Assessing the stability of membrane proteins to detect ligand binding using differential static light scattering.
Protein stabilization upon ligand binding has frequently been used to identify ligands for soluble proteins. Methods such as differential scanning fluorimetry (DSF) and differential static light scattering (DSLS) have been employed in the 384-well format and have been useful in identifying ligands that promote crystallization and 3D structure determination of proteins. However, finding a generic method that is applicable to membrane proteins has been a challenge as the high hydrophobicity of membrane proteins and the presence of detergents essential for their solubilization interfere with fluorescence-based detections. Here the authors used MsbA (an adenosine triphosphate binding cassette transporter), CorA (a Mg(++) channel), and CpxA (a histidine kinase) as model proteins and show that DSLS is not sensitive to the presence of detergents or protein hydrophobicity and can be used to monitor thermodenaturation of membrane proteins, assess their stability, and detect ligand binding in a 384-well format.
Structural Genomics Consortium, Toronto, Ontario, Canada.
Guillermo A
GA
Senisterra
Hamed
H
Ghanei
Galina
G
Khutoreskaya
Elena
E
Dobrovetsky
Aled M
AM
Edwards
Gilbert G
GG
Privé
Masoud
M
Vedadi
D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D001681:Biological Assay / Q000379:methods*; D003902:Detergents / Q000494:pharmacology; D029968:Escherichia coli Proteins / Q000378:metabolism; D005960:Glucosides / Q000378:metabolism; D008024:Ligands; D008027:Light; D008565:Membrane Proteins / Q000378:metabolism*; D050505:Mutant Proteins / Q000378:metabolism; D011489:Protein Denaturation / Q000187:drug effects; D011494:Protein Kinases / Q000378:metabolism; D055550:Protein Stability / Q000187:drug effects; D015203:Reproducibility of Results; D012542:Scattering, Radiation; D054852:Small Molecule Libraries / Q000032:analysis; D013696:Temperature
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D003902:Detergents; D029968:Escherichia coli Proteins; D005960:Glucosides; D008024:Ligands; D008565:Membrane Proteins; C079726:MsbA protein, Bacteria; D050505:Mutant Proteins; D054852:Small Molecule Libraries; C040358:dodecyl maltoside; D011494:Protein Kinases; C490475:CpxA protein, E coli