Lisa A Della Ripa1, Zoe A Petros1, Alexander G Cioffi2, Dennis W Piehl2, Joseph M Courtney1, Martin D Burke3, Chad M Rienstra4. 1. Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA. 2. Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA. 3. Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA; Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA; Carle-Illinois College of Medicine, University of Illinois at Urbana-Champaign, Champaign, IL 61820, USA. Electronic address: mdburke@illinois.edu. 4. Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA; Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA; Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA. Electronic address: rienstra@illinois.edu.
Abstract
Cholesterol (Chol) is vital for cell function as it is essential to a myriad of biochemical and biophysical processes. The atomistic details of Chol's interactions with phospholipids and proteins is therefore of fundamental interest, and NMR offers unique opportunities to interrogate these properties at high resolution. Towards this end, here we describe approaches for examining the structure and dynamics of Chol in lipid bilayers using high levels of 13C enrichment in combination with magic-angle spinning (MAS) methods. We quantify the incorporation levels and demonstrate high sensitivity and resolution in 2D 13C-13C and 1H-13C spectra, enabling de novo assignments and site-resolved order parameter measurements obtained in a fraction of the time required for experiments with natural abundance sterols. We envision many potential future applications of these methods to study sterol interactions with drugs, lipids and proteins.
Cholesterol (n class="Chemical">Chol) is vital for cell function as it is essential to a myriad of biochemical and biophysical processes. The atomistic details of Chol's interactions with phospholipids and proteins is therefore of fundamental interest, and NMR offers unique opportunities to interrogate these properties at high resolution. Towards this end, here we describe approaches for examining the structure and dynamics of Chol in lipid bilayers using high levels of 13C enrichment in combination with magic-angle spinning (MAS) methods. We quantify the incorporation levels and demonstrate high sensitivity and resolution in 2D 13C-13C and 1H-13C spectra, enabling de novo assignments and site-resolved order parameter measurements obtained in a fraction of the time required for experiments with natural abundance sterols. We envision many potential future applications of these methods to study sterol interactions with drugs, lipids and proteins.
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