BSI: A Widely Applicable Molecular Interaction Platform
Prof. Darryl Bornhop, Department of Chemistry, Vanderbilt University, Nashville
While membrane-associated proteins are ubiquitous within all living organisms and represent the majority of drug targets, a general label-free method for the direct measurement of membrane-bound protein-ligand binding in the native bilayer environment has not been reported. Here it will be shown that back-scattering interferometry (BSI) is a generally applicable, label free and free solution methodology for quantifying binding affinities of membrane-associated protein-ligand interactions of biochemical and pharmacological interest, over a wide range of binding affinities. Measuring minute changes in the refractive index of a solution allowed equilibrium binding constants values to be obtained by BSI for small- and large-molecule interactions with receptors in vesicles derived from synthetic lipids and from cell extracts. These values were found to be in good agreement with those previously obtained by indirect methods. We will also show that it is possible to measure changes in interfacial and kinetic parameters on in vitro reconstituted extruded lipid vesicles and purified protein with BSI. These studies characterized the molecular mechanism of inhibition for a recently identified class of potent and isoformselective small molecules that block Phospolipase D (PLD) activity and compared activity to other known small molecule inhibitors of mammalian PLD. Our preliminary results confirm that BSI is a useful nanoscale technique to measure Ki and Kd, indicating that these new compounds disrupt interfacial binding and bulk lipid binding disruption (Ks) can be quantified with BSI.
Other unique capabilities of BSI will be presented, showing it can be used in either the tethered or free-solution mode, provides femtomolar sensitivity, shows promise for use for in vitro diagnostics, quantifies allosteric effects and can be used to measure formation, breaking and quantify hydrogen bonding strength in non-aqueous media.
1) "Label-free quantification of membrane-ligand interactions using backscattering interferometry" M. M. Baksh, et.al., Nat. Biotech., 29, 357-360, (2011). (The Economist, June 4-10, 2011)
2) “Universal Sensing by Transduction of Antibody Binding using Backscattering Interferometry” A. Kussrow, et.al., ChemBioChem, 12, 367-70, (2011).
3) “The potential of Back-scattering Interferometry as an in vitro Clinical Diagnostic Tool for the Serological Diagnosis of Infectious Disease.” A. Kussrow, et.al., Analyst, 135, 1535-7, (2010).
4) "Study of Hydrogen Binding Interactions in Acetonitrile using Back-scattering Interferometry", E. N. Presciotta, D. J. Bornhop, and R. A. Flowers, Organic Letters, 13, 2654-7 (2011).