Fast Rotational Motion Research Articles

Fluorescence depolarization study of lamellar liquid crystalline to inverted cylindrical micellar phase transition of phosphatidylethanolamine

The orientational order and rotational dynamics of 2-[3-(diphenyl-hexatrienyl) propanoyl]-3-palmitoyl-L-alpha- phosphatidylcholine (DPH-PC) embedded in dioleoylphosphatidyl-ethanolamine (DOPE) were studied by fluorescence depolarization technique. Upon increasing the temperature, the calculated wobbling diffusion constant D perpendicular of the fluorescent probe was found to decrease at the lamellar (L alpha) to inverted cylindrical (H II) phase transition (10 degrees C). This suggested that the increased gauche rotamers of the alkene chains in the HII phase imposes a constraint in the wobbling motion of the fluorophore. The calculated ratio of order parameter in the L alpha phase to that in the HII phase was 1.7 and different from the theoretical value of 2.0 as predicted from the change in packing symmetry. This result can be explained by a slightly higher local order parameter of the fluorophore or by the fast rotational diffusion motion of the fluorophore around the symmetry axis of the cylindrical tubes in the HII phase.

Molecular dynamics of vinylidenefluoride-trifluoroethylene copolymers

We have studied the molecular dynamics of the vinylidenefluoride-trifluoroethylene (VF 2/F 3E) ferroelectric copolymers with 60/40 and 80/20 molar compositions by incoherent quasielastic neutron scattering. In the ferroelectric phase we did not observe any motion whereas in the paraelectric and liquid phase a fast rotational motion and slow diffusion were detected.

Conformational change in thrombospondin induced by removal of bound Ca 2+ A spin label approach

The effect of removal of Ca 2+ bound to thrombospondin (TSP) on the protein structure in solution has been investigated using ESR spin-label techniques. A maleimide spin label was selectively attached to the free thiol group presumably near the carboxyl-terminal domain in which Ca 2+-binding sites are situated. The ESR spectra of spin-labeled TSP showed that the bound label undergoes a relatively fast rotational motion with an effective rotational correlation time in the nanosecond time regimes. Removal of bound Ca 2+ in TSP by dialyzing spin-labeled TSP from a Ca 2+-containing buffer into an EDTA-containing buffer resulted in an increase in the mobility of the bound label by a factor of 2.3. The data suggest that EDTA chelation of bound Ca 2+ in TSP induces a conformational change of TSP at least near the site of spin labeling.

Conformational flexibility of neurophysin as investigated by local motions of fluorophores. Relationships with neurohypophyseal hormone binding

Flexibility of various structural domains of neurophysin and neurophysin-neurohypophyseal hormone complexes has been investigated through the fast rotational motion of fluorophores in highly viscous medium. Despite seven intrachain disulfide links, it is shown that some domains of neurophysin remain highly flexible. Dimerization of neurophysin does not affect the structural integrity of the individual subunits, each subdomain being conformationally equivalent within each protomer of the unliganded dimer. The absence of heterogeneous fluorescence anisotropy precludes the existence of a dimer tautomerization equilibrium. Binding of the hormonal ligands to neurophysin dimer promotes a large conformational change over the whole protein structure as assessed by differential alterations of the flexibility-rigidity and intrasegmental interaction properties of domains that do not participate directly to the dimerization/binding areas. The order of free-energy coupling between ligand binding and protein subunit association has been evaluated. Data are consistent with a model in which the first mole of bound ligand stabilizes the dimer by increasing the intersubunit contacts while the second mole of ligand induces most of the described conformational change. Accordingly, the positive cooperativity between the two dimeric binding sites is linked mainly to the binding of the second ligand. The induced structural change is perceived differently by each subunit as assessed by opposite local motions of Tyr49 in each liganded protomer and leads to the formation of a dimeric complex with a global pseudospherical symmetry although containing domains of local asymmetry.

Phase transitions in phosphatidylcholine multibilayers.

The (2)H NMR spectrum of a multilamellar dispersion of 1-myristoyl-2-[14,14,14-(2)H(3)]myristoyl-sn-glycero-3-phosphocholine with 1 mol% cholesterol in excess water has been recorded at temperatures between -15 degrees C and 36 degrees C. Motionally averaged quadrupole coupling constants nu(Q) and motionally induced asymmetry parameters eta are obtained by spectral analysis. Values of these quantities indicate that, at temperatures below -4 degrees C, any rotational motion of the molecules about their molecular long axis is slow on the NMR time scale. At temperatures immediately above the pretransition these same parameters show that a fast-rotational motion is occurring about the molecular long axis. This rotational motion is hindered in that the molecules flip about a twofold symmetry axis. Between -4 degrees C and the pretransition, spectra appear as the superposition of two powder patterns, one corresponding to the pattern observed below -4 degrees C and the other to the pattern above the pretransition. The relative contribution of the latter increases with temperature until the pretransition is reached. These data have been interpreted in two ways: either the sample between -4 degrees C and the pretransition contains two populations of rapidly and slowly rotating molecules, or there is only a single population of molecules undergoing a 180 degrees flipping motion on the time scale of the NMR measurement. The latter interpretation is more consistent with other experimental findings. At the temperature of the main transition the hydrocarbon chains melt. In the absence of cholesterol, spectra are more complex in that the line shape is reproduced by the superposition of three spectral powder patterns between -4 degrees C and the pretransition and by the superposition of two spectral patterns above the pretransition. It is postulated that these two patterns observed above the pretransition are in direct correspondence to the two ripple structures observed by freeze-fracture electron microscopy in the absence of cholesterol.

The measurement of subnanosecond fluorescence decay of flavins using time-correlated photon counting and a mode-locked Ar ion laser

A system is described consisting of a mode-locked Ar ion laser and time-resolved photon-counting electronics. The system is capable of measuring fluorescence lifetimes in the subnanosecond time domain. The Ar ion laser is suitable for the excitation of flavins, since the available laser wavelengths encompass the first absorption band of the yellow chromophore. Due to the high radiation density and the short pulse, both the time and wavelength resolution of the fluorescence of very weakly emitting compounds can be measured. Experiments have been described for flavin models exhibiting single and multiple modes of decay. In these examples lifetimes were determined both from deconvolved decay curves and from direct analysis of the tail of the curve, where no interference of the exciting pulse is encountered. Both determinations showed very good agreement. Due to the highly polarized laser light the decay of the emission anisotropy could be measured directly after the exciting pulse. In principle, fast rotational motions might be detected. An anisotropy measurement conducted with a flavoprotein with a noncovalently attached FAD is presented.