A graphical method for the analysis of anisotropic rotational diffusion in proteins

A graphical method for the analysis of relaxation data is presented. It allows a fast estimation of the range of values of the components of the axially symmetric rotational diffusion tensor that are compatible with the experimental relaxation data. The graphical method clearly shows the contribution of different experimental relaxation parameters to the measured anisotropy. In particular, for proteins with moderate anisotropy, data from at least two N-H bonds forming angles close to 0° and 90° with respect to the principal axis of the rotational diffusional tensor are needed. For very anisotropic systems, combination of different relaxation parameters from a single residue is enough to characterize the local anisotropy.     

Interpretation of 15N NMR relaxation data of globular proteins using hydrodynamic calculations with HYDRONMR

HYDRONMR is an implementation of state of the art hydrodynamic modeling to calculate the spectral density functions for NH or C-H vectors in a rigid protein structure starting from an atomic level representation. Thus HYDRONMR can be used to predict NMR relaxation times from a rigid model and to compare them with the experimental results. HYDRONMR contains a single adjustable parameter, the atomic element radius. A protocol to determine the value that gives the best agreement between calculated and experimental T₁/T₂values is described. For most proteins, the value of the atomic element radius ranges between 2.8 Å and 3.8 Å with a distribution centered at 3.3 Å. Deviations from the usual range towards larger values are associated to aggregation in several proteins. Deviations to lower values may be related to large-scale motions or inappropriate model structures.If the average structure is correct, deviations between experimental T₁/T₂values and those calculated with HYDRONMR can be used to distinguish residues affected by anisotropic motion from those that are involved in chemical exchange.     

Rolling properties of rGPIbalpha-conjugated phospholipid vesicles with different membrane flexibilities on vWf surface under flow conditions

The recombinant fragment of the platelet membrane glycoprotein, rGPIbalpha, was conjugated to phospholipid vesicles with the average diameter of ca. 1 microm using N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). We used five kinds of rGPIbalpha-vesicles with different fluorescent anisotropies of 1,6-diphenyl-1,3,5-hexatriene (DPH) to study the rolling properties of the vesicles on the von Willebrand factor (vWf)-immobilized surface. Under flow conditions, the rolling velocity of the rGPIbalpha-vesicles decreased with the increasing membrane flexibility. It is considered that the vesicles with a high membrane flexibility have a high deformability and can be flattened to a high degree during rolling on the vWf surface, thus resulting in the large contact area. We obtained a recipe to control the rolling velocity of artificial platelets by membrane flexibility.     

Antimetabolite incorporation into DNA: structural and thermodynamic basis for anticancer activity

Antimetabolites are a class of effective anticancer drugs that structurally resemble naturally occurring biochemicals and interfere in essential biochemical processes. In this review, the recent literature describing investigations of the structural and thermodynamic basis for the anticancer activity of three antipyrimidines [1-beta-D-arabinofuranosyl cytidine (AraC). 2',2'-difluoro deoxycytidine (dFdC), and 5-fluoro-2'-deoxyuridine (FdUrd)] is summarized. Our laboratory, and others, have shown that misincorporation of any of these three antipyrimidines into DNA perturbs the structure and decreases the stability of duplex DNA. These data are useful for rationalizing the effects of antipyrimidine misincorporation on the activities of proteins required for DNA replication and repair such as DNA topoisomerase 1 and DNA polymerases. The studies completed to date and summarized in this review demonstrate the utility of investigations into the structure-function relationships between antipyrimidine-substituted DNA complexed with DNA-modifying proteins for the purpose of understanding the basis for effective antipyrimidine cancer chemotherapy and the future design of novel anticancer drugs.     

Effect of a 125 mT static magnetic field on the kinetics of voltage activated Na+ channels in GH3 cells

Voltage activated Na(+) channels were examined in GH3 cells, using the whole cell patch clamp method. Channel currents were recorded before, during, and after a 150 s exposure to a 125 mT static magnetic field. There was a slight shift in the current-voltage relationship and a less than 5% reduction in peak current during magnetic field exposure. More pronounced, however, was an increase in the activation time constant, tau(m), during and for at least 100 s following exposure to the field. This change in tau(m) was seen primarily at lower activation voltages. No change was noted in the inactivation time constant, tau(h). Changes were clearly temperature dependent, being evident only at and above 35 degrees C. These findings are consistent with the hypothesis that reorientation of diamagnetic anisotropic molecules in the cell membrane are capable of distorting imbedded ion channels sufficiently to alter their function. The temperature dependence of this phenomenon is probably due to the greater ease with which a liquid crystal membrane can be deformed.     

Angular dependence of dipole-dipole-Curie-spin cross-correlation effects in high-spin and low-spin paramagnetic myoglobin

The ¹⁵N-HSQC spectra of low-spin cyano-met-myoglobin and high-spin fluoro-met-myoglobin were assigned and dipole-dipole-Curie-spin cross-correlated relaxation rates measured. These cross-correlation rates originating from the dipolar ¹H-¹⁵N interaction and the dipolar interaction between the ¹H and the Curie spin of the paramagnetic center contain long-range angular information about the orientation of the ¹H-¹⁵N bond with respect to the iron-¹H vector, with information measurable up to 11 Å from the metal for the low-spin complex, and between 10 to 25 Å for the high-spin complex. Comparison of the experimental data with predictions from crystal structure data showed that the anisotropy of the magnetic susceptibility tensor in low spin cyano-met-myoglobin significantly influences the cross-correlated dipole-dipole-Curie-spin relaxation rates.     

Cross correlations between 13C-1H dipolar interactions and 15N chemical shift anisotropy in nucleic acids

Two sets of cross-correlated relaxation rates involving chemical shift anisotropy and dipolar interactions have been measured in an RNA kissing complex. In one case, both the CSA and dipolar interaction tensors are located on the same nucleotide base and are rigidly fixed with respect to each other. In the other case, the CSA tensor is located on the nucleotide base whereas the dipolar interaction is located on the adjoining ribose unit. Analysis of the measured rates in terms of isotropic or anisotropic rotational diffusion has been carried out for both cases. A marked difference between the two models is observed for the cross-correlation rates involving rigidly fixed spin interactions. The influence of internal motions about the glycosidic linkage between the nucleotide base and the ribose unit on cross-correlated relaxation rates has been estimated by applying a model of restricted rotational diffusion. Local motions seem to have a more pronounced effect on cross-correlated relaxation rates when the two spin interactions are not rigidly fixed with respect to each other.     

γ-Ray footprinting and fluorescence polarization anisotropy of a 30-mer synthetic DNA fragment with one 2′-deoxy-7-hydro-8- oxoguanosine lesion

The influence of the oxidative lesion 2'-deoxy-7-hydro-8-oxoguanosine (8-oxodG) on some conformational properties of DNA has been studied. Four 30-mer duplexes of the form [5'-GATCCTCTAGAGTC[G(*) or G]ACCTGCAGGCATGCA-3']:[3'-CTAGGAGATCTCAG[C or A]TGGACGTCCGTACGT-5'], in which G(*) is the 8-oxodG lesion, were synthesized in order to compare the effect of the GA mismatch and of the damaged G(*)C and G(*)A forms with the normal GC. Spectroscopic measurements performed by means of UV denaturation and circular dichroism experiments do not show gross changes of stability and overall structure in the damaged and mismatched samples. The control DNA and the samples containing GA mismatch show very similar gamma-rays cutting patterns, indicating that the introduction of the GA mismatch does not perturb the phosphate backbone geometry. In the samples containing the 8-oxodG there are some variations of the cleavage pattern near G(*) which are extended for almost one helical turn. Some differences are observed between G(*)C and G(*)A duplexes. In particular, in the G(*)C sample the reduced accessibility to OH radicals at the G15 site, observed in the control, spreads on the intrastrand adjacent bases and in the G(*)A sample a shift of the minimum is observed. The hydrodynamic radius R(h) derived by fluorescence polarization anisotropy decay exhibits a constant value of 11.4 +/- 0.2 A between 5 and 40 degrees C, in all the samples. The torsional constant alpha of each oligomer decreases when the temperature is raised and the alpha values of the damaged samples are higher than those of the normal ones.