Thomas L. James

DOCK 6: Combining techniques to model RNA–small molecule complexes

With an increasing interest in RNA therapeutics and for targeting RNA to treat disease, there is a need for the tools used in protein-based drug design, particularly DOCKing algorithms, to be extended or adapted for nucleic acids. Here, we have compiled a test set of RNA-ligand complexes to validate the ability of the DOCK suite of programs to successfully recreate experimentally determined binding poses. With the optimized parameters and a minimal scoring function, 70% of the test set with less than seven rotatable ligand bonds and 26% of the test set with less than 13 rotatable bonds can be successfully recreated within 2 A heavy-atom RMSD. When DOCKed conformations are rescored with the implicit solvent models AMBER generalized Born with solvent-accessible surface area (GB/SA) and Poisson-Boltzmann with solvent-accessible surface area (PB/SA) in combination with explicit water molecules and sodium counterions, the success rate increases to 80% with PB/SA for less than seven rotatable bonds and 58% with AMBER GB/SA and 47% with PB/SA for less than 13 rotatable bonds. These results indicate that DOCK can indeed be useful for structure-based drug design aimed at RNA. Our studies also suggest that RNA-directed ligands often differ from typical protein-ligand complexes in their electrostatic properties, but these differences can be accommodated through the choice of potential function. In addition, in the course of the study, we explore a variety of newly added DOCK functions, demonstrating the ease with which new functions can be added to address new scientific questions.

A theoretical study of distance determinations from NMR. Two-dimensional nuclear overhauser effect spectra

Abstract In principle, two-dimensional nuclear Overhauser effect NMR experiments can be used to establish internuclear distances and thus to determine molecular structure in solution. Theoretical calculations have been carried out for the 2D NOE experiment addressing the questions of (l) how sensitive 2D NOE cross-peak intensities are to spatial location of nuclear spins, and (2) how well the motional characteristics (and, therefore, spectral densities) of the molecule must be known in order to determine accurate distances. Theoretical values for cross-peak intensities obtained in 2D NOE spectra were calculated for a set of three-proton spins and a set of four-proton spins. The relaxation rate matrix whose elements establish the cross-peak intensities was diagonalized using a numerical procedure which enables accurate calculations of cross-peak intensities in large spin systems at any mixing time. Several calculations were performed; each calculation entailed a different set of circumstances. Specifically, the transformation of cross-peak intensities into proton-proton distances was examined for 21 configurations of the three-proton system and 7 configurations of the four-proton spin system. Additionally the influence of specific types of overall and internal motion on the calculated cross-peak intensities and the distances determined from them was tested. The conclusions from these calculations are as follows. With the experimental signal-to-noise ratio usually attained for biomolecular concentrations M , interproton distances up to 5 A should lead to detectable cross-peaks in the 2D NOE spectrum with mixing times within a few hundred milliseconds. Recording cross-peak intensities at several mixing times is important for obtaining accurate distances from them. Simple motional models assuming only a single isotropic motion with an effective correlation time may be used to obtain the spectral densities needed to calculate cross-peak intensities even though the actual motional dynamics of the molecule may be more complicated. Using the simplified models introduces approximately 10% error in the distance determination. Finally, it is shown how the cross-peak between a proton and each of its neighbors may increase the accuracy of the determination and help in locating the position of the proton. Our calculations indicate that distances with an accuracy of ±0.5 A should be attainable with knowledge of the overall molecular reorientation rate or individual proton relaxation times.

Perspectives on NMR in drug discovery: a technique comes of age

In the past decade, the potential of harnessing the ability of nuclear magnetic resonance (NMR) spectroscopy to monitor intermolecular interactions as a tool for drug discovery has been increasingly appreciated in academia and industry. In this Perspective, we highlight some of the major applications of NMR in drug discovery, focusing on hit and lead generation, and provide a critical analysis of its current and potential utility.

MARDIGRAS-A procedure for matrix analysis of relaxation for discerning geometry of an aqueous structure

Abstract An accurate method for estimating distances from the two-dimensional nuclear Overhauser effect spectrum is described. The method entails a back-calculation of the relaxation matrix from the measured 2D NOE intensity matrix. While a straightforward back-calculation of distances will fail in the absence of all cross- and diagonal-peak intensities, the MARDIGRAS procedure supplements the observed intensities with calculated values from an arbitrary model and is less prone to the mathematical singularities encountered with an incomplete data set. Several model structures were used for generating the initial matrix of intensities for embedding the observed intensities, and the method is found to be relatively insensitive to the model structure.

COMATOSE, a method for constrained refinement of macromolecular structure based on two-dimensional nuclear overhauser effect spectra

Abstract COMATOSE ( co mplete m atrix a nalysis t orsion o ptimized s tructur e ) is a structure-refinement program based on the quantitative calculation of 2D NOE intensities taking into account the effects of network relaxation and spin diffusion (J. W. Keepers and T. L. James, J. Magn. Reson. 57 , 404 (1984)). The macromolecular structure is defined by fixed bondlengths and two-bond angles, leaving only torsion angles and residue orientations as variable parameters. The performance of this structure refinement algorithm with idealized and experimental 2D NOE intensities for oligonucleotides is discussed. The structural information available from COMATOSE is compared with the distances obtained by analysis of the 2D NOE intensities, and with the distances available from the direct solution of the intensity eigenvalue problem. The most commonly used calculation of distances relies on the assumption that the intensities can be approximated by assuming that the individual spin pairs are essentially isolated from all other protons. This approach systematically results in underestimation of distances. Direct solution of the eigenvalue problem when all intensities (i.e., relaxation pathways) are not accounted for results in large errors especially at longer distances (greater than ∼3 A). COMATOSE is capable of optimizing the structure in favorable cases and, at least, yielding a set of reliable proton-proton distances (relative error ∼10% ). In most practical applications, an iterative approach to the development of the structure should probably be taken. An initial trial structure is subjected to COMATOSE to obtain accurate distance estimates from the 2D NOE. These in turn can be used as constraints in energy minimization of the structure to arrive at an improved structure for recycling into COMATOSE.

Relaxation matrix analysis of 2D NMR data

In this article we will survey some of the approaches taken to derive structural information from the 2D NOE experiment. The underlying physical basis for the methods will be discussed and their limitations will be highlighted. In so doing, we will try to develop guidelines for obtaining the most accurate and realistic structural parameters from the spectral data

PHYSICOCHEMICAL CHARACTERIZATION OF LARGE UNILAMELLAR PHOSPHOLIPID-VESICLES PREPARED BY REVERSE-PHASE EVAPORATION

Properties of large unilamellar vesicles (LUV), composed of phosphatidylcholine and prepared by reverse-phase evaporation and subsequent extrusion through Unipore polycarbonate membranes, have been investigated and compared with those of small unilamellar vesicles (SUV) and of multilamellar vesicles (MLV). The unilamellar nature of the LUV is shown by 1H-NMR using Pr3+ as a shift reagent. The gel to liquid-crystalline phase transition of LUV composed of dipalmitoylphosphatidylcholine (DPPC) monitored by differential scanning calorimetry, fluorescence polarization of diphenylhexatriene and 90 degrees light scattering, occurs at a slight lower temperature (40.8 degrees C) than that of MLV (42 degrees C) and is broadened by about 50%. The phase transition of SUV is shifted to considerably lower temperatures (mid-point, 38 degrees C) and extends over a wide temperature range. In LUV a well-defined pretransition is not observed. The permeability of LUV (DPPC) monitored by leakage of carboxyfluorescein, increases sharply at the phase transition temperature, and the extent of release is greater than that from MLV. Leakage from SUV occurs in a wide temperature range. Freeze-fracture electron microscopy of LUV (DPPC) reveals vesicles of 0.1-0.2 micron diameter with mostly smooth fracture faces. At temperatures below the phase transition, the larger vesicles in the population have angled faces, as do extruded MLV. A banded pattern, seen in MLV at temperatures between the pretransition and the main transition, is not observed in the smaller LUV, although the larger vesicles reveal a dimpled appearance.

ACCF/AHA/AMA-PCPI 2011 performance measures for adults with coronary artery disease and hypertension: a report of the American College of Cardiology Foundation/American Heart Association Task Force on Performance Measures and the American Medical Association-Physician Consortium for Performance Improvement.

Eric D. Peterson, MD, MPH, FACC, FAHA, Chair; Frederick A. Masoudi, MD, MSPH, FACC, FAHA[†††][1]; Elizabeth DeLong, PhD; John P. Erwin III, MD, FACC; Gregg C. Fonarow, MD, FACC, FAHA; David C. Goff, Jr., MD, PhD, FAHA, FACP; Kathleen Grady, PhD, RN, FAHA, FAAN; Lee A. Green, MD, MPH; Paul A.

A molecular switch underlies a human telomerase disease

Telomerase is a ribonucleoprotein (RNP) required for maintenance of telomeres. Although up-regulated telomerase activity is closely linked to the cellular immortality characteristic of late stage carcinogenesis, recently, mutations in the telomerase RNA gene in humans have been associated with dyskeratosis congenita and aplastic anemia, both typified by impaired haemopoietic function. These mutations include base changes in a highly conserved putative telomerase RNA pseudoknot. Here, by using in vitro telomerase assays, NMR, and UV absorbance melting analyses of model oligonucleotides designed to form a “trans-pseudoknot,” we describe functional, structural, and energetic properties of this structure. We demonstrate that the pseudoknot domain exists in two alternative states of nearly equal stability in solution: one is the previously proposed pseudoknot formed by pairing P3 with the loop domain of P2b, and the other is a structured P2b loop alone. We show that the two-base mutation (GC107/8 → AG) present in one gene copy in a family with dyskeratosis congenita abrogates telomerase activity. This mutation hyperstabilizes the P2b intraloop structure, blocking pseudoknot formation. Conversely, when the P3 pseudoknot pairing is hyperstabilized by deleting a conserved bulge in P3, telomerase activity also decreases. We propose that the P2b/P3 pseudoknot domain acts as a molecular switch, and interconversion between its two states is important for telomerase function. Phylogenetic covariation in the P2b and P3 sequences of 35 species provides a compelling set of “natural” compensatory base pairing changes supporting the existence of the crucial molecular switch.

Measurement of the self-diffusion coefficient of each component in a complex system using pulsed-gradient fourier transform NMR☆

Abstract The self-diffusion coefficient of each component in a multicomponent system may be determined by obtaining the Fourier transform of the spin echo using the pulsed gradient, spin echo technique. The pulsed gradient Fourier transform technique also has the individual advantages of Fourier transform NMR and the pulsed gradient technique leading to enhanced signal-to-noise ratio and improved resolution. The method is illustrated for a water(dimethylsulfoxide solution.