Lov-Trap: A Broadly Applicable, Genetically Encoded System to Control Protein Activity with Light through Controlled Sequestration at Membranes

Abstract

The kinetics and subcellular localization of protein activity is precisely controlled to regulate protein interactions and to create specific signaling networks. To study this important aspect of cell signaling, genetically encoded methods are being developed to control protein activity with light in living cells and animals (1-4). Here we describe a new approach that promises to be broadly applicable and is simple to apply. Through an in vitro selection from a protein domain library with more than 10∧13 unique sequences using mRNA display, we developed Zdark, a small protein based on the Z domain (5) that binds only to the dark state of the LOV2 domain from Avena sativa phototropin (Figure 1). Zdark binds to LOV2 with a Kd of ∼100nM in the dark, but shows no detectable binding upon irradiation with blue light. In our new method (dubbed LOV-TRAP for LOV trapping and release of active protein) the LOV domain is anchored at an intracellular membrane (here mitochondrial) and the protein of interest is fused to Zdark. In the dark, the Zdark-protein fusion is sequestered at a membrane where it cannot interact with its targets. Upon irradiation, Zdark-protein fusion is released rapidly (< 0.5s) and reversibly from the sequestering site. To date, we have completed caging of VAV2, Rac1 and RhoA, and shown effects of these proteins on oscillation frequencies at the cell edge. Progress with other targets will be described, as will the use of the approach to control protein dimerization. A crystal structure will be presented to illuminate the Zdark-LOV interaction.