Protein Delivery into Plant Cells: Toward In vivo Structural Biology.

Cesyen Cedeño,Kris Pauwels,Peter Tompa

doi:10.3389/fpls.2017.00519

Abstract

Understanding the biologically relevant structural and functional behavior of proteins inside living plant cells is only possible through the combination of structural biology and cell biology. The state-of-the-art structural biology techniques are typically applied to molecules that are isolated from their native context. Although most experimental conditions can be easily controlled while dealing with an isolated, purified protein, a serious shortcoming of such in vitro work is that we cannot mimic the extremely complex intracellular environment in which the protein exists and functions. Therefore, it is highly desirable to investigate proteins in their natural habitat, i.e., within live cells. This is the major ambition of in-cell NMR, which aims to approach structure-function relationship under true in vivo conditions following delivery of labeled proteins into cells under physiological conditions. With a multidisciplinary approach that includes recombinant protein production, confocal fluorescence microscopy, nuclear magnetic resonance (NMR) spectroscopy and different intracellular protein delivery strategies, we explore the possibility to develop in-cell NMR studies in living plant cells. While we provide a comprehensive framework to set-up in-cell NMR, we identified the efficient intracellular introduction of isotope-labeled proteins as the major bottleneck. Based on experiments with the paradigmatic intrinsically disordered proteins (IDPs) Early Response to Dehydration protein 10 and 14, we also established the subcellular localization of ERD14 under abiotic stress.

Highlights

When we want to study the conformations of plant proteins, their interactions and their functions in their native intracellular localization, we need to rely on a combination of molecular biophysics and cell biology
It is reported that both endogenous ERD14 and ERD10 are overexpressed in planta under abiotic stress, like low temperature and drought (Hundertmark and Hincha, 2008)
We established by fluorescent fusion proteins that under our experimental conditions ERD14 and ERD10 are cytosolic proteins that do not change their intracellular localization under abiotic stress conditions

Summary

Introduction

When we want to study the conformations of plant proteins, their interactions and their functions in their native intracellular localization, we need to rely on a combination of molecular biophysics and cell biology. The conventional structural biology approaches that aim to elucidate the structure of proteins, such as X-ray crystallography and nuclear magnetic resonance (NMR), traditionally rely on samples of isolated, stable and folded proteins. These samples are the product of elaborate and sometimes tedious purification protocols. Solutionstate biomolecular NMR offers an orthogonal approach to crystallographic methods, because the final experiment is not done in solid state, but with a protein that freely diffuses in an aqueous environment. Despite sample limitations in terms of the size, solubility and stability of the protein, NMR does not provide a single structural snapshot in the solid state, rather it provides

Methods

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Conclusion