A high-throughput differential filtration assay to screen and select detergents for membrane proteins.
Structural studies on integral membrane proteins are routinely performed on protein-detergent complexes (PDCs) consisting of purified protein solubilized in a particular detergent. Of all the membrane protein crystal structures solved to date, a subset of only four detergents has been used in more than half of these structures. Unfortunately, many membrane proteins are not well behaved in these four detergents and/or fail to yield well-diffracting crystals. Identification of detergents that maintain the solubility and stability of a membrane protein is a critical step and can be a lengthy and "protein-expensive" process. We have developed an assay that characterizes the stability and size of membrane proteins exchanged into a panel of 94 commercially available and chemically diverse detergents. This differential filtration assay (DFA), using a set of filtered microplates, requires sub-milligram quantities of purified protein and small quantities of detergents and other reagents and is performed in its entirety in several hours.
Vergis
James M
JM
Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA 22908, USA.
Purdy
Michael D
MD
Wiener
Michael C
MC
D020346:Aquaporins / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry; D003460:Crystallization; D003902:Detergents / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry; D005374:Filtration / Q000379:methods*; D006639:Histidine / Q000737:chemistry / Q000276:immunology; D008565:Membrane Proteins / Q000737:chemistry*; D009842:Oligopeptides / Q000737:chemistry / Q000276:immunology; D015221:Potassium Channels / Q000737:chemistry; D011485:Protein Binding; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry; D012995:Solubility
D020346:Aquaporins; D001426:Bacterial Proteins; D003902:Detergents; D029968:Escherichia coli Proteins; C471213:His-His-His-His-His-His; D008565:Membrane Proteins; D009842:Oligopeptides; D015221:Potassium Channels; D011994:Recombinant Proteins; C106425:aqpZ protein, E coli; C107790:prokaryotic potassium channel; D006639:Histidine