NMR of membrane proteins in micelles and bilayers: the FXYD family proteins.
Determining the atomic resolution structures of membrane proteins is of particular interest in contemporary structural biology. Helical membrane proteins constitute one-third of the expressed proteins encoded in a genome, many drugs have membrane-bound proteins as their receptors, and mutations in membrane proteins result in human diseases. Although integral membrane proteins provide daunting technical challenges for all methods of protein structure determination, nuclear magnetic resonance (NMR) spectroscopy can be an extremely versatile and powerful method for determining their structures and characterizing their dynamics, in lipid environments that closely mimic the cell membranes. Once milligram amounts of isotopically labeled protein are expressed and purified, micelle samples can be prepared for solution NMR analysis, and lipid bilayer samples can be prepared for solid-state NMR analysis. The two approaches are complementary and can provide detailed structural and dynamic information. This paper describes the steps for membrane protein structure determination using solution and solid-state NMR. The methods for protein expression and purification, sample preparation and NMR experiments are described and illustrated with examples from the FXYD proteins, a family of regulatory subunits of the Na,K-ATPase.
Franzin
Carla M
CM
The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA.
Gong
Xiao-Min
XM
Thai
Khang
K
Yu
Jinghua
J
Marassi
Francesca M
FM
D000595:Amino Acid Sequence; D002461:Cell Line, Transformed; D003902:Detergents; D015870:Gene Expression; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics; D008051:Lipid Bilayers / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D008823:Micelles; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000737:chemistry / Q000235:genetics; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics; D010759:Phosphorus Isotopes; D010957:Plasmids; D015221:Potassium Channels / Q000737:chemistry / Q000235:genetics; D017434:Protein Structure, Tertiary; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000235:genetics
D003902:Detergents; C484265:Fxyd3 protein, mouse; C094337:Fxyd4 protein, rat; D047908:Intracellular Signaling Peptides and Proteins; D008051:Lipid Bilayers; D008565:Membrane Proteins; D008823:Micelles; D009363:Neoplasm Proteins; D009587:Nitrogen Isotopes; D010750:Phosphoproteins; D010759:Phosphorus Isotopes; D015221:Potassium Channels; C069141:phospholemman; C416502:FXYD2 protein, human; D000254:Sodium-Potassium-Exchanging ATPase