title: Temperature-resistant bicelles for structural studies by solid-state NMR spectroscopy.
abstract: Three-dimensional structure determination of membrane proteins is important to fully understand their biological functions. However, obtaining a high-resolution structure has been a major challenge mainly due to the difficulties in retaining the native folding and function of membrane proteins outside of the cellular membrane environment. These challenges are acute if the protein contains a large soluble domain, as it needs bulk water unlike the transmembrane domains of an integral membrane protein. For structural studies on such proteins either by nuclear magnetic resonance (NMR) spectroscopy or X-ray crystallography, bicelles have been demonstrated to be superior to conventional micelles, yet their temperature restrictions attributed to their thermal instabilities are a major disadvantage. Here, we report an approach to overcome this drawback through searching for an optimum combination of bicellar compositions. We demonstrate that bicelles composed of 1,2-didecanoyl-sn-glycero-3-phosphocholine (DDPC) and 1,2-diheptanoyl-sn-glycero-3-phosphocholin (DHepPC), without utilizing additional stabilizing chemicals, are quite stable and are resistant to temperature variations. These temperature-resistant bicelles have a robust bicellar phase and magnetic alignment over a broad range of temperatures, between -15 and 80 °C, retain the native structure of a membrane protein, and increase the sensitivity of solid-state NMR experiments performed at low temperatures. Advantages of two-dimensional separated-local field (SLF) solid-state NMR experiments at a low temperature are demonstrated on magnetically aligned bicelles containing an electron carrier membrane protein, cytochrome b5. Morphological information on different DDPC-based bicellar compositions, varying q ratio/size, and hydration levels obtained from (31)P NMR experiments in this study is also beneficial for a variety of biophysical and spectroscopic techniques, including solution NMR and magic-angle-spinning (MAS) NMR for a wide range of temperatures.
authors.affiliation: Department of Chemistry and Biophysics, University of Michigan , 930 N. University Ave., Ann Arbor, Michigan 48109-1055, United States.
authors.forename: Kazutoshi
authors.initials: K
authors.lastname: Yamamoto
authors.forename: Paige
authors.initials: P
authors.lastname: Pearcy
authors.forename: Dong-Kuk
authors.initials: DK
authors.lastname: Lee
authors.forename: Changsu
authors.initials: C
authors.lastname: Yu
authors.forename: Sang-Choul
authors.initials: SC
authors.lastname: Im
authors.forename: Lucy
authors.initials: L
authors.lastname: Waskell
authors.forename: Ayyalusamy
authors.initials: A
authors.lastname: Ramamoorthy
mesh_terms: D008565:Membrane Proteins / Q000737:chemistry*; D008823:Micelles; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011487:Protein Conformation; D013696:Temperature
chemical_list: D008565:Membrane Proteins; D008823:Micelles