Abstract
Encapsulation of proteins can be beneficial for food and biomedical applications. To study their biophysical properties in complex coacervate core micelles (C3Ms), we previously encapsulated enhanced green fluorescent protein (EGFP) and its monomeric variant, mEGFP, with the cationic-neutral diblock copolymer poly(2-methyl-vinyl-pyridinium)n-b-poly(ethylene-oxide)m (P2MVPn-b-PEOm) as enveloping material. C3Ms with high packaging densities of fluorescent proteins (FPs) were obtained, resulting in a restricted orientational freedom of the protein molecules, influencing their structural and spectral properties. To address the generality of this behavior, we encapsulated seven FPs with P2MVP41-b-PEO205 and P2MVP128-b-PEO477. Dynamic light scattering and fluorescence correlation spectroscopy showed lower encapsulation efficiencies for members of the Anthozoa class (anFPs) than for Hydrozoa FPs derived from Aequorea victoria (avFPs). Far-UV CD spectra of the free FPs showed remarkable differences between avFPs and anFPs, caused by rounder barrel structures for avFPs and more elliptic ones for anFPs. These structural differences, along with the differences in charge distribution, might explain the variations in encapsulation efficiency between avFPs and anFPs. Furthermore, the avFPs remain monomeric in C3Ms with minor spectral and structural changes. In contrast, the encapsulation of anFPs gives rise to decreased quantum yields (monomeric Kusabira Orange 2 (mKO2) and Tag red fluorescent protein (TagRFP)) or to a pKa shift of the chromophore (FP variant mCherry).
Highlights
Fluorescent proteins (FPs) are nowadays indispensable in life sciences [1,2,3,4]
We investigated seven differently colored FPs: four FPs derived from Aequorea victoria green fluorescent protein (GFP) (avFPs: strongly enhanced blue fluorescent protein 2 (SBFP2), mTurquoise2, mEGFP, and strongly enhanced yellow fluorescent protein 2 (SYFP2), from class Hydrozoa) and three FPs from class Anthozoa (anFPs: monomeric Kusabira Orange 2, Tag red fluorescent protein (TagRFP), and mCherry)
We have studied the encapsulation efficiency of SBFP2, mTurquoise2, mEGFP, SYFP2, monomeric Kusabira Orange 2 (mKO2), TagRFP, and mCherry and determined their spectral and structural properties as protein free in solution and upon encapsulation with P2MVP41-b-PEO205 or P2MVP128-b-PEO477
Summary
Fluorescent proteins (FPs) are nowadays indispensable in life sciences [1,2,3,4]. The discovery of FPs started in the early 1960s with studies on the identification of the glow of jellyfish from Aequorea victoria by Osamu Shimomura [5]. There were no GFP variants with emission maxima above 527 nm [4] This limitation was overcome by cloning of GFP homologs from non-bioluminescent reef corals of the Anthozoa class [8,9,10,11]. From this class, a palette of FPs became available emitting at longer wavelengths. The number of applications of FPs has exploded, which is mainly because they can be genetically introduced into cells, tissues or whole organisms This allows using FPs for multicolor imaging and for studying protein interactions [12,13,14]. Besides using FPs as fusion tags and biosensors, they have been used as model proteins in encapsulation studies [15,16,17,18]
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