title: Resolving the challenge of measuring ligand binding to membrane proteins by combining analytical ultracentrifugation and light scattering photometry.
abstract: Membrane proteins are attractive therapeutic targets, however the presence of detergents complicates biophysical binding measurements. Difficulties in determining quantitative dissociation constants for problematic membrane proteins were addressed by combining analytical ultracentrifugation and classical light scattering techniques. Validation of the algorithm used to calculate dissociation constants from sedimentation equilibrium experiments was demonstrated by analyzing binding data of the inhibitor Y-27632 to rho-kinase (ROCK). Kd's of 1.3 ± 0.7 and 52 ± 27 µM were calculated for ROCK constructs (S6-R415) and (M71-E379) respectively, consistent with previously published Ki's of 1.4 ± 0.1 and > 30 µM. Extension of the algorithm to membrane proteins required the collection of light scattering data to determine the partial specific volume, ν, for the membrane protein-detergent complex. Vitamin B12 binding to the bacterial protein btuB in octyl β-D-glucopyranoside (β-OG) illustrates the applicability of the method. A ν of 0.781 ml/g was determined for the btuB-β-OG complex. Incorporating this value into the algorithm generated a Kd of 7.0 ± 1.5 µM for the vitamin B12-btuB affinity. A Kd of 9.7 ± 2.7 µM was determined by equilibrium dialysis under similar experimental conditions. Successfully applying AUC to quantifying small-molecule ligand affinities to membrane proteins represents a significant advance to the field.
authors.affiliation: Vertex Pharmaceuticals Incorporated, 130 Waverly St, Cambridge, MA 02139-4211, USA. john doran@vrtx.com
authors.forename: J D
authors.initials: JD
authors.lastname: Doran
authors.forename: A K
authors.initials: AK
authors.lastname: Mohanty
authors.forename: T
authors.initials: T
authors.lastname: Fox
mesh_terms: D000465:Algorithms; D000577:Amides / Q000737:chemistry / Q000378:metabolism; D019540:Area Under Curve; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D003902:Detergents / Q000737:chemistry; D003956:Dialysis / Q000379:methods; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D005960:Glucosides / Q000737:chemistry / Q000378:metabolism; D008024:Ligands; D008027:Light; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism; D008970:Molecular Weight; D010783:Photometry / Q000379:methods*; D011485:Protein Binding; D011725:Pyridines / Q000737:chemistry / Q000378:metabolism; D012542:Scattering, Radiation; D014155:Transcobalamins / Q000737:chemistry / Q000378:metabolism; D014461:Ultracentrifugation / Q000379:methods*; D014805:Vitamin B 12 / Q000737:chemistry / Q000378:metabolism; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism
chemical_list: D000577:Amides; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; C044592:BtuB protein, E coli; D003902:Detergents; D029968:Escherichia coli Proteins; D005960:Glucosides; D008024:Ligands; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D011725:Pyridines; D014155:Transcobalamins; C108830:Y 27632; C018619:octyl-beta-D-glucoside; D054460:rho-Associated Kinases; D014805:Vitamin B 12