Luminescence resonance energy transfer between genetically encoded donor and acceptor for protein-protein interaction studies in the molecular chaperone HSP70/HSP90 complexes

Kaushik Bhattacharya,Lilia Bernasconi,Didier Picard

doi:10.1038/s41598-018-21210-6

Kaushik Bhattacharya, Lilia Bernasconi + Show 1 more

Open Access

https://doi.org/10.1038/s41598-018-21210-6

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Journal: Scientific Reports	Publication Date: Feb 12, 2018
Citations: 18	License type: open-access

Affiliation: University of Geneva

Abstract

Complex patterns of protein-protein interactions (PPInts) are involved in almost all cellular processes. This has stimulated the development of a wide range of methods to characterize PPInts in detail. Methods with fluorescence resonance energy transfer can be technically challenging and suffer from several limitations, which could be overcome by switching to luminescence resonance energy transfer (LRET) with lanthanide ions such as Tb3+. With LRET, energy transfer between PPInt partners works over a larger distance and with less topological constraints; moreover, the long-lived luminescence of lanthanides allows one to bypass the short-lived background fluorescence. We have developed a novel LRET method to investigate PPInts between partners expressed as fusion proteins with genetically encoded donor and acceptor moieties. Upon UV excitation of a tryptophan within a lanthanide binding peptide, the Tb3+ luminescence is harnessed to excite either a green or a red fluorescent protein. We demonstrate the usefulness of the LRET assay by applying it to analyze the interactions of the molecular chaperones HSP70 and HSP90 with their common co-chaperone HOP/Sti1. We recapitulate the previously described interaction specificities between the HSP70/HSP90 C-termini and tetratricopeptide repeat domains of HOP/Sti1 and demonstrate the impact of single point mutants on domain-domain interactions.

Highlights

A detailed knowledge of protein complexes and protein-protein interactions (PPInts) is essential to understand cellular processes
For luminescence resonance energy transfer (LRET) assays, the lanthanide luminescence can be induced with UV at 280 nm because of a properly positioned tryptophan residue in the lanthanide binding polypeptide tag (LBT)
To establish a novel flexible method based on LRET to analyze PPInts in vitro, we set out to develop the appropriate toolbox

Summary

Introduction

A detailed knowledge of protein complexes and protein-protein interactions (PPInts) is essential to understand cellular processes. Tb3+ may not passively cross mammalian cell membranes and apparently cannot use a specific ion channel either This particular version of the technique with LBTs has so far only been applied to mammalian cells to investigate interactions outside of the cells, notably the structural rearrangements of a Na+/K+ ATPase pump and of a large-conductance Ca2+-and voltage-activated K+ channel with fluorescently labeled ouabain and iberiotoxin as acceptors, respectively[11,12]. Similar experiments have been done in bacterial cells to study the interaction between membrane-bound oligosaccharyl transferase and its substrates[13] These proof of concept experiments had established LBTs as useful components of LRET assays, but they had not yet been combined with fluorescent proteins as acceptors to investigate PPInts of full-length proteins or individual domains. HOP, known as STIP1 in mammals, is a co-chaperone, which acts as an adaptor molecule binding HSP90 and HSP70 simultaneously, and thereby facilitating substrate transfer from HSP70 to HSP90 in the process of protein folding and maturation[22,23,24,25]

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