Biomedical subjects

Catherine Royer

Publications and source records attributed to Catherine Royer.

8 recordsLinked to original sources

PubMed2006

Pressure perturbation calorimetric studies of the solvation properties and the thermal unfolding of proteins in solution--experiments and theoretical interpretation.

We used pressure perturbation calorimetry (PPC), a relatively new and efficient technique, to study the solvation and volumetric properties of amino acids and peptides as well as of proteins in their native and unfolded state. In PPC, the coefficient of thermal expansion of the partial volume of the protein is deduced from the heat consumed or produced after small isothermal pressure jumps, which strongly depends on the interaction of the protein with the solvent or cosolvent at the protein-solvent interface. Furthermore, the effects of various chaotropic and kosmotropic cosolvents on the volume and expansivity changes of proteins were measured over a wide concentration range with high precision. Depending on the type of cosolvent and its concentration, specific differences were found for the solvation properties and unfolding behaviour of the proteins, and the volume change upon unfolding may even change sign. To yield a molecular interpretation of the different terms contributing to the partial protein volume and its temperature dependence, and hence a better understanding of the PPC data, molecular dynamics computer simulations on SNase were also carried out and compared with the experimental data. The PPC studies introduced aim to obtain more insight into the basic thermodynamic properties of protein solvation and volume effects accompanying structural transformations of proteins in various cosolvents on one hand, as these form the basis for understanding their physiological functions and their use in drug designing and formulations, but also to initiate further valuable applications in studies of other biomolecular and chemical systems.

Calorimetry↗

PubMed2005

A high-throughput fluorescence polarization assay specific to the CD4 binding site of HIV-1 glycoproteins based on a fluorescein-labelled CD4 mimic.

The three-dimensional structure of CD4M33, a mimic of the host-cell receptor-antigen CD4 and a powerful inhibitor of CD4-gp120 (viral envelope glycoprotein 120) interaction and HIV-1 entry into cells [Martin, Stricher, Misse, Sironi, Pugniere, Barthe, Prado-Gotor, Freulon, Magne, Roumestand et al. (2003) Nat. Biotechnol. 21, 71-76], was solved by 1H-NMR and its structure was modelled in its complex with gp120. In this complex, CD4M33 binds in a CD4-like mode and inserts its unnatural and prominent Bip23 (biphenylalanine-23) side-chain into the gp120 interior 'Phe43 cavity', thus filling its volume. CD4M33 was specifically labelled with fluorescein and shown by fluorescence anisotropy to bind to different gp120 glycoproteins with dissociation constants in the nanomolar range. Fluorescent CD4M33 was also used in a miniaturized 384-well-plate assay to study direct binding to a large panel of gp120 glycoproteins and in a competition assay to study binding of CD4 or other ligands targeting the CD4 binding site of gp120. Furthermore, by using the fluorescently labelled CD4M33 and the [Phe23]M33 mutant, which possesses a natural Phe23 residue and thus cannot penetrate the gp120 Phe43 cavity, we show that a recently discovered small-molecule-entry inhibitor, BMS-378806, does not target the CD4 binding site nor the Phe43 cavity of gp120. The fluorescently labelled CD4M33 mimic, its mutants and their derivatives represent useful tools with which to discover new molecules which target the CD4 binding site and/or the Phe43 cavity of gp120 glycoproteins in a high-throughput fluorescence-polarization assay and to characterize their mechanism of action.

CD4 Antigens↗

PubMed2004

Structural basis for the co-activation of protein kinase B by T-cell leukemia-1 (TCL1) family proto-oncoproteins.

Chromosomal translocations leading to overexpression of p14(TCL1) and its homologue p13(MTCP1) are hallmarks of several human T-cell malignancies (1). p14(TCL1)/p13(MTCP1) co-activate protein kinase B (PKB, also named Akt) by binding to its pleckstrin homology (PH) domain, suggesting that p14(TCL1)/p13(MTCP1) induce T-cell leukemia by promoting anti-apoptotic signals via PKB (2, 3). Here we combined fluorescence anisotropy, NMR, and small angle x-ray-scattering measurements to determine the affinities, molecular interfaces, and low resolution structure of the complex formed between PKBbeta-PH and p14(TCL1)/p13(MTCP1). We show that p14(TCL1)/p13(MTCP1) target PKB-PH at a site that has not yet been observed in PH-protein interactions. Located opposite the phospholipid binding pocket and distal from known protein-protein interaction sites on PH domains, the binding of dimeric TCL1 proteins to this site would allow the crosslinking of two PKB molecules at the cellular membrane in a preactivated conformation without disrupting certain PH-ligand interactions. Thus this interaction could serve to strengthen membrane association, promote trans-phosphorylation, hinder deactivation of PKB, and involve PKB in a multi-protein complex, explaining the array of known effects of TCL1. The binding sites on both proteins present attractive drug targets against leukemia caused by TCL1 proteins.

Amino Acid Sequence↗

PubMed2003

Ligands differentially modulate the protein interactions of the human estrogen receptors alpha and beta.

The interactions of human estrogen receptor subtypes ERalpha and ERbeta with DNA and a 210 amino acid residue fragment of the coactivator protein SRC-1 bearing three nuclear receptor interaction motifs were investigated quantitatively using fluorescence anisotropy in the presence of agonist and antagonist ligands. ERalpha and ERbeta were found to bind in a similar manner to DNA, and both salt and temperature affected the affinity and/or stoichiometry of these interactions. The agonist ligands estradiol, estrone and estriol did not modify the binding of ERalpha to the fluorescein-labeled target estrogen response element. However, in the case of ERbeta, these ligands led to the formation of some higher-order protein-DNA complexes and a small decrease in affinity. The partial agonist 4-hydroxytamoxifen had little effect on either ER subtype, whereas the pure antagonist ICI 182,780 led to the cooperative formation of protein-DNA complexes of higher order than dimer, as further demonstrated by competition experiments and gel mobility-shift assays. In addition to DNA binding, the interaction of both ER subtypes with the Alexa488-labeled SRC-1 coactivator fragment was investigated by fluorescence anisotropy. The agonist ligands estrone, estradiol, estriol, genistein and ethynyl estradiol exhibited distinct capacities for inducing the recruitment of SRC-1 that were not correlated with their affinity for the receptor. Moreover, estrone and genistein exhibited subtype specificity in that they induced SRC-1 recruitment to ERbeta with much higher efficiency than in the case of ERalpha. The differential coactivator recruitment capacities of the ER agonists and their receptor subtype coactivator recruitment specificity may be linked to the molecular structure of the agonists with respect to their interactions with a specific histidine residue located at the back of the ligand-binding pocket. Altogether, these quantitative in vitro studies of ER interactions reveal the complex energetic and stoichiometric consequences of changes in the chemical structures of these proteins and their ligands.

DNA↗

PubMed2003

Time-resolved fluorescence anisotropy studies show domain-specific interactions of calmodulin with IQ target sequences of myosin V.

Single cysteine mutants of calmodulin, Cam(S38C) and Cam(N111C), have been specifically labelled with Alexa488 maleimide to study the effects of calcium on the structural dynamics of calmodulin complexed with IQ3, IQ4 and IQ34 target peptide motifs of mouse unconventional myosin-V. Using phase fluorometry, the time-resolved anisotropy shows well-separated global and segmental correlation times. The calcium-sensitive global motion of either calmodulin domain can be independently monitored in domain-specific interactions of either apo- or Ca(4).calmodulin with IQ3 or IQ4 peptides. C-domain interactions predominate, and apo-N-domain interactions are unexpectedly weak. The 1:1 complex of Ca(4).calmodulin with IQ34 behaves as a compact globular species. The results demonstrate novel dynamic aspects of calmodulin-IQ interactions relating to the calcium regulation of motility of unconventional myosin.

Amino Acid Sequence↗

PubMed2002

Effect of solvent diffusion on the apomyoglobin-water interface.

Few techniques can identify interactions between proteins and individual water molecules when the protein is in solution. The present work has sought to bridge the gap between the molecular level studies and the search for a physical property of the solution (bathing the proteins) that would regulate the protein hydration level. The properties of the solution were varied by adding nondenaturing solutes and solvents to the protein solutions and then studying their effect on the intrinsic fluorescence of apomyoglobin. The resolution of the tryptophan emission into the two component spectra corresponding to tryptophans W7 (accessible to the solvent) and W14 (buried in the protein matrix) has allowed us to probe two specific parts of the protein. Whereas W14 is not affected when the medium is altered, the analysis of W7 fluorescence has shown that cosolvent diffusion plays a dominant role in the mobility of water molecules near the protein surface.

Ampholyte Mixtures↗

PubMed2002

Equilibrium and pressure-jump relaxation studies of the conformational transitions of P13MTCP1.

The conformational transitions of a small oncogene product, p13(MTCP1), have been studied by high-pressure fluorescence of the intrinsic tryptophan emission and high-pressure 1D and 2D 1H-15N NMR. While the unfolding transition monitored by fluorescence is cooperative, two kinds of NMR spectral changes were observed, depending on the pressure range. Below approximately 200 MPa, pressure caused continuous, non-linear shifts of many of the 15N and 1H signals, suggesting the presence of an alternate folded conformer(s) in rapid equilibrium (tau<<ms) with the basic native structure. Above approximately 200 MPa, pressure caused a sharp decrease in the intensity of the folded proteins signals, while the peaks corresponding to disordered structures increased, yielding a free energy of unfolding change of 6.0 kcal/mol and associated volume change of -100 ml/mol, in agreement with the fluorescence result. Differential scanning calorimetry also reveals two transitions between 21 and 65 degrees C, confirming the existence of an additional species under mildly denaturing conditions. We report here a real-time observation of pressure-jump unfolding kinetics by 2D NMR spectroscopy on P13MTCP1 made possible due to its very long relaxation times at high pressure revealed by fluorescence studies. Within the dead-time after the pressure-jump, the NMR spectra of the native conformer changed to those of the transient conformational species, identified in the equilibrium studies, demonstrating the equivalence between a transient species and an equilibrium excited state. After these rapid spectral changes, the intensities of all of the individual 15N-1H cross-peaks decreased gradually, and those of the disordered structure increased, consistent with the slow relaxation to the unfolded form at this pressure. Rate constants of unfolding monitored at individual amide sites within the beta-barrel were similar to those obtained from fluorescence and from side-chain protons in the hydrophobic core region, consistent with nearly cooperative unfolding. However, some heterogeneity in the apparent unfolding rate constants is apparent across the sequence and can be understood as non-uniform effects of pressure on the unfolding rate constant due to non-uniform hydration.

Humans↗

PubMed2002

Equilibrium binding assays reveal the elevated stoichiometry and salt dependence of the interaction between full-length human sex-determining region on the Y chromosome (SRY) and DNA.

In an effort to better define the molecular mechanism of the functional specificity of human sex-determining region on the Y chromosome (SRY), we have carried out equilibrium binding assays to study the interaction of the full-length bacterial-expressed protein with a DNA response element derived from the CD3epsilon gene enhancer. These assays are based on the observation of the fluorescence anisotropy of a fluorescein moiety covalently bound to the target oligonucleotide. The low anisotropy value due to the fast tumbling of the free oligonucleotide in solution increases substantially upon binding the protein to the labeled target DNA. Our results indicate that the full-length human wild-type SRY (SRY(WT)) forms a complex of high stoichiometry with its target DNA. Moreover, we have demonstrated a strong salt dependence of both the affinity and specificity of the interaction. We have also addressed the DNA bending properties of full-length human SRY(WT) in solution by fluorescence resonance energy transfer and revealed that maximal bending is achieved with a protein to DNA ratio significantly higher than the classical 1:1. Oligomerization thus appears, at least in vitro, to be tightly coupled to SRY-DNA interactions. Alteration of protein-protein interactions observed for the mutant protein SRY(Y129N), identified in a patient presenting with 46,XY sex reversal, suggests that oligomerization may play an important role in vivo as well.

DNA↗