Article

Label-free quantification of membrane-ligand interactions using backscattering interferometry

Department of Chemistry, The Scripps Research Institute, La Jolla, California, USA.
Nature Biotechnology (Impact Factor: 39.08). 03/2011; 29(4):357-60. DOI: 10.1038/nbt.1790
Source: PubMed

ABSTRACT Although membrane proteins are ubiquitous within all living organisms and represent the majority of drug targets, a general method for direct, label-free measurement of ligand binding to native membranes has not been reported. Here we show that backscattering interferometry (BSI) can accurately quantify ligand-receptor binding affinities in a variety of membrane environments. By detecting minute changes in the refractive index of a solution, BSI allows binding interactions of proteins with their ligands to be measured at picomolar concentrations. Equilibrium binding constants in the micromolar to picomolar range were obtained for small- and large-molecule interactions in both synthetic and cell-derived membranes without the use of labels or supporting substrates. The simple and low-cost hardware, high sensitivity and label-free nature of BSI should make it readily applicable to the study of many membrane-associated proteins of biochemical and pharmacological interest.

Download full-text

Full-text

Available from: Darryl J Bornhop, May 07, 2014
0 Followers
 · 
179 Views
  • Source
    • "Culture-based quantitative proteomics. It is of note that these protein-protein interactions can also be measured by label-free techniques (Baksh et al. 2011), such as backscattering interferometry, SPR spectroscopy and isothermal titration calorimetry. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Abstract Membrane proteins control fundamental processes that are inherent to nearly all forms of life such as transport of molecules, catalysis, signaling, vesicle fusion, sensing of chemical and physical stimuli from the environment, and cell-cell interactions. Membrane proteins are harbored within a non-equilibrium fluid-like environment of biological membranes that separate cellular and non-cellular environments, as well as in compartmentalized cellular organelles. One of the classes of membrane proteins that will be specifically treated in this article are transport proteins of plant origin, that facilitate material and energy transfer at the membrane boundaries. These proteins import essential nutrients, export cellular metabolites, maintain ionic and osmotic equilibriums and mediate signal transduction. The aim of this article is to report on the progress of membrane protein functional and structural relationships, with a focus on producing stable and functional proteins suitable for structural and biophysical studies. We interlink membrane protein production primarily through wheat-germ cell-free protein synthesis (WG-CFPS) with the growing repertoire of membrane mimicking environments in the form of lipids, surfactants, amphipathic surfactant polymers, liposomes and nanodiscs that keep membrane proteins soluble. It is hoped that the advancements in these fields could increase the number of elucidated structures, in particular those of plant membrane proteins, and contribute to bridging of the gap between structures of soluble and membrane proteins, the latter being comparatively low.
    Molecular Membrane Biology 01/2013; 30(3). DOI:10.3109/09687688.2012.762125 · 1.73 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: PKR (double-stranded RNA-activated protein kinase) is an important component of the innate immunity, antiviral, and apoptotic pathways. Recently, our group found that palmitate, a saturated fatty acid, is involved in apoptosis by reducing the autophosphorylation of PKR at the Thr451 residue; however, the molecular mechanism by which palmitate reduces PKR autophosphorylation is not known. Thus, we investigated how palmitate affects the phosphorylation of the PKR protein at the molecular and biophysical levels. Biochemical and computational studies show that palmitate binds to PKR, near the ATP-binding site, thereby inhibiting its autophosphorylation at Thr451 and Thr446. Mutation studies suggest that Lys296 and Asp432 in the ATP-binding site on the PKR protein are important for palmitate binding. We further confirmed that palmitate also interacts with other kinases, due to the conserved ATP-binding site. A better understanding of how palmitate interacts with the PKR protein, as well as other kinases, could shed light onto possible mechanisms by which palmitate mediates kinase signaling pathways that could have implications on the efficacy of current drug therapies that target kinases.
    Biochemistry 02/2011; 50(6):1110-9. DOI:10.1021/bi101923r · 3.01 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: An optical spectroscopy method can measure the affinities of ligands to membrane receptors that are embedded in their native membranes.
    Nature Biotechnology 04/2011; 29(4):328-9. DOI:10.1038/nbt.1820 · 39.08 Impact Factor
Show more