Rotational Tumbling Time Research Articles

How Much Entropy Is Contained in NMR Relaxation Parameters?

Solution-state NMR relaxation experiments are the cornerstone to study internal protein dynamics at an atomic resolution on time scales that are faster than the overall rotational tumbling time τR. Since the motions described by NMR relaxation parameters are connected to thermodynamic quantities like conformational entropies, the question arises how much of the total entropy is contained within this tumbling time. Using all-atom molecular dynamics simulations of the T4 lysozyme, we found that entropy buildup is rather fast for the backbone, such that the majority of the entropy is indeed contained in the short-time dynamics. In contrast, the contribution of the slow dynamics of side chains on time scales beyond τR on the side-chain conformational entropy is significant and should be taken into account for the extraction of accurate thermodynamic properties.

ChemInform Abstract: Towards Polymetallic Lanthanide Complexes as Dual Contrast Agents for Magnetic Resonance and Optical Imaging

AbstractReview: 106 refs.

Detecting enzyme activities with exogenous MRI contrast agents.

This review focuses on exogenous magnetic resonance imaging (MRI) contrast agents that are responsive to enzyme activity. Enzymes can catalyze a change in water access, rotational tumbling time, the proximity of a (19)F-labeled ligand, the aggregation state, the proton chemical-exchange rate between the agent and water, or the chemical shift of (19)F, (31)P, (13)C or a labile (1)H of an agent, all of which can be used to detect enzyme activity. The variety of agents attests to the creativity in developing enzyme-responsive MRI contrast agents.

Lanthanide(III) Complexes of Diethylenetriaminepentaacetic Acid (DTPA)–Bisamide Derivatives as Potential Agents for Bimodal (Optical/Magnetic Resonance) Imaging

AbstractDiethylenetriaminepentaacetic acid (DTPA)–bisamide derivatives functionalized with p‐toluidine, 6‐aminocoumarin, 1‐naphthalene methylamine and 4‐ethynylaniline were synthesized and fully characterized by mass spectrometry, NMR spectroscopy, FTIR spectroscopy and elemental analysis. LnIII complexes (Ln = Gd, Eu, Tb, Y) of the ligands DTPA–bis‐p‐toluidineamide (DTPA–BTolA), DTPA–bis‐6‐coumarinamide (DTPA–BCoumA), DTPA–bis‐1‐naphthylmethylamide (DTPA–BNaphA) and DTPA–bis‐4‐ethynylphenylamide (DTPA–BEthA) were prepared and studied for their bimodal magnetic resonance imaging/optical properties. EuIII and TbIII derivatives in aqueous solutions exhibit characteristic red and green emission, respectively, with quantum yields of 0.73 % for EuIII–DTPA–BNaphA and 2.5 % for TbIII–DTPA–BEthA. Ligand‐centred photophysical properties of the GdIII complexes were investigated to gain insight into energy‐transfer processes that take place in these systems. The GdIII complexes were also analyzed by nuclear magnetic relaxation dispersion (NMRD) techniques. The relaxivity (r1) at 20 MHz and 310 K equals 4.1 s–1 mM–1 for Gd–DTPA–BTolA, 5.1 s–1 mM–1 for Gd–DTPA–BCoumA, 6.4 s–1 mM–1 for Gd–DTPA–BNaphA and 5.7 s–1 mM–1 for Gd–DTPA–BEthA. These values are higher than the value of 3.8 s–1 mM–1 for Gd–DTPA (Magnevist). The improved relaxivity is due to the increase in the rotational tumbling time τR with a factor of 1.6 for Gd–DTPA–BTolA, 2.1 for Gd–DTPA–BCoumA, 3.1 for Gd–DTPA–BNaphA and 6.5 for Gd–DTPA–BEthA. In a 4 % human serum albumin solution, the apparent relaxivity at 20 MHz increases to values of 13.9 and 19.1 s–1 mM–1 for Gd–DTPA–BNaphA and Gd–DTPA–BEthA, respectively. All these features assist the search for optimal bimodal optical and magnetic resonance imaging probes.

Recombinant proteins incorporating short non-native extensions may display increased aggregation propensity as detected by high resolution NMR spectroscopy

The use of a recombinant protein to investigate the function of the native molecule requires that the former be obtained with the same amino acid sequence as the template. However, in many cases few additional residues are artificially introduced for cloning or purification purposes, possibly resulting in altered physico-chemical properties that may escape routine characterization. For example, increased aggregation propensity without visible protein precipitation is hardly detected by most analytical techniques but its investigation may be of great importance for optimizing the yield of recombinant protein production in biotechnological and structural biology applications.In this work we show that bile acid binding proteins incorporating the common C-terminal LeuValProArg extension display different hydrodynamic properties from those of the corresponding molecules without such additional amino acids. The proteins were produced enriched in nitrogen-15 for analysis via heteronuclear NMR spectroscopy. Residue-specific spin relaxation rates were measured and related to rotational tumbling time and molecular size. While the native-like recombinant proteins show spin-relaxation rates in agreement with those expected for monomeric globular proteins of their mass, our data indicate the presence of larger adducts for samples of proteins with very short amino acid extensions. The used approach is proposed as a further screening method for the quality assessment of biotechnological protein products.

A new metallostar complex based on an aluminum(iii) 8-hydroxyquinoline core as a potential bimodal contrast agent

A ditopic DTPA monoamide derivative containing an 8-hydroxyquinoline moiety was synthesized and the corresponding gadolinium(III) complex ([Gd(H5)(H(2)O)](-)) was prepared. After adding aluminum(III), the 8-hydroxyquinoline part self-assembled into a heteropolymetallic triscomplex [(Gd5)(3)Al(H(2)O)(3)](3-). The magnetic and optical properties of this metallostar compound were investigated in order to classify it as a potential in vitro bimodal contrast agent. The proton nuclear magnetic relaxation dispersion measurements indicated that the relaxivity r(1) of [Gd(H5)(H(2)O)](-) and [(Gd5)(3)Al(H(2)O)(3)](3-) at 20 MHz and 310 K equaled 6.17 s(-1) mM(-1) and 10.9 s(-1) mM(-1) per Gd(III) ion respectively. This corresponds to a relaxivity value of 32.7 s(-1) mM(-1) for the supramolecular complex containing three Gd(III) ions. The high relaxivity value is prominently caused by an increase of the rotational tumbling time τ(R) by a factor of 2.7 and 5.5 respectively, in comparison with the commercially used MRI contrast agent Gd(III)-DTPA (Magnevist®). Furthermore, upon UV irradiation, [(Gd5)(3)Al(H(2)O)(3)](3-) exposes green broad-band emission with a maximum at 543 nm. Regarding the high relaxivity and the photophysical properties of the [(Gd5)(3)Al(H(2)O)(3)](3-) metallostar compound, it can be considered as a lead compound for in vitro bimodal applications.

Prediction of the Rotational Tumbling Time for Proteins with Disordered Segments

For well-structured, rigid proteins, the prediction of rotational tumbling time (tau(c)) using atomic coordinates is reasonably accurate, but is inaccurate for proteins with long unstructured sequences. Under physiological conditions, many proteins contain long disordered segments that play important regulatory roles in fundamental biological events including signal transduction and molecular recognition. Here we describe an ensemble approach to the boundary element method that accurately predicts tau(c) for such proteins by introducing two layers of molecular surfaces whose correlated velocities decay exponentially with distance. Reliable prediction of tau(c) will help to detect intra- and intermolecular interactions and conformational switches between more ordered and less ordered states of the disordered segments. The method has been extensively validated using 12 reference proteins with 14 to 103 disordered residues at the N- and/or C-terminus and has been successfully employed to explain a set of published results on a system that incorporates a conformational switch.

An overview of responsive MRI contrast agents for molecular imaging

This review focuses on MR contrast agents that are responsive to a change in physiological environment. The "response" mechanisms are dependent on 6 physicochemical phenomena, including the accessibility of water to the agent, rotational tumbling time, proton exchange rate, electron spin state, MR frequency, or local field inhomogenieties caused by the agent. These phenomena can be affected by the physiological environment, including changes in concentrations or activities of proteins, enzymes, nucleic acids, metabolites, oxygen and metal ions, and changes in pH and temperature. A total of 52 examples are presented, which demonstrate the variety and creativity of different approaches used to create responsive MRI contrast agents.

Evaluating Rotational Diffusion from Protein MD Simulations

It is now feasible to carry out molecular dynamics simulations of proteins in water that are long compared to the overall tumbling of the molecule. Here, we examine rotational diffusion in four small, globular proteins (ubiquitin, binase, lysozyme, and fragment B3 of protein G) with the TIP3P, TIP4P/EW, and SPC/E water models, in simulations that are 6 to 60 times as long as the mean rotational tumbling time. We describe a method for extracting diffusion tensors from such simulations and compare the results to experimental values extracted from NMR relaxation measurements. The simulation results accurately follow a diffusion equation, even for spherical harmonic correlation functions with l as large as 8. However, the best-fit tensors are significantly different from experiment, especially for the commonly used TIP3P water model. Simulations that are 20 to 100 times longer than the rotational tumbling times are needed for good statistics. A number of residues exhibit internal motions on the nanosecond time scale, but in all cases examined here, a product of internal and overall time-correlation functions matches the total time-correlation function well.

Interaction of Ochratoxin A with Human Serum Albumin. Preferential Binding of the Dianion and pH Effects

Ochratoxin A (OTA), a fungal metabolite produced by several strains of Aspergillus and Penicillium, binds to serum albumin with high affinity only in the completely deprotonated form (dianion). The pKa of the phenolic group of OTA decreased by more than three units when it was bound to human serum albumin (HSA). Optical spectroscopy provided evidence that HSA has at least two binding sites for OTA, each being able to accommodate one dianion. These two sites were characterized by the binding constants of 5.2 × 106 and 1.0 × 105 M-1. The binding constant for the monoanion of OTA was estimated to be ∼103 M-1. Fluorescence polarization spectroscopy confirmed weak interaction of the monoanion with the protein in the F and E forms (pH < 4) and showed lower affinity of the dianion to the B form of HSA (pH > 8) compared to the N form (pH ∼ 7). Fluorescence anisotropy decay of the dianion of OTA bound to HSA (36.6 ns) was much longer than its emission lifetime (5.2 ns) and was close to reported values for the rotational tumbling time of the HSA molecule. Results of chemical denaturation with 9 M urea or 5 M guanidine hydrochloride established that high-affinity binding of OTA only occurred to the native protein. Efficient energy transfer from the single tryptophan residue of HSA (Trp214) to bound OTA was observed. Analysis of fluorescence data provided an estimate of the distance between the dianion of OTA in its highest-affinity site and the Trp214 residue, which was on the order of 16 Å. The results are discussed with respect to recent crystallographic data for HSA−ligand complexes and pH-dependent conformation of HSA.

Metal binding sites of oxovanadium(IV) in native and modified soluble collagen

Oxovanadium(IV) coordination to native and modified soluble collagen flora calf skin, has been studied by EPR spectroscopy on aqueous and lyophilized samples at physiological pH. At room temperature, the isotropic spectrum g 0=1.967, A 0=95.61×10 −4 cm −1 of a VO(IV)-collagen complex, weakly immobilized in solution has been seen, with vanadyl rotational correlation time τ r≈6×10 −9 s shorter than rotational tumbling time τ≈10 −7 s of the whole protein. This is probing a local environment for the VO(IV) site and vanadyl monocoordination to the protein. Simultaneously, an anisotropic spectrum with axial symmetry has been detected from VO(IV)-native collagen and VO(IV)-derivative collagen complexes in solution at 77 K and, in the solid fibrous state at both r.t. and 77 K. The apparent g 0 and A 0 values calculated for the powder spectra are comparable with those of solution spectra at r.t. of VO(IV)-insulin in the A site and VO(IV)-(N 2O 2) model compounds. However, oxovanadium(IV) seems to interact with two nitrogens and two water molecules on the equatorial plane. The two protein nitrogen ligands could be two imidazole nitrogens, or one imidazole nitrogen and one amino group, or two amino groups. By increasing the vanadyl concentration, the metal-ion interaction with tropocollagen molecules loses its specificity, to give a mixture of VO(IV)-collagen compounds.

Estimation of the distance between the two binding sites of ovotransferrin

Ovotransferrin (conabulmin, M.wt. 76,000) has been found in large quantities in egg white. Its physiological function is not known with certainty. Because of its large affinity for iron, it is an effective antimicrobial agent which could be important in the protection of the developing chick embryo [1]. In the presence of carbonate, or a difunctional anion, metal ions bind transferrin at two specific sites [2]. The binding of metal ions to the second specific site takes after the first site is filled, i.e. a sequential rather than a random binding process takes place [3]. In this communication the results of the addition of Gd(III) ion in the presence of malonate, using high resolution nuclear magnetic resonance spectroscopy, are reported. To 1.23 m M solution of protein containing 12.3 m M malonate, kept at pH 6.00 ± 0.02 by the presence of 50 m M trideuteroacetate buffer, one and two equivalents of Gd(III) per protein, dissolved in D 2O, were added. In each case, the spectrum was recorded using a Bruker-270 MHz spectrometer (Fig. 1). The spectrum of the protein solution containing one equivalent of Gd(III) shows that all histidine C 2-H resonances have been broadened. In fact the signals of the six histidines which were assigned to be involved in the binding sites [3] have nearly disappeared, indicating that these resonances broaden by at least 80 Hz which could make them undistinguishable from noise. On addition of the two equivalents, ▪ all the histidine C2-H resonances have disappeared, Fig. 1. Theoretical consideration of the line broadening by Gd(III): The line-width of the NMR signals is given by, ▪ where Δ v is the line-width (in Hz) at the half height and T 2M is the transverse relaxation time of the protein. In the presence of Gd(III) ions T 2M is given by the Solomon-Bloembergen equation [4]: ▪ where τ c is the correlation time which modulates the interaction, and r is the distance between the proton and the paramagnetic centre. Assuming τ c is the rotational tumbling time of the macromolecule (τ R, then from Stockes law, ▪ where M is the molecular weight V is the partial specific volume, η is the viscosity of the solvent, T is the temperature and R is the gas constant. Using the above equation, a value of τ R = 2.2 × 10 −8 s −1 could be obtained for ovotransferrin. At 270 MHz eqn. (1) reduces to ▪ giving r = 21 Å for an increase of 80 Hz in the line-width due to the presence of Gd(III). This is the upper limit of the distance between the first binding site of ovotransferrin and the C2-H protons in the second binding site. Thus we conclude from the broadening of the spectrum of the whole protein, when it contains two equivalents of Gd(III), that all the C2-H protons of histidines in ovotransferrin lie in a sphere of radius not exceeding 31.5 Å.

Magnetic-field-dependent water proton spin-lattice relaxation rates of hemoglobin solutions and whole blood

The spin-lattice relaxation rates of solvent water protons in solutions of normal and sickle cell hemoglobin, normal red blood cells, and whole blood have been measured at magnetic fields between 5 Oe and 12 kOe. The field-dependent data have been analyzed to obtain the rotational tumbling time of the hemoglobin molecules and a measure of water-protein interaction. Comparison of the protein tumbling time in concentrated hemoglobin solutions to the tumbling time of hemoglobin inside red blood cells indicates that encapsulation of hemoglobin within the cell membrane has little effect on its molecular motion. The relaxation rates of solutions of deoxygenated sickle cell hemoglobin indicate that when the solution forms a gel at 10°C, approximately 90% of the hemoglobin molecules remain in solution and are freely tumbling, but at 35°C, virtually all of the hemoglobin has been incorporated into the gel structure.