Alteration of Membrane Protein Function Through the Photo-Activation of the Hydrophobic Probe Iodonaphtylazide

Abstract

Iodonaphtylazide (INA) has been developed 30 years ago to determine the penetration of proteins into biological membranes. Due to its very high partition coefficient into lipidic bilayers and the sensitivity of its detection, 125INA has long been used to label and identify membrane proteins, to study membrane dynamics and fusion and to detect protein-membrane interactions. The labeling of membrane proteins is mediated by the azido moiety of INA that can be activated by near UV light. Upon excitation, a nitrene radical is formed leading to the covalent binding of membrane proteins in the surroundings. Besides labeling, this binding results in specific alterations of the hydrophobic domains of proteins. When applied to enveloped viruses, the treatment resulted in a complete loss of infectivity. While the overall integrity of the virus is preserved, the ability of the viral envelope glycoprotein to promote full fusion is impaired. In the case of influenza, hemifusion was not affected by the treatment indicating a blockage at the late stage of fusion. We also tested the effect of hydrophobic labeling on the function of cellular transmembrane receptors. The lateral mobility of chemokine receptors, which are G coupled receptors, was reduced and CXCR4 lost its ability to signal in response to external stimuli. However, the activity of a tyrosine kinase receptor (IGF1) was increased. The activity of a multi drug resistance transporter MRP1 was blocked by the hydrophobic treatment. Overall, photo-activation of INA in various cell lines, including those over-expressing the multi-drug resistance transporters MRP1 or Pgp, leads to apoptosis.