Microscale fluorescent thermal stability assay for membrane proteins.
Systematic efforts to understand membrane protein stability under a variety of different solution conditions are not widely available for membrane proteins, mainly due to technical problems stemming from the presence of detergents necessary to keep the proteins in the solubilized state and the background that such detergents usually generate during biophysical characterization. In this report, we introduce an efficient microscale fluorescent stability screen using the thiol-specific fluorochrome N-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]maleimide (CPM) for stability profiling of membrane proteins under different solution and ligand conditions. The screen uses the chemical reactivity of the native cysteines embedded in the protein interior as a sensor for the overall integrity of the folded state. The thermal information gained by thorough investigation of the protein stability landscape can be effectively used to guide purification and biophysical characterization efforts including crystallization. To evaluate the method, three different protein families were analyzed, including the Apelin G protein-coupled receptor (APJ).
Alexandrov
Alexander I
AI
Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Mileni
Mauro
M
Chien
Ellen Y T
EY
Hanson
Michael A
MA
Stevens
Raymond C
RC
D000075244:Apelin Receptors; D002021:Buffers; D003902:Detergents / Q000494:pharmacology; D005453:Fluorescence; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D008954:Models, Biological; D008958:Models, Molecular; D009994:Osmolar Concentration; D011489:Protein Denaturation; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000378:metabolism; D013501:Surface-Active Agents / Q000737:chemistry; D013816:Thermodynamics; D044366:Transition Temperature
C104575:APLNR protein, human; D000075244:Apelin Receptors; D002021:Buffers; D003902:Detergents; D008565:Membrane Proteins; D043562:Receptors, G-Protein-Coupled; D013501:Surface-Active Agents