Order parameters from deuterium NMR are often used to validate or calibrate molecular dynamics simulations. This paper gives a short overview of the literature in which experimental order parameters from (2)H NMR are compared to those calculated from MD simulations. The different ways in which order parameters from experiment are used to calibrate and validate simulations are reviewed. In the second part of this review, a case study of cholesterol in a DMPC bilayer is presented. It is concluded that the agreement between experimental data and simulation is favorable in the hydrophobic region of the membrane, for both the phospholipids and cholesterol. In the interfacial region the agreement is less satisfactory, probably because of the high polarity of this region which makes the correct computation of the electrostatics more complex.

Proton decoupled deuterium NMR spectra of oriented bilayers made of DMPC and 30 mol % deuterated cholesterol acquired at 76.8 MHz (30 degreesC) have provided a set of very accurate quadrupolar splitting for eight C-D bonds of cholesterol. Due to the new precision of the experimental data, the original analysis by. Biochemistry. 23:6062-6071) had to be reconsidered. We performed a systematic study of the influence on the precision and uniqueness of the data-fitting procedure of: (i) the coordinates derived from x-ray, neutron scattering, or force field-minimized structures, (ii) internal mobility, (iii) the axial symmetry hypothesis, and (iv) the knowledge of some quadrupolar splitting assignments. Good agreement between experiment and theory could be obtained only with the neutron scattering structure, for which both axial symmetry hypothesis and full order parameter matrix analysis gave satisfactory results. Finally, this work revealed an average orientation of cholesterol slightly different from those previously published and, most importantly, a molecular order parameter equal to 0.95 +/- 0.01, instead of 0.79 +/- 0.03 previously found for the same system at 30 degreesC. Temperature dependence in the 20-50 degreesC range shows a constant average orientation and a monotonous decrease of cholesterol Smol, with a slope of -0.0016 K-1. A molecular order parameter of 0.89 +/- 0.01 at 30 degreesC was determined for a DMPC/16 mol % of cholesterol.

CD1-restricted lipid-specific T lymphocytes are primed during infection with Mycobacterium tuberculosis, the causative agent of tuberculosis. Here we describe the antigenicity of glycerol monomycolate (GroMM), which stimulates CD1b-restricted CD4(+) T cell clones. Chemical characterization of this antigen showed that it exists as two stereoisomers, one synthetic isomer being more stimulatory than the other. The hydroxyl groups of glycerol and the mycolic acid length are critical for triggering the T cell responses. GroMM was presented by M. tuberculosis-infected dendritic cells, demonstrating that the antigen is available for presentation during natural infection. Ex vivo experiments showed that GroMM stimulated T cells from vaccinated or latently infected healthy donors but not cells from patients with active tuberculosis, suggesting that GroMM-specific T cells are primed during infection and their detection correlates with lack of clinical active disease.

Backbone dynamics of trp repressor, a 25 kDa DNA binding protein, have been studied using 15N relaxation data measured by proton-detected two-dimensional 1H-15N NMR spectroscopy. 15N spin-lattice relaxation time (T1), spin-spin relaxation time (T2), and heteronuclear NOEs were determined for all visible backbone amide 15N nuclei. Monte Carlo simulations of the amplitudes of backbone motions led to the conclusion that a wobbling in a cone model with consideration of the anisotropic reorientation of the molecule was appropriate to describe the underlying motions, allowing us to derive the semiangle of the cone (alpha) and the effective correlation time for internal motions (tau e) for each N-H bond vector. The final optimized rotational diffusion coefficients parallel (D parallel) and perpendicular (D perpendicular) to the unique axis of the molecule were found to be 1.48 +/- 0.06 x 10(7) and 1.15 +/- 0.05 x 10(7) s-1, respectively. The average semiangle of the cone (alpha) describing the amplitude of NH vector motions on the picosecond time scale was found to be 20.9 +/- 5.7 degrees. Large amplitude motions on the picosecond time scale are found at both the N and C termini but are restricted in both the hydrophobic core and DNA-binding regions.

THAP1, the founding member of a previously uncharacterized large family of cellular proteins (THAP proteins), is a sequence-specific DNA-binding factor that has recently been shown to regulate cell proliferation through modulation of pRb/ E2F cell cycle target genes. THAP1 shares its DNA-binding THAP zinc finger domain with Drosophila P element transposase, zebrafish E2F6, and several nematode proteins interacting genetically with the retinoblastoma protein pRb. In this study, we report the three-dimensional structure and structurefunction relationships of the THAP zinc finger of human THAP1. Deletion mutagenesis and multidimensional NMR spectroscopy revealed that the THAP domain of THAP1 is an atypical zinc finger of ϳ80 residues, distinguished by the presence between the C2CH zinc coordinating residues of a short antiparallel ␤-sheet interspersed by a long loop-helix-loop insertion. Alanine scanning mutagenesis of this loop-helix-loop motif resulted in the identification of a number of critical residues for DNA recognition. NMR chemical shift perturbation analysis was used to further characterize the residues involved in DNA binding. The combination of the mutagenesis and NMR data allowed the mapping of the DNA binding interface of the THAP zinc finger to a highly positively charged area harboring multiple lysine and arginine residues. Together, these data represent the first structure-function analysis of a functional THAP domain, with demonstrated sequence-specific DNA binding activity. They also provide a structural framework for understanding DNA recognition by this atypical zinc finger, which defines a novel family of cellular factors linked to cell proliferation and pRb/E2F cell cycle pathways in humans, fish, and nematodes.Zinc finger proteins represent the most abundant class of DNA-binding proteins in the human genome. Zinc fingers have been defined as small, functional, independently folded domains that require coordination of a zinc atom to stabilize their structure (1). The zinc finger superfamily includes the C2H2-type zinc finger, a compact ϳ30-amino acid DNA-binding module repeated in multiple copies in the protein structure (2, 3), the C4-type zinc finger found in the GATA family of transcription factors (4), and the zinc-coordinating DNA-binding domain of nuclear hormone receptors (5). We recently described an atypical zinc finger motif, characterized by a large C2CH module (Cys-X 2-4 -Cys-X 35-53 -Cys-X 2 -His) with a spacing of up to 53 amino acids between the zinc-coordinating C2 and CH residues (6). This motif, designated THAP domain or THAP zinc finger, defines a previously uncharacterized large family of cellular factors with more than 100 distinct members in the animal kingdom (6, 7). We showed that the THAP domain of THAP1, the prototype of the THAP family (8), possesses zinc-dependent sequence-specific DNA binding activity and recognizes a consensus DNA target sequence of 11 nucleotides (THABS, for the THAP1 binding sequence) (7), considerably larger than the 3-4 nucleotides motif typ...

A new solid-state NMR-based strategy is established for the precise and efficient analysis of orientation and dynamics of transmembrane peptides in fluid bilayers. For this purpose, several dynamically averaged anisotropic constraints, including (13)C and (15)N chemical shift anisotropies and (13)C-(15)N dipolar couplings, were determined from two different triple-isotope-labeled WALP23 peptides ((2)H, (13)C, and (15)N) and combined with previously published quadrupolar splittings of the same peptide. Chemical shift anisotropy tensor orientations were determined with quantum chemistry. The complete set of experimental constraints was analyzed using a generalized, four-parameter dynamic model of the peptide motion, including tilt and rotation angle and two associated order parameters. A tilt angle of 21 degrees was determined for WALP23 in dimyristoylphosphatidylcholine, which is much larger than the tilt angle of 5.5 degrees previously determined from (2)H NMR experiments. This approach provided a realistic value for the tilt angle of WALP23 peptide in the presence of hydrophobic mismatch, and can be applied to any transmembrane helical peptide. The influence of the experimental data set on the solution space is discussed, as are potential sources of error.

BackgroundOne strategy to increase the stability of proteins is to reduce the area of water-accessible hydrophobic surface.ResultsIn order to test it, we replaced 14 solvent-exposed hydrophobic residues of acetylcholinesterase by arginine. The stabilities of the resulting proteins were tested using denaturation by high temperature, organic solvents, urea and by proteolytic digestion.ConclusionAltough the mutational effects were rather small, this strategy proved to be successful since half of the mutants showed an increased stability. This stability may originate from the suppression of unfavorable interactions of nonpolar residues with water or from addition of new hydrogen bonds with the solvent. Other mechanisms may also contribute to the increased stability observed with some mutants. For example, introduction of a charge at the surface of the protein may provide a new coulombic interaction on the protein surface.

There is rising evidence that cancer development is associated from its earliest stages with DNA replication stress, a major source of spontaneous genomic instability. However, the origin of these replication defects has remained unclear. We have investigated the consequences of upregulating error-prone DNA polymerases (pol) beta and kappa on chromosomal DNA replication. These enzymes are misregulated in different types of cancers and induce major chromosomal instabilities when overexpressed at low levels. Here, we have used DNA combing to show that a moderate overexpression of pol beta or pol kappa is sufficient to impede replication fork progression and to promote the activation of additional replication origins. Interestingly, alterations of the normal replication program induced by excess error-prone polymerases were not detected by the replication checkpoint. We therefore propose that upregulation of error-prone DNA polymerases induces a checkpoint-blind replication stress that contributes to genomic instability and to cancer development.