Marise Ouellet

Interaction of the Neuropeptide Met-Enkephalin with Zwitterionic and Negatively Charged Bicelles as Viewed by 31P and 2H Solid-State NMR

The interaction of the neuropeptide methionine-enkephalin (Menk) with bicelles was investigated by solid-state NMR. Bicelles composed of dimyristoylphosphatidylcholine (DMPC) and dicaproylphosphatidylcholine (DCPC) were modified to investigate the effect of the lipid headgroup and electrostatic charges on the association with Menk. A total of 10 mol % of DMPC was replaced by zwitterionic phosphatidylethanolamine (DMPE), anionic phosphatidylglycerol (DMPG), or phosphatidylserine (DMPS). The preparation of DMPE-doped bicelles (Bic/PE) is reported for the first time. The (31)P and (2)H NMR results revealed changes in the lipid dynamics when Menk interacts with the bicellar systems. (2)H NMR experiments showed a disordering effect of Menk on the lipid chains in all the bicelles except Bic/PG, whereas the study of the choline headgroups indicated a decreased order of the lipids only in Bic/PE and Bic/PG. Our results suggest that the insertion depth of Menk into bicelles is modulated by their composition, more specifically by the balance between hydrophobic and electrostatic interactions. Menk would be buried at the lipid polar/apolar interface, the depth of penetration into the hydrophobic membrane core following the scaling Bic > Bic/PE > Bic/PS at the slightly acidic pH used in this study. The peptide would not insert into the bilayer core of Bic/PG and would rather remain at the surface.

Membrane interactions and dynamics of a 21-mer cytotoxic peptide: a solid-state NMR study.

We have investigated the membrane interactions and dynamics of a 21-mer cytotoxic model peptide that acts as an ion channel by solid-state NMR spectroscopy. To shed light on its mechanism of membrane perturbation, (31)P and (2)H NMR experiments were performed on 21-mer peptide-containing bicelles. (31)P NMR results indicate that the 21-mer peptide stabilizes the bicelle structure and orientation in the magnetic field and perturbs the lipid polar head group conformation. On the other hand, (2)H NMR spectra reveal that the 21-mer peptide orders the lipid acyl chains upon binding. (15)N NMR experiments performed in DMPC bilayers stacked between glass plates also reveal that the 21-mer peptide remains at the bilayer surface. (15)N NMR experiments in perpendicular DMPC bicelles indicate that the 21-mer peptide does not show a circular orientational distribution in the bicelle planar region. Finally, (13)C NMR experiments were used to study the 21-mer peptide dynamics in DMPC multilamellar vesicles. By analyzing the (13)CO spinning sidebands, the results show that the 21-mer peptide is immobilized upon membrane binding. In light of these results, we propose a model of membrane interaction for the 21-mer peptide where it lies at the bilayer surface and perturbs the lipid head group conformation.

Chapter 1 - Structure and Membrane Interactions of Antimicrobial Peptides as Viewed by Solid-State NMR Spectroscopy

Abstract Solid-state NMR spectroscopy is a well-suited technique to study the membrane interactions of antimicrobial peptides by taking advantage of the orientational dependence of nuclear spin interactions. This paper discusses several solid-state NMR experiments to extract information on the peptide structure and dynamics as well as on the effect of antimicrobial peptides on model membranes. More specifically, studies of peptide dynamics by 13C and 15N CP MAS and static experiments are reported. Also, the peptide orientation and location in membranes can be extracted from 15N 1D NMR spectra and spin diffusion NMR, whereas PISEMA experiments that correlate 15N chemical shifts and 15N–1H dipolar couplings allow the complete determination of membrane topology by specifying the peptide orientation and tilt angle. In addition, examples of peptide structure determination by isotropic chemical shifts, internuclear distance and torsion angle measurements are described. Finally, 31P NMR and 2H NMR experiments are commonly used to obtain information on both the polar region and the hydrophobic core of phospholipid bilayers. 31P NMR spectra reflect the nature of lipid phases and the conformation of the phospholipid polar headgroup, whereas 2H NMR spectra are indicative of acyl chain orientational order.

Tonal distinctions between emphatic stress and pretonic lengthening in Quebec French

The study compares the tonal structures of stressed penultimate syllables in Quebec French and emphatic stress in the same spoken variety. Two main experiments have been conducted: the first was designed to highlight the tonal characteristics of emphatic stress in read and spontaneous speech. The second was concerned with the phonetic and tonal description of stressed, penultimate syllables as a result of a possible stress shift. Our results do not confirm the common assumption that penultimate, stressed syllables in Quebec French are the result of emphatic prominence. Emphatic stress is characterized by a LH tone on the target syllable followed by a more or less abrupt fall covering a particular domain depending on the speech style. By contrast, penultimate stressed syllables are characterized by a falling F0 modulation covering the lengthened syllable. The hypothesis that the tonal anchoring happens on the penultimate syllable could explain the variety of tonal patterns observed on the final syllables in those precise cases.

From segmental duration properties to rhythmic structure: a study of interactions between high and low level constraints

The study deals with the temporal structure of utterances in a variety of Canadian French spoken in the province of Quebec. In order to illustrate the way segment intrinsic durations could influence the temporal structure on which prosody takes place in Quebec French, data from read utterances and spontaneous speech were gathered and compared to results from a previous study on the variety spoken in France. The observations confirmed the preservation of a length distinction in spontaneous speech in Quebec French and showed how segmental features are apt to change the durational frame of a stress group. By so doing, the results brought up the question of the merits of using general claims about French stress patterning, segmental phonology, and syllable division for the study of Canadian French prosody. We demonstrated that the stress group, instead of the lexical units, can be used to capture segment intrinsic duration properties.