Engineering color variants of green fluorescent protein (GFP) for thermostability, pH-sensitivity, and improved folding kinetics

Abstract

A number of studies have been conducted to improve chromophore maturation, folding kinetics, thermostability, and other traits of green fluorescent protein (GFP). However, no specific work aimed at improving the thermostability of the yellow fluorescent protein (YFP) and of the pH-sensitive, yet thermostable color variants of GFP has so far been done. The protein variants reported in this study were improved through rational multiple site-directed mutagenesis of GFP (ASV) by introducing up to ten point mutations including the mutations near and at the chromophore region. Therefore, we report the development and characterization of fast folder and thermo-tolerant green variant (FF-GFP), and a fast folder thermostable yellow fluorescent protein (FFTS-YFP) endowed with remarkably improved thermostability and folding kinetics. We demonstrate that the fluorescence intensity of this yellow variant is not affected by heating at 75 °C. Moreover, we have developed a pH-unresponsive cyan variant AcS-CFP, which has potential use as part of in vivo imaging irrespective of intracellular pH. The combined improved properties make these fluorescent variants ideal tools to study protein expression and function under different pH environments, in mesophiles and thermophiles. Furthermore, coupling of the FFTS-YFP and AcS-CFP could potentially serve as an ideal tool to perform functional analysis of live cells by multicolor labeling.

Folding latency of fluorescent proteins affects the mitochondrial localization of fusion proteins

“…It might be possible that once fast-folding FPs are folded, they become stable in the folded state and are resistant to unfolding by the mitochondrial protein import machinery. Indeed, it has been reported that GFP39N (F99S/M153T/V163A) displays higher thermostability than wild-type aqGFP (Aliye et al, 2015). Another possibility is that folded FPs prevent the binding of FFPs to mitochondrial-targeting factors or chaperones in the cytosol, which guide them to the mitochondrial surface.…”

Novel Thermostable Flavin‐binding Fluorescent Proteins from Thermophilic Organisms

“…However, some recent studies reported the development of more thermostable derivatives of GFP and also LOV‐based fluorescent proteins. For example, Aliye and coworkers developed a fast folding and thermostable YFP (FFTS‐YFP) that is stable at 75°C in vitro . Kiss et al .…”

Enhancing the thermal tolerance of a cis-epoxysuccinate hydrolase via combining directed evolution with various semi-rational redesign methods

“…To manipulate proteins more efficiently, rational design was invented. Rational design involves creating new molecules with certain functionalities by predicting how the molecule's structure will affect its behavior through physical models [12]. Although generally considered fast and accurate in modifying proteins, rational design can be more painstaking than directed evolution.…”