Determination of the orientation and dynamics of ergosterol in model membranes using uniform 13C labeling and dynamically averaged 13C Chemical Shift Anisotropies as experimental restraints
Olivier Soubias 1, Franck Jolibois 1, Stéphane Massou 1, Alain Milon 1 and Valérie Reat 1*
1 Université Paul Sabatier - CNRS
* To whom correspondence should be addressed. E-mail: reat{at}ipbs.fr.
Submitted on February 7, 2005
Revised on April 6, 2005
Accepted on 19 May 2005
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Abstract |
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A new strategy was established to determine the average orientation and dynamics of ergosterol in DMPC model membranes. It is based on the analysis of CSAs averaged by the molecular dynamics. Static 13C CSA tensors were computed by quantum chemistry, using GIAO approach within HF theory. Uniformly 13C labeled ergosterol was purified from P. Pastoris cells grown on labeled methanol. After reconstitution into DMPC lipids, the complete 1H and 13C assignment of ergosterol's resonances was performed using a combination of MAS 2D experiments. Dynamically averaged CSA's were determined by standard side band intensity analysis for isolated 13C resonances (C3 and ethylenic carbons) and by OMAS experiments for other carbons. A set of 18 constraints was thus obtained, from which the sterol's molecular order parameter and average orientation could be precisely defined. The validity of using computed CSAs in this strategy was verified on cholesterol model systems. This new method allowed us to quantify ergosterol's dynamics at three molar ratio: 16 mol% (Ld phase), 30 mol% (Lo phase) and 23 mol% (mixed phases). Contrary to cholesterol, ergosterol's molecular diffusion axis makes an important angle with the inertial axis of the rigid four ring system (14°).
Key Words:
DMPC, chemical shift anisotropy, magic angle spinning, quantum Chemistry, solid state NMR, sterols
