Identification and Characterisation of a pH-stable GFP.

Tania Michelle Roberts,Andreas Meyer,Fabian Rudolf,Ellis Whitehead,Sven Panke,Rene Pellaux,Martin Held

doi:10.1038/srep28166

Tania Michelle Roberts, Andreas Meyer + Show 5 more

Open Access

https://doi.org/10.1038/srep28166

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Abstract

Green fluorescent proteins (GFPs) are invaluable tools for modern cell biology. Even though many properties of GFP have been successfully engineered, a GFP retaining brightness at low pH has not emerged. This limits the use of GFP in quantitative studies performed in fluctuating or acidic conditions. We report the engineering and characterisation of tandem dimer GFP (pH-tdGFP), a bright and stable GFP that can be efficiently excited and maintains its fluorescence properties in acidic conditions. Therefore, pH-tdGFP could act as a quantitative marker for cellular processes that occur at low pH, such as endocytosis, autophagy or starvation.

Highlights

Fluorescent proteins are important markers for qualitative and quantitative analysis of biological processes
We describe pH-tdGFP, a Green fluorescent proteins (GFPs) variant, whose fluorescence is stable at pH as low as 5.5
Careful examination of the identified mutations revealed that the concomitant introduction of N149Y and Q204H resulted in a pH-stable GFP and led to dimerisation of the protein

Summary

Introduction

Fluorescent proteins are important markers for qualitative and quantitative analysis of biological processes. The pH sensitivity of all GFPs (excited at 488 nm) is attributed to the protonation of the electron-rich, light-absorbing part of the chromophore, which occurs at sub-physiological pH2 This sensitivity prevents the use of GFP as a quantitative tool for monitoring acidic intracellular compartments such as lysosomes and vacuoles, organelles involved in the fundamental processes of receptor-mediated endocytosis and autophagy or localisation changes under changing cytoplasmic pH conditions. N149Y and Q204H, resulted in the greatest increase in pH and environmental stability As both amino acids lie within the dimer interface of GFP and led to the dimerisation of the variant, the resulting protein is rendered impractical as a tag for in vivo studies. We generated an intradimerising construct of two sfGFPs containing N149Y and Q204H, separated by a flexible linker, termed pH-tdGFP (pH-stable tandem dimer GFP). pH-tdGFP behaves as a monomer in vivo and in vitro, performs as predicted for in vivo applications at acidic pH and is more stable than sfGFP over the in vitro-tested pH range (3.75 to 8.50)

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