Investigating magnetically aligned phospholipid bilayers with EPR spectroscopy at 94 GHz.

In this paper, we report our initial results on studying magnetically aligned phospholipid bilayers (bicelles) at high magnetic fields (approximately 3.4 T) with electron paramagnetic resonance (EPR) spectroscopy at 95 GHz (W-band). In order to characterize this system for W-band EPR studies, we have utilized the nitroxide spin probe 3beta-doxyl-5alpha-cholestane to demonstrate the effects of macroscopic bilayer alignment. At W-band due to the increase in magnetic field strength (when compared to X-band studies at 9.5 GHz) (S. M. Garber et al., J. Am. Chem. Soc. 121, 3240-3241 (1999)), we were able to examine magnetically aligned phospholipid bilayers at two orientations with the bilayer normal oriented either perpendicular or parallel (upon addition of YbCl3) with respect to the direction of the static magnetic field. Additionally, at a magnetic field of 3.4 T (g=2 resonance at W-band), we were able to study the parallel alignment with a lower concentration of Yb3+, thereby eliminating the possible unwanted effects associated with lanthanide-protein interactions and paramagnetic shifts and/or line broadening induced by the lanthanide ions. The development of this new spin label alignment technique will open up a whole new area of investigation for phospholipid bilayer systems and membrane protein EPR studies at high magnetic fields.

Nuclear magnetic resonance study of doxorubicin binding to cardiolipin containing magnetically oriented phospholipid bilayers.

Doxorubicin (DOX) is a potent anthracycline cancer drug whose interaction with anionic membrane phospholipids, such as cardiolipin (CL), is thought to contribute to its cytotoxicity as well as induce cardiotoxic side effects. We have studied the interaction of DOX with a CL containing model membrane system using high resolution, oriented sample (31)P and (2)H NMR. The model membrane system is composed of a bilayer forming phospholipid and a detergent that breaks the extended bilayers into disc-shaped micelles (bicelles) that can orient in a magnetic field. The effects of DOX on the phospholipid bilayer were monitored using samples containing dimyristoylphosphatidylcholine (DMPC), selectively deuterated in either headgroup or acyl chain positions, and measuring the changes in (2)H quadrupolar splittings as DOX was added. Changes in quadrupolar splittings upon DOX addition provide evidence for interaction with both surface and buried sites within the membrane bilayer.

Pegylation of magnetically oriented lipid bilayers.

We report NMR data for magnetically oriented phospholipid bilayers which have been doped with a lipid derivatized with a polyethylene glycol polymer headgroup to stabilize samples against aggregation. (13)C, (31)P, and (2)H NMR data indicate that the incorporation of PEG2000-PE (1% molar to DMPC) does not interfere with the orientation properties of bicelles prepared at 25% w/v with or without the presence of lanthanide. Bicelles prepared at 10% w/v are also shown to orient when PEG2000-PE is added. The addition of PEG2000-PE to cholesterol-containing, lanthanide-flipped bicelles is shown to inhibit sample phase separation and improve spectral quality. Furthermore, the addition of PEG2000-PE to high w/v bicelles (40% w/v) is demonstrated to lead to an increase in overall sample order.

Conformation of sulfoquinovosyldiacylglycerol bound to a magnetically oriented membrane system.

The conformation of uniformly 13C-labeled sulfoquinovosyldiacylglycerol (SQDG) is studied in both membrane and solution environments using NMR spectroscopy. Analysis in a membrane-like environment is based on the measurement of dipolar interactions between 13C-13C and 1H-13C spin pairs and on the measurement of 13C chemical shift anisotropy offsets, which appear in magnetically oriented phospholipid-based membrane fragments. Potential energy maps for glycosidic torsions, phi, psi and theta 1, are calculated with a membrane interaction energy and are used in the interpretation of experimental data. The membrane-bound description for SQDG is most consistent with a set of low-energy conformations that extends the headgroup of SQDG away from the membrane surface. Analysis of the conformation of SQDG in CD3OD solution is based on measured 3JCH scalar couplings. The description of the solution conformation is modeled as a mixture of low-energy conformers predicted in the absence of a membrane interaction term and involves more extensive motional averaging than the model for SQDG embedded in the lipid matrix.

Electrostatic forces contribute to interactions between trp repressor dimers.

The trp repressor of Escherichia coli (TR), although generally considered to be dimeric, has been shown by fluorescence anisotropy of extrinsically labeled protein to undergo oligomerization in solution at protein concentrations in the micromolar range (Fernando, T., and C. A. Royer 1992. Biochemistry. 31:3429-3441). Providing evidence that oligomerization is an intrinsic property of TR, the present studies using chemical cross-linking, analytical ultracentrifugation, and molecular sieve chromatography demonstrate that unmodified TR dimers form higher order aggregates. Tetramers and higher order species were observed in chemical cross-linking experiments at concentrations between 1 and 40 microM. Results from analytical ultracentrifugation and gel filtration chromatography were consistent with average molecular weight values between tetramer and dimer, although no plateaus in the association were evident over the concentration ranges studied, indicating that higher order species are populated. Analytical ultracentrifugation data in presence of corepressor imply that corepressor binding destabilizes the higher order aggregates, an observation that is consistent with the earlier fluorescence work. Through the investigation of the salt and pH dependence of oligomerization, the present studies have revealed an electrostatic component to the interactions between TR dimers.

Torsion angle analysis of glycolipid order at membrane surfaces.

A method is presented for determining the average glycosidic torsion angles and motion about those angles for a glycolipid headgroup at a model membrane surface. Dipolar and quadrupolar coupling constants were previously collected on the headgroup of beta-dodecyl glucoside embedded in phospholipid/detergent bilayers which orient in a magnetic field (Sanders, C.R., and J.H. Prestegard. 1991. J. Am. Chem. Soc. 113:1987-1996). These observables are expressed as averages of second order spherical harmonics, and Wigner rotation matrices are used here to transform the spherical harmonics from the laboratory frame to a set of frames which allow motional averaging to be described as the result of simple bond rotations. Euler angles corresponding to rotations about glycosidic torsion angles phi and psi are chosen to best reproduce experimental coupling constants, using models which have varying degrees of motional averaging. These models include a rigid headgroup, axially symmetric headgroup motion, and independent motion about each torsion angle in a square well potential. The square well model proves to be significantly better than the rigid model in reproducing experimental observations and it offers a more physically meaningful description of motion than the axially symmetric model. The structures obtained, assuming a square well potential, are compared to potential energy maps for the glycolipid torsional angles to illustrate the need for inclusion of the membrane interface in energetic modeling of glycolipid conformations.

Computer modelling of glycolipids at membrane surfaces.

Interactions of membrane anchored molecules such as glycolipids with a membrane surface are important in determining headgroup conformation. It is therefore essential to represent these membrane surface interactions in molecular modeling studies of glycolipids and other membrane bound molecules. We introduce here an energy term that represents the interaction of molecules with a membrane bilayer. This membrane interaction energy term has been added to the potential energy function of a molecular dynamics and mechanics program and has been parameterized using partition coefficients between an aqueous solution and a vesicular membrane for two model glycolipids.

Choosing to do obstetrics in practice: factors affecting the decisions of third-year family practice residents.

To investigate factors influencing a resident's decision to participate in obstetrical care, a survey was undertaken of program directors and third-year residents from the 16 Michigan family practice residency programs. Responses indicated that the majority of third-year residents (55%) intended to practice obstetrics on graduation. Rural practice plans were significantly correlated with a decision to practice obstetrics, but suburban practice plans were negatively correlated. Residents who reported the presence of a good family practice obstetric role model were significantly more likely to practice obstetrics; residents deciding against obstetrical practice expressed concerns about legal liability and malpractice fees. These findings have important implications for family practice training programs if obstetrics is to remain a central part of comprehensive family care.