site, shows variable dynamics that reflect the different dimerization propensities of the domains. These results provide a basis for understanding target recognition by BRD4.

Negative Thermal Expansion and Disorder-to-Order Collapse of an Intrinsically Disordered Protein under Marginally Denaturing Conditions.

Recent work with intrinsically disordered proteins (IDPs) has projected a myriad of their survival instincts based mainly on the total charge content, the abundance of polar residues, and the paucity of hydrophobic amino acids. This work uses a plant IDP AtPP16-1 (Arabidopsis thaliana phloem protein class 16-1), whose solution NMR structure was determined by us recently, to show legitimate negative thermal expansion (NTE) of the native state. The thermal expansion continues to be negative even as the tertiary structure is perturbed by ultralow levels of urea up to 0.4 M. The NTE of these subdenatured states is called apparent NTE because they are prone to undergo conformational changes with temperature. Hydrodynamic shrinkage of the NTE IDP is also observed by dynamic light scattering (DLS) and NMR-measured global rotational correlation time (τc). The protein with denatured tertiary structure but otherwise preserved native-state secondary structure collapses to a dynamically rigid state. The data are mainly based on thermal coefficients of chemical shift and nuclear relaxation measured by heteronuclear NMR. The hydrodynamic shrinkage and collapse under marginally varying solvent compositions that may arise from unstable tertiary structure and dynamic disorder of chain segments across the backbone could be a generic property of IDPs.

Secondary Structure and Folding of Amino Acid Transporter from Outer Chloroplast Membrane

Solving high-resolution structures for membrane proteins continues to be a daunting challenge in the structural biology community. OEP16 is a transmembrane amino acid selective transporter with a molecular mass of 16 kDa [1]. Here we report high-resolution NMR results for OEP16 solubilized in SDS detergent micelles. Three-dimensional NMR experiments were performed on U-15N, 13C- labeled and 80% perdeuterated protein to assign protein backbone resonances and obtain secondary structure information. To understand the protein dynamics and function 15N relaxation, amino acid titration, and 2H exchange experiments were performed. The chemical shift data for 15N, 1HN, 13CO, 13Cα and 13Cβ nuclei obtained from three-dimensional NMR experiments were used to assign 95% of the backbone amino acid residues. Chemical shift data were analyzed using the TALOS+ program to detect the secondary structure elements and to estimate 1H-15N bond orientational order parameters. Our analysis leads to three important conclusions: the tertiary structure of OEP16 involves four transmembrane helices connected by flexible loop regions, it is likely to be monomeric in SDS micelles, and the ligand binding sites could be located. The α-helix content calculated in this method (55%) fits well with previous measurements (50%) [1]. Our 15N relaxation data supports the predicted secondary structure and helps in estimating the isotropic global rotational correlation time (τm) [2]. The value of τm indicates that OEP16 is a monomer in SDS micelles. The 2H exchange data revealed solvent exposed regions in the protein that aid in understanding the tertiary structural folding of the protein. The present study provides a basis for a high resolution structural determination of this membrane protein in future NMR experiments. [1] D. Ni et al. (2011) Protein Expression Purif. 80, 157-168. [2] M. F. Brown (1982) J. Chem. Phys. 77, 1576-1599.

Observation and relaxation properties of individual fast-relaxing proton transitions in [13CH3]-methyl-labeled, deuterated proteins

A pair of NMR experiments is developed for separation of individual fast-relaxing transitions in 13CH3 methyl groups of methyl-protonated, highly deuterated proteins, and the measurement of their relaxation rates. Intra-methyl 1H–1H/1H–13C dipole–dipole cross-correlated spin relaxation that differentiates the rates of the fast-relaxing transitions depending on the state of 13C spins, is measured in the selectively [13CH3]-methyl-labeled, deuterated ubiquitin at 10, 27, and 40°C. In contrast with previous observations, the 1H–1H/1H–13C cross-correlated relaxation rates measured from relaxation rates of single-quantum proton transitions serve as good measures of side-chain order even in proteins with global rotational correlation times significantly less than 10ns.

Carboxymethylation of Cysteines Impedes Aggregation of Hen Lysozyme in Alkaline pH

In our previous work, we have shown that hen lysozyme spontaneously forms globular and fibrillar aggregates which are stabilized later through formation of intermolecular disulphide bond in pH 12.2. Therefore, focus of the present work was on suppression of HEWL aggregation by carboxymethylation of free -SH groups with iodoacetamide that inhibits further formation of disulphide bonds. Changes in structure and dynamics of aggregates were monitored using various biophysical techniques like fluorescence spectroscopy and atomic force microscopy (AFM). Thioflavin T (ThT) fluorescence showed that in comparison to free thiol containing HEWL control, carboxymethylated HEWL was unable to form fibrils. However presence of moderate ThT intensity in modified HEWL indicated presence of oligomers. Increased fluorescence intensity and marginal red shift of ANS spectra in modified HEWL compared to unblocked control revealed that modified HEWL possessed exposed hydrophobic residues compared to free -SH containing HEWL. Fluorescence anisotropy of carboxymethylated dansyl-labeled HEWL were significantly lower compared to control. The fluorescence anisotropy decay kinetics revealed that carboxymethylated HEWL-dansyl conjugates possessed shorter global rotational correlation time (∼11.7 ns) compare to control (∼22 ns) after 30 hours of incubation in pH 12.2 at room temperature. Taken together, these investigations suggest that blocking thiol groups in HEWL suppresses formation of amyloid fibrils and bigger aggregates but not oligomers, in pH 12.2 at room temperature. This work can be helpful for food industries in making aggregate-free lysozyme preservatives in future.Authors acknowledge Department of Science and Technology, New Delhi and University Grants Commision New Delhi for financial support.

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.

PAMAM Dendrimers Conjugated with an Uncharged Gadolinium(III) Chelate with a Fast Water Exchange: The Influence of Chelate Charge on Rotational Dynamics

A bifunctional ligand, DO3A-py(NO-C) (DO3A-py(NO-C) = 10-[(4-carboxy-1-oxidopyridin-2-yl)methyl]-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid), was attached to different generations (G0, G1, G2, and G4) of ethylenediamine-cored PAMAM dendrimers (PAMAM = polyamidoamine). The gadolinium(III) complex of this ligand possesses one molecule of water in its first coordination sphere and has a unique combination of a short water residence lifetime (tau(M) = 34 ns), a neutral overall charge, and a possibility for rigid attachment to molecules bearing primary amino groups. These favorable properties predestine the ligand for constructions of highly efficient nanosized contrast agents for magnetic resonance imaging (MRI). The coupling reaction between the carboxylic group on the pyridine-N-oxide moiety of the protected ligand molecule and primary amines in the dendrimers was achieved by an active ester method under nonaqueous conditions using the coupling agent TBTU. This reaction afforded conjugates with high loadings (80-100% of the theoretically available primary amines) and of high purity. The gadolinium(III) complexes of the conjugates were studied by variable-temperature 17O NMR and 1H NMRD measurements. The water residence lifetime (tau(M) = 55 ns) found in the largest conjugate G4-[Gd(H2O)(do3a-py(NO-C))]57, though somewhat longer compared to the "monomeric" complex, is still short enough not to limit the relaxivity. Surprisingly, compared with analogous conjugates with negatively charged chelates, the prepared uncharged compounds displayed much faster global rotational correlation times (tau(g)) and lower relaxivities. This phenomenon can be explained on the basis of Coulomb interactions. The motion of the charged chelates is restrained due to interactions with their counterions and the chelates themselves, while the uncharged chelates are not affected. Comparison of the PAMAM-based conjugates bearing uncharged and (1-)-charged chelates based on relaxometric data, 1H DOSY spectra, and SAXS measurements reveals that tau(g) reflects the rotational motion of large segments (dendrons) of the conjugates rather than that of the whole macromolecule.

Probing the dynamics of domain III of human serum albumin entrapped in sol–gel derived silica using a Sudlow’s site II specific fluorescent ligand

Previous studies from our lab reported on the use of time-resolved fluorescence anisotropy (TRFA) to probe the dynamics of domains I and II within the model protein, human serum albumin (HSA), in solution and when entrapped into sol–gel derived silica. In order to further our understanding of the dynamics within this multi-domain protein, TRFA was used to measure the dynamics of domain III of the protein. For this purpose, the fluorescence ligand dansylsarcosine (DS), which has a 400-fold higher emission intensity in the bound state relative to the free state and an emission lifetime of >22 ns when bound to Sudlow’s site II (domain III) in HSA, was selected. This probe is able to accurately report on slow rotational motions (up to 300 ns correlation time) and the bound form of the probe can be selectively measured at 475 nm, ensuring that the dynamics reflect only the properly folded form of the protein. The mobility of HSA with bound dansylsarcosine (HSA–DS) was evaluated in solution and after entrapment in sol–gel derived silica prepared from sodium silicate under varying ionic strength and pH conditions. The results here show that (1) the 43 ns global rotational correlation time of HSA in buffered solution can be accurately measured via labeling with DS with no interference from faster local or segmental motions; (2) the global motion of HSA in silica is greatly hindered immediately after encapsulation, with no correlation time faster than 300 ns discernable, indicative of strong templating of the silica around domain III of the native protein; and (3) the addition of salt and variation of pH have essentially no effect on HSA mobility, ruling out electrostatics as the primary interaction restricting HSA motion. The results from this study are compared to past studies using intrinsic tryptophan fluorescence (domain II) or fluorescein-labeled HSA (domain I), and demonstrate that motion observed using such probes likely reflects differential mobility of the three domains, consistent with domain III of HSA adsorbing to or templating with silica upon entrapment while the other domains protrude into the pore. Restricted motion of domain III of HSA was also observed in silica materials derived from diglycerylsilane or tetraethylorthosilicate, showing that templating is not dependent on the silica precursor or processing conditions.

Fluorescence studies of the replication initiator protein RepA in complex with operator and iteron sequences and free in solution

RepA, the replication initiator protein from the Pseudomonas plasmid pPS10, regulates plasmid replication and copy number. It is capable of autorepression, in which case it binds as a dimer to the inverted repeat operator sequence preceding its own gene. RepA initiates plasmid replication by binding as a monomer to a series of four adjacent iterons, which contain the same half-repeat as found in the operator sequence. RepA contains two domains, one of which binds specifically to the half-repeat. The other is the dimerization domain, which is involved in protein-protein interactions in the dimeric RepA-operon complex, but which actually binds DNA in the monomeric RepA-iteron complex. Here, detailed fluorescence studies on RepA and an N-(iodoacetyl)aminoethyl-8-naphthylamine-1-sulfonic acid-labeled single-cysteine mutant of RepA (Cys160) are described. Using time-resolved fluorescence depolarization measurements, the global rotational correlation times of RepA free in solution and bound to the operator and to two distinct iteron dsDNA oligonucleotides were determined. These provide indications that, in addition to the monomeric RepA-iteron complex, a stable dimeric RepA-iteron complex can also exist. Further, Förster resonance energy transfer between Trp94, located in the dimerization domain, and N-(iodoacetyl)aminoethyl-8-naphthylamine-1-sulfonic acid-Cys160, located on the DNA-binding domain, is observed and used to estimate the distance between the two fluorophores. This distance may serve as an indicator of the orientation between both domains in the unbound protein and RepA bound to the various cognate DNA sequences. No major change in distance is observed and this is taken as evidence for little to no re-orientation of both domains upon complex formation.

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+)).

Helix mobility and recognition function of the rat thyroid transcription factor 1 homeodomain - hints from 15N-NMR relaxation studies

The backbone dynamics of the (15)N-labeled homeodomain of the rat thyroid transcription factor 1 has been studied by 2D NMR spectroscopy. Longitudinal (R(1)) and transverse (R(2)) (15)N relaxation rate constants and steady-state {(1)H}-(15)N NOEs were measured at 11.7 T. These data were analyzed by both the model-free formalism and the reduced spectral density mapping (RSDM) approaches. The global rotational correlation time, tau(m), of the thyroid transcription factor 1 homeodomain in aqueous solution at 286 K was found to be 10.51 +/- 0.05 ns by model-free formalism and 9.85 +/- 1.79 ns by RSDM calculation. The homogeneity of the values of the overall correlation time calculated from the individual (R(2)/R(1)) ratios suggested a good degree of isotropy of the global molecular motion, consistent with the similar global tau(m) results obtained with the two different methods. Tyr25 was found to undergo slow conformational exchange by both methods, whereas this contribution was identified also for Lys21, Gln22, Ile38 and His52 only by RSDM. With both methods, the C-terminal fragment of helix III was found to be more flexible than the preceding N-terminal portion, with slightly different limits between rigid and mobile moieties. Additionally, Arg53 appeared to be characterized by an intermediate motional freedom between the very flexible N-terminal and C-terminal residues and the structured core of the molecule, suggesting the occurrence of a hinge point. Finally, slow-time-scale motions observed at the end of helix I, at the end of helix II and within helix III appear to be consistent with typical fraying transitions at helical C-termini.

The Structure of the C-Terminal KH Domains of KSRP Reveals a Noncanonical Motif Important for mRNA Degradation

The AU-rich element (ARE) RNA-binding protein KSRP (K-homology splicing regulator protein) contains four KH domains and promotes the degradation of specific mRNAs that encode proteins with functions in cellular proliferation and inflammatory response. The fourth KH domain (KH4) is essential for mRNA recognition and decay but requires the third KH domain (KH3) for its function. We show that KH3 and KH4 behave as independent binding modules and can interact with different regions of the AU-rich RNA targets of KSRP. This provides KSRP with the structural flexibility needed to recognize a set of different targets in the context of their 3'UTR structural settings. Surprisingly, we find that KH4 binds to its target AREs with lower affinity than KH3 and that KSRP's mRNA binding, and mRNA degradation activities are closely associated with a conserved structural element of KH4.

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.

Measurement of 15N relaxation in the detergent-solubilized tetrameric KcsA potassium channel

A set of TROSY-HNCO (tHNCO)-based 3D experiments is presented for measuring (15)N relaxation parameters in large, membrane-associated proteins, characterized by slow tumbling times and significant spectral overlap. Measurement of backbone (15)N R (1), R (1rho), (15)N-{(1)H} NOE, and (15)N CSA/dipolar cross correlation is demonstrated and applied to study the dynamic behavior of the homotetrameric KcsA potassium channel in SDS micelles under conditions where this channel is in the closed state. The micelle-encapsulated transmembrane domain, KcsA(TM), exhibits a high degree of order, tumbling as an oblate ellipsoid with a global rotational correlation time, tau(c) = 38 +/- 2.5 ns, at 50 degrees C and a diffusion anisotropy, Dparallel/Dperpendicular = 0.79+/-0.05, corresponding to an aspect ratio a/b >/= 1.4. The N- and C-terminal intracellular segments of KcsA exhibit considerable internal dynamics (S (2) values in the 0.2-0.45 range), but are distinctly more ordered than what has been observed for unstructured random coils. Relaxation behavior in these domains confirms the position of the C-terminal helix, and indicates that in SDS micelles, this amphiphilic helix does not associate into a stable homotetrameric helical bundle. The relaxation data indicate the absence of elevated backbone dynamics on the ps-ns time scale for the 5-residue selectivity filter, which selects K(+) ions to enter the channel.

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.

PAMAM Dendrimeric Conjugates with a Gd−DOTA Phosphinate Derivative and Their Adducts with Polyaminoacids: The Interplay of Global Motion, Internal Rotation, and Fast Water Exchange

A series of dendrimeric conjugates based on a PAMAM (polyamidoamine) backbone with macrocyclic Gd-DO3A-P(ABn) complexes (monophosphinated analogue of DOTA) was prepared. The chelates were covalently attached to the G1-, G2-, and G4-PAMAM dendrimers through a thiourea linker in high loads (>90%). The prepared conjugates G1-(Gd-DO3A-P(BnN{CS}))(8), G2-(Gd-DO3A-P(BnN{CS}))(16), and G4-(Gd-DO3A-P(BnN{CS}))(59) showed relaxivities of 10.1, 14.1, and 18.6 s(-)(1) mM(-)(1) at 20 MHz and 37 degrees C and pH = 7.5, respectively. A variable-pH study (range 2-12) revealed up to 30% increase in the relaxivity at low pH for the G2-(Gd-DO3A-P(BnN{CS}))(16) conjugate. As confirmed by (1)H NMR titration of the unmodified G2 dendrimer, this is due to protonation of core tertiary amines leading to a more open and rigid structure. The variable-temperature (17)O NMR and (1)H NMRD relaxometric studies confirmed that the relaxivity is not controlled by water exchange but by rotational dynamics. A multiparametrical data evaluation using the Lipari-Szabo approach revealed that the water residence lifetime, (298)tau(M), for the conjugates studied was ca. 45-70 ns, which is longer than the value found for the monomeric model compound Gd-DO3A-P(ABn) (16 ns) but short enough so as not to limit the relaxivity. The global rotational correlation time, (298)tau(Rg), varied from 1.5 to 3.1 ns and seemed to indicate a sufficiently slow molecular tumbling to achieve the high relaxivities measured; however, the rigidity factor S(2) (approximately 0.26), describing the internal flexibility, was far from optimum. The overall relaxivity was significantly increased (e.g. by a factor of 1.8 for the G1-(Gd-DO3A-P(BnN{CS}))(8) conjugate) when a positively charged polyaminoacid like poly(Arg) or poly(Lys) was added to the conjugate solutions. The electrostatic interactions partially "freeze" the internal mobility of the conjugate and also slow down global motion. This assumption was confirmed by an evaluation of (1)H relaxometric data obtained for the G2-(Gd-DO3A-P(BnN{CS}))(16)-poly(Lys)(59) adduct. Importantly, it was proved that the adduct formation did not hamper the water exchange process.

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)).

NMR structure and backbone dynamics of the extended second transmembrane domain of the human neuronal glycine receptor alpha1 subunit.

The structure and backbone dynamics of an extended second transmembrane segment (TM2e) of the human neuronal glycine receptor alpha(1) subunit in sodium dodecyl sulfate micelles were studied by (1)H and (15)N solution-state NMR. The 28-amino acid segment contained the consensus TM2 domain plus part of the linker between the second and third transmembrane domains. The presence of a well-structured helical region of at least 13 amino acids long and an unstructured region near the linker was evident from the proton chemical shifts and the pattern of midrange nuclear Overhauser effects (NOE). (15)N relaxation rate constants, R(1) and R(2), and (15)N-[(1)H] NOE indicated restricted internal motions in the helical region with NOE values between 0.6 and 0.8. The squared order parameter (S(2)), the effective correlation time for fast internal motions (tau(e)), and the global rotational correlation time (tau(m)) were calculated for all TM2e backbone N-H bonds using the model-free approach. The S(2) values ranged about 0.75-0.86, and the tau(e) values were below 100 ps for most of the residues in the helical region. The tau(m) value, calculated from the dynamics of the helical region, was 5.1 ns. The S(2) values decreased to 0.1, and the tau(e) values sharply increased up to 1.2 ns at the linker near the C-terminus, indicating that the motion of this region is unrestricted. The results suggest a relatively high degree of motional freedom of TM2e in micelles and different propensities of the N- and C-terminal moieties of the transmembrane domain to assume stable helical structures.

From monomers to micelles: investigation of the parameters influencing proton relaxivity.

17O NMR and (1)H NMRD studies have been performed on a series of Gd(III) 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) derivatives as potential liver-specific magnetic resonance imaging (MRI) contrast agents. They bear aliphatic side chains which make them capable of micellar self-organization. The compounds differ in the length (C10-C18) and in the chemical nature (alkyl or monoamide-alkyl) of their lipophilic chain. We have established a convenient method to determine the critical micellar concentration (cmc) of paramagnetic surfactants by (1)H relaxivity measurements. This technique can be easily used over a large temperature range; thus, it can find wide application outside the field of MRI contrast agents. The knowledge of the cmc allowed us to determine the parameters governing the water proton relaxivity of the Gd(III) chelates in both nonaggregated and aggregated micellar forms. The relaxation data of the micellar complexes have been interpreted with the Lipari-Szabo approach. This model allows a local motion to be separated from the global tumbling of the whole micelle (modulated by a local, tau(l), and a global, tau(g), rotational correlation time, respectively). The aggregation substantially affects the rotational dynamics and thus increases the proton relaxivity of the Gd(III) chelates. The global rotational correlation times increase with increasing length of the side chain (500-2800 ps for C10-C18). Local motions are also influenced by the length and by the hydrophobicity of the side chain. The analysis of the relaxation data reveals considerable flexibility for these micellar aggregates. The rate of water exchange obtained for these chelates is identical to that for [Gd(DOTA)(H(2)O)](-) (k(ex)(298)= 4.8 x 10(6)s(-1))and is not sensitive either to micellization or to differences in the aliphatic chain. A relaxivity gain in such systems could be attained by simultaneously optimizing the water exchange by modifications of the chelate and increasing the micelle rigidity by using water-soluble surfactants with more hydrophobic side chains.