Resolving the Challenge of Measuring Ligand Binding to Membrane Proteins by Combining Analytical Ultracentrifugation and Light Scattering Photometry

• Published in: Journal of pharmaceutical sciences 2012-01, Vol.101 (1), p.92-101
• Main Authors: Doran, J.D., Mohanty, A.K., Fox, T.
• Format: Article
• Language: English
• Subjects: Algorithms; Amides - chemistry; Amides - metabolism; analytical ultracentrifugation; Area Under Curve; Bacterial Outer Membrane Proteins - chemistry; Bacterial Outer Membrane Proteins - metabolism; Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; Biological and medical sciences; Detergents - chemistry; Dialysis - methods; dissociation constants; Escherichia coli - metabolism; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - metabolism; General pharmacology; Glucosides - chemistry; Glucosides - metabolism; kinetics; Ligands; Light; light-scattering (static); Medical sciences; membrane proteins; Membrane Proteins - chemistry; Membrane Proteins - metabolism; Membrane Transport Proteins - chemistry; Membrane Transport Proteins - metabolism; micelles; Molecular Weight; Pharmaceutical technology. Pharmaceutical industry; Pharmacology. Drug treatments; Photometry - methods; Protein Binding; Pyridines - chemistry; Pyridines - metabolism; refractive index; rho-Associated Kinases - antagonists & inhibitors; rho-Associated Kinases - chemistry; rho-Associated Kinases - metabolism; Scattering, Radiation; small-molecule ligand binding; Transcobalamins - chemistry; Transcobalamins - metabolism; Ultracentrifugation - methods; Vitamin B 12 - chemistry; Vitamin B 12 - metabolism
• ISSN: 0022-3549; 1520-6017
• DOI: 10.1002/jps.22755

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. © 2011 Wiley Periodicals, Inc. and the American Pharmacists Association.