Daniel Genest

Pulse fluorimetry of 1,6-diphenyl-1,3,5-hexatriene incorporated in membranes of mouse leukemic L 1210 cells.

During the past few years, a very large number of investigations was devoted to the dynamics proper- ties of cell membranes. Dynamic studies of lipid bilayers were done either by the use of spin label or fluo- rescence depolarization techniques. Amongst various fluorescent probes, 1,6-diphenyl-1,3,5-hexatriene [1] was the most widely used. DPH is easily introduced into membrane bilayers from aqueous dispersions, in which it is not fluorescent [2]. On the basis of phase fluorometric measurements in a homogeneous refer- ence oil, this probe was reported to exhibit a single exponential decay of the emission and a linear Perrin plot [3] e.g., fluorescence intensity and lifetime are in a constant ratio when temperature changes [4]. These results were confirmed by direct measurements of the decay of both intensity and emission anisotropy of DPH in a different oil, using a pulse fluorimeter [5]. However, very recently, nanosecond pulse fluo- rescence studies of DPH incorporated into L-a- dimyristoyl lecithin vesicles [6] and into DL-a- dipalrnitoylphosphatidyl choline vesicles [7] showed that the decay of the total fluorescent emission and the decay of the emission anisotropy could not be described adequately in terms of single exponential decay laws. Therefore, when DPH is embedded in vesicles, the Perrin relationship is no longer valid, and steady state polarization measurements cannot be interpreted in terms of microviscosity. The complex decay could be interpreted either in terms of microheterogeneity of sites for the probe in + To whom correspondence should be

Detailed description of an alpha helix-->pi bulge transition detected by molecular dynamics simulations of the p185c-erbB2 V659G transmembrane domain.

Molecular dynamics simulations of a 29-residue peptide including the transmembrane domain of the V659G mutant of the c-erbB2 protein demonstrate important dynamical behavior. Although the alpha helix is the structure commonly assumed for transmembrane hydrophobic segments, we found that hydrogen bond rearrangements can occur, giving rise to a structural deformation termed pi bulge stabilized by successive hydrogen bonds of pi helix type. A series of simulations enables us to give a detailed description, at the atomic level, of the alpha helix->pi bulge transition. The major consequence of this deformation covering one and a half turn of helix results in a noticeable shift around the helix axis of the C-Terminal residues relatively to those of the N-terminus. Such a deformation closely related to structural motifs described in the literature, induces a change in the distribution of the residues along the helix faces which could modulate the protein activity mediated by a dimerization process.

Fluorescence anisotropy decay due to rotational brownian motion of ethidium intercalated in double strand DNA

The transient fluorescence of solutions of ethidium bromide . DNA complexes has been measured by pulse fluorimetry at different temperatures and in solvents containing various amounts of sucrose. The molar ratio of ethidium to nucleotides was low. Under these conditions the anisotropy decay was due to the Brownian motion of ethidium molecules intercalated in the double strand DNA molecules. This anisotropy decay could be described by a sum of 3 exponential terms, with correlation times 01, 02, 03 which were linear functions of the ratio of the solvent viscosity to the absolute temperature (n/T). The amplitude of the exponential term characterized by the shortest correlation time (01) has been found to depend on temperature while the ratio of the amplitude of the two other terms (characterized by 02 and 03) was independent of temperature. These results were interpreted as follows: 01 corresponds to a fast motion of the dye in its site. 02 and 03 describe a tortional motion of the ethidium bromide. DNA complex, involving several nucleotide pairs.

Molecular modeling of c‐erbB2 receptor dimerization: Coiled‐coil structure of wild and oncogenic transmembrane domains—Stabilization by interhelical hydrogen bonds in the oncogenic form

Dimerization models of c-erbB2 transmembrane domains (Leu651-Ile675) are studied by molecular mechanics and molecular dynamics simulations. Both wild and Glu mutated transmembrane helices exhibit the same relative orientation for favorable associations and dimerize preferentially in left-handed coiled-coil structures. The mutation point 659 belongs to the interfacing residues, and in the transforming domain, symmetric hydrogen bonds between Glu carboxylic groups stabilize the dimeric structure. The same helix packing found for the wild dimers, except side-chain-side-chain hydrogen bonds, suggests that the transmembrane domains dimerize according to similar process. Structural and energetical characterization of the models are presented.

Molecular Dynamics Study of the Base Pair Opening Process in the Self-Complementary Octanucleotide d(CTGATCAG)

We report an analysis of a 200 ps Molecular Dynamics simulation of the double stranded oligonucleotide d(CTGATCAG) in the presence of 1534 water molecules and 14 Na+ ions. We focus on the opening process of Thymine 5, by analyzing in detail the glycosidic bond rotational motion about the helix axis. The present analysis is mainly based on autocorrelation functions and on mean square displacements. We show that the opening of the base has a Brownian character and we find a rotational diffusion coefficient of 4.7 rad2s-1. Furthermore we estimate the DNA torsional constant to be about 0.5 10(-18) J.rad-2 and the RMS of the angular displacement to be 8.3 degrees. All these values are in fair agreement with those determined experimentally by fluorescence polarization of DNA-Ethidium bromide complexes. This shows that the rotational motions of the bases detected in the range 10(-9)-10(-7) s. by fluorescence techniques are the same as those analyzed in the present study (10(-12)-2 10(-10) s).

Étude des transferts d'énergie dans le complexe DNA-bromure d'éthidium au moyen du déclin de l'anisotropie de fluorescence

In a recent investigation (Ph. Wahl et al., Proc. Natl. Acad. Sci. U.S., 65 (1971) 417), the decay of fluorescence anisotropy of the ethidium bromide-DNA complex has been studied for high values of PD (ratio of molar concentrations of DNA and bound ethidium bromide). Evidence for a brownian motion of oscillation has been established, the angular amplitude of which is 35°. In the present work we study the decay of anisotropy for different values of PD. It is observed that the slope of this decay is increased when PD decreases. We attributed this phenomenon, to the existence of energy transfers between molecules of ethidium bromide bound to the same molecule of DNA. To describe quantitatively this behaviour, we try to use Jablonskys and Forsters theory. The expression of anisotropy decay depends, in particular, on the angular distribution of the molecules. We have considered successively an isotropic distribution, and a distribution in which chromophores have a direction perpendicular to the DNA molecule axis. This last distribution corresponds to the generally admitted hypothesis of intercalation of ethidium bromide between the DNA base pairs. The best fit has been obtained using Forsters theory with the chromophores perpendicular to the axis of DNA. The fit is good in the range of small concentrations of ethidium bromide. For higher concentrations the fit is good only in the initial part of the decay curve. The deviation can be explained by the fact that only transfers between two molecules are considered in the theory. No good fit can be obtained with the Forsters formula corresponding to an isotropic distribution of the chromophores. Furthermore, the shapes of the decay curves predicted by Jablonskys theory differ markedly from the experimental curves. Our measurements suggest strongly, for the first time, that the decay of fluorescence anisotropy of a solution of chromophores is better described by a Forster type theory than by the Jablonsky theory.

Influence of a Mutation in the Transmembrane Domain of the pl85c-erbB2 Oncogene-Encoded Protein Studied by Molecular Dynamics Simulations

The c-erbB2 proto-oncogene encodes for a protein of 185kDa (p185) which becomes transforming upon the Val-->Glu transmembrane amino acid substitution. The transforming ability seems to be due to a substitution-resulting constitutive activation of the tyrosine kinase cytosolic domain of the protein. These observations prompted us to evaluate the structural and dynamical behavior of the transmembrane region of the wild and transforming p185 protein in order to understand the role of this region in the transduction mechanism. 160 ps molecular dynamics simulations in vacuo have been performed on two peptides corresponding to the sequence [651-679] of p185c-erbB2 protein and its transforming mutant Val659-->Glu659. These two sequences include the transmembrane domain and are initially postulated to be in an alpha-helix conformation. Noticeable differences in the flexibility of the two peptides are shown. The nontransforming sequence seems rather flexible and several conformational changes are detected at the junction of the mutation point [658-659] and at position Val665-Val666 during the 160 ps simulations. On the contrary, no transitions were observed for the mutated sequence which adopts a stable alpha-helix conformation. This difference in flexibility could be hypothesized as a factor involved in the regulation of the tyrosine kinase activity of p185c-erbB2.

Canonical analysis of correlated atomic motions in DNA from molecular dynamics simulation

We report a method for analyzing atomic correlated motions in biopolymers from trajectories obtained by molecular dynamics simulation. A correlation coefficient based on the canonical analysis of data is defined which is independent on the relative orientation of atomic displacement. To illustrate the method we studied correlation between positional fluctuations of protons in the double-stranded self complementary oligonucleotide d(CTGAT-CAG), deduced from a 200 ps molecular dynamics simulation in the presence of explicit water molecules and counterions. It is found that on this time scale the motions of protons belonging to different residues are poorly coupled while the motion of a base proton is correlated to the motion of the sugar ring protons of the same nucleotide. Such a method may be generalized to study correlated motions of two distinct domains of a macro-molecule.

Rigid-Body Motions of Sub-Units in DNA: A Correlation Analysis of a 200 ps Molecular Dynamics Simulation

A 200 ps molecular dynamics simulation of the B-form double stranded self-complementary octanucleotide d(CTGATCAG) is analyzed in terms of correlated motions using the canonical analysis approach. Each nucleotide is decomposed in three sub-units corresponding to the base, the sugar ring and the backbone respectively. The correlation between the full dynamics of two sub-units was found to decrease as their mutual distance increases. The interpretation of the full dynamics of sub-units as the superimposition of rigid-body motions (translation and orientation) and deformation shows that the main source of correlation is rigid-body motions. Correlation between sub-units deformation is weak and practically vanishes for sub-units belonging to non-adjacent nucleotides. It is also shown that the correlation is much more important for sub-units of the same strand than of opposite strands. We conclude that the internal dynamics of the octanucleotide may be well described by rigid-body motions, the sub-units deformation having only local influence whereas sub-units translation and rotation have repercussion to long distances. The results presented in this study suggest how the number of degrees of freedom may be reduced for simulating long-time dynamics of oligonucleotides.

How long does DNA keep the memory of its conformation? A time-dependent canonical correlation analysis of molecular dynamics simulation.

The time dependence of the correlation between motions of different parts of DNA is analyzed from a 200 ps molecular dynamics simulation of the double-stranded self-complementary d(CTGATCAG) in the B form. Each nucleotide is decomposed into three subunits corresponding to the furanose ring (SU), the base (BA), and the backbone (SK). The motion of each subunit is considered as the superimposition of rigid body translation, rigid body rotation, and internal deformation. Canonical time-dependent correlation functions calculated with coordinates describing the different components of the subunits motion are defined and computed. This allows us to probe how long a particular type of motion of one subunit influences the other types of motions of other subunits (cross correlation functions) or how long a particular subunit keeps the memory of its own conformation or location (autocorrelation functions). From autocorrelation analysis it is found that deformation decorrelates within a few tenths of picoseconds, rotational correlation times are on the order of 8 ps, while translational motions are long-time correlated. The deformation of a subunit is not correlated to the deformation of another one (at the 200 ps time scale of our simulation), but influences slightly their translation and orientation as time increases.