title: Synthesis, purification, and characterization of single helix membrane peptides and proteins for NMR spectroscopy.
abstract: Membrane proteins function as receptors, channels, transporters, and enzymes. These proteins are generally difficult to express and purify in a functional form due to the hydrophobic nature of their membrane spanning sequences. Studies on membrane proteins with a single membrane spanning helix have been particularly challenging. Single-pass membrane proteins will often form dimers or higher order oligomers in cell membranes as a result of sequence motifs that mediate specific transmembrane helix interactions. Understanding the structural basis for helix association provides insights into how these proteins function. Nevertheless, nonspecific association or aggregation of hydrophobic membrane spanning sequences can occur when isolated transmembrane domains are reconstituted into membrane bilayers or solubilized into detergent micelles for structural studies by solid-state or solution NMR spectroscopy. Here, we outline the methods used to synthesize, purify, and characterize single transmembrane segments for structural studies. Two synthetic strategies are discussed. The first strategy is to express hydrophobic peptides as protein chimera attached to the maltose binding protein. The second strategy is by direct chemical synthesis. Purification is carried out by several complementary chromatography methods. The peptides are solubilized in detergent for solution NMR studies or reconstituted into model membranes for solid-state NMR studies. We describe the methods used to characterize the reconstitution of these systems prior to NMR structural studies to establish if there is nonspecific aggregation.
authors.affiliation: Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY, USA.
authors.forename: Miki
authors.initials: M
authors.lastname: Itaya
authors.forename: Ian C
authors.initials: IC
authors.lastname: Brett
authors.forename: Steven O
authors.initials: SO
authors.lastname: Smith
mesh_terms: D002850:Chromatography, Gel; D002942:Circular Dichroism; D057927:Hydrophobic and Hydrophilic Interactions; D058619:Maltose-Binding Proteins / Q000378:metabolism; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010452:Peptide Biosynthesis; D010455:Peptides / Q000737:chemistry* / Q000302:isolation & purification*; D011487:Protein Conformation; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D058619:Maltose-Binding Proteins; D008565:Membrane Proteins; D010455:Peptides