Monomerization of the photoconvertible fluorescent protein SAASoti by rational mutagenesis of single amino acids

Ilya D Solovyev,Alexey I Chizhik,Jörg Enderlein,Aditya S Katti,Alexandra V Gavshina,Grigory D Lapshin,Maria G Khrenova,Leonid M Vinokurov,Ingo Gregor,Tatiana V Ivashina,Igor I Kireev,Alexander P Savitsky

doi:10.1038/s41598-018-33250-z

Abstract

Photoconvertible fluorescent proteins (PCFPs) are widely used as markers for the visualization of intracellular processes and for sub-diffraction single-molecule localization microscopy. Although wild type of a new photoconvertible fluorescent protein SAASoti tends to aggregate, we succeeded, via rational mutagenesis, to obtain variants that formed either tetramers or monomers. We compare two approaches: one is based on the structural similarity between SAASoti and Kaede, which helped us to identify a single point mutation (V127T) at the protein’s hydrophobic interface that leads to monomerization. The other is based on a chemical modification of amino groups of SAASoti with succinic anhydride, which converts the protein aggregates into monomers. Mass-spectrometric analysis helped us to identify that the modification of a single ε-amino group of lysine K145 in the strongly charged interface AB was sufficient to convert the protein into its tetrameric form. Furthermore, site-directed mutagenesis was used to generate mutants that proved to be either monomeric or tetrameric, both capable of rapid green-to-red photoconversion. This allows SAASoti to be used as a photoconvertible fluorescent marker for in vivo cell studies.

Highlights

Fluorescent proteins are widely used for the specific fluorescent labeling of cells, organelles, and individual proteins[1,2]
A monomeric FP conformation is preferable for sub-diffraction single-molecule localization microscopy, since the precise position of the fluorophore is determined by fitting the center position of its image, and a larger label size can affect the localization accuracy[24]
We decided to determine the role of these amino acid residues in the oligomerization by succinilating SAASoti amino groups with succinic anhydride, because that should mask the charge of these groups and disrupt electrostatic interaction

Summary

Introduction

Fluorescent proteins are widely used for the specific fluorescent labeling of cells, organelles, and individual proteins[1,2]. Wild type fluorescent proteins obtained from corals are prone to aggregation Expression of such proteins in cells results in the formation of fluorescent granules[11]. The overall structure of the tetramer looks like a dimer formed by dimers[16] These properties suggest a strategy for inducing monomerization of a protein by using rational mutagenesis, where the amino acids in the interface region are prime targets[8,28,29]. The first monomeric red fluorescent protein was obtained by introducing 33 mutations, 13 of them were located in the tetramer interfaces[28]. The aim of the work presented here is to determine the oligomeric state of this protein, to obtain a monomeric variant and to improve its fluorescent properties to make it useful for single-molecule localization microscopy

Methods

Results

Conclusion