AUTHOR=Jinno Yuka , Shoda Keiko , Rial-Verde Emiliano , Yuste Rafael , Miyawaki Atsushi , Tsutsui Hidekazu TITLE=Engineering a genetically-encoded SHG chromophore by electrostatic targeting to the membrane JOURNAL=Frontiers in Molecular Neuroscience VOLUME=7 YEAR=2014 URL=https://www.frontiersin.org/journals/molecular-neuroscience/articles/10.3389/fnmol.2014.00093 DOI=10.3389/fnmol.2014.00093 ISSN=1662-5099 ABSTRACT=

Although second harmonic generation (SHG) microscopy provides unique imaging advantages for voltage imaging and other biological applications, genetically-encoded SHG chromophores remain relatively unexplored. SHG only arises from non-centrosymmetric media, so an anisotropic arrangement of chromophores is essential to provide strong SHG signals. Here, inspired by the mechanism by which K-Ras4B associates with plasma membranes, we sought to achieve asymmetric arrangements of chromophores at the membrane-cytoplasm interface using the fluorescent protein mVenus. After adding a farnesylation motif to the C-terminus of mVenus, nine amino acids composing its β-barrel surface were replaced by lysine, forming an electrostatic patch. This protein (mVe9Knus-CVIM) was efficiently targeted to the plasma membrane in a geometrically defined manner and exhibited SHG in HEK293 cells. In agreement with its design, mVe9Knus-CVIM hyperpolarizability was oriented at a small angle (~7.3°) from the membrane normal. Genetically-encoded SHG chromophores could serve as a molecular platform for imaging membrane potential.