Colorful packages : fluorescent proteins in complex coacervate core micelles

This thesis explores the encapsulation of fluorescent proteins (FPs) into complex coacervate core micelles (C3Ms) and features the impact of this encapsulation on the biophysical properties of the FPs. In total eight different FPs were investigated originating from two different classes (Hydrozoa: SBFP2, mTurquoise2, EGFP, mEGFP, and SYFP2; and Anthozoa: mKO2, mCherry, and TagRFP), thereby covering the whole visible spectrum. As enveloping material the diblock copolymer poly(2-methyl-vinyl-pyridinium)n-b-poly(ethylene-oxide)m (P2MVPn-b-PEOm) of two different lengths (P2MVP41-b-PEO205 and P2MVP128-b-PEO477) was used. The research was focused on the formation, composition, dynamics, and stability of the FP-containing C3Ms, but it also gave us insights into the structural and spectral properties of the encapsulated FPs. We showed the successful encapsulation of about 500 EGFP molecules per C3M using both diblock copolymers. This high amount of FPs per C3M promoted dimerization of EGFP, resulting in a somewhat stronger acid character of its chromophore. By using seven other FPs, the effect of encapsulation on the structure and spectral properties of these proteins was systematically investigated. Hydrozoa FPs were more efficiently encapsulated than Anthozoa FPs, and the latter proteins were subject to di- or tetramerization in C3Ms. Finally, fast exchange dynamics of C3Ms were detected using FRET. Combining the insights presented in this thesis with sophisticated protein engineering and bioconjugation procedures, may lead in the near future to C3M-based protein nanoparticles that can be used for food and pharmaceutical applications.</p

Revealing heterogeneity in correlation times of EGFP encapsulated in complex coacervate core micelles by analysis of fluorescence anisotropies

Encapsulation of enhanced green fluorescent protein (EGFP) in complex coacervate core micelles (C3Ms) can be established by mixing EGFP with diblock polymers at equal charge ratio. It has previously been shown that this encapsulation system is highly dynamic, implying existence of different populations; GFP free in solution or complexed with polymers (small complexes) and EGFP encapsulated in C3Ms. We performed time resolved fluorescence anisotropy experiments to determine the relative populations of EGFP encapsulated in C3Ms using three different fluorescence anisotropy decay analysis methods. First, Maximum Entropy Method (MEM) data analysis was employed for five different EGFP concentrations in C3Ms that were mixed with dark fluorescent proteins (10, 20, 30, 40 and 50% EGFP, respectively). In all cases, correlation-time distributions between 0.1 and 100 ns (on a logarithmic timescale) are clearly visible showing bimodal distribution. The distribution between 0.1 and 2.0 ns is due to homo-FRET between EGFP molecules packed in micelles and the distribution between 8 and 30 ns coincides with the correlation-time distribution of free EGFP in solution. The fraction of homo-FRET distribution linearly increases with increase of relative micellar EGFP concentrations. These MEM results were corroborated by two different analysis methods: global population analysis of all five fluorescence anisotropy decays arising from EGFP in micelles together with the one of free EGFP (direct analysis of anisotropies) and global associative population analysis of anisotropies by fitting parallel and perpendicular fluorescence decay components. In contrast to global analyses approaches, the MEM method directly reveals distributions of correlation times without any prior information about the sample. However, global associative analysis of anisotropies by fitting parallel and perpendicular fluorescence decay components is the only method that allows to estimate accurately fractions of free fluorophores in solution and encapsulated fluorophores

Colorful packages : Encapsulation of fluorescent proteins in complex coacervate core micelles

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)

FRET Reveals the Formation and Exchange Dynamics of Protein-Containing Complex Coacervate Core Micelles

The encapsulation of proteins into complex coacervate core micelles (C3Ms) is of potential interest for a wide range of applications. To address the stability and dynamic properties of these polyelectrolyte complexes, combinations of cyan, yellow, and blue fluorescent proteins were encapsulated with cationic-neutral diblock copolymer poly(2-methyl-vinyl-pyridinium)128-b-poly(ethylene-oxide)477. Förster resonance energy transfer (FRET) allowed us to determine the kinetics of C3M formation and of protein exchange between C3Ms. Both processes follow first-order kinetics with relaxation times of ±100 s at low ionic strength (I = 2.5 mM). Stability studies revealed that 50% of FRET was lost at I = 20 mM, pointing to the disintegration of the C3Ms. On the basis of experimental and theoretical considerations, we propose that C3Ms relax to their final state by association and dissociation of near-neutral soluble protein-polymer complexes. To obtain protein-containing C3Ms suitable for applications, it is necessary to improve the rigidity and salt stability of these complexes.</p

Visualization of BRI1 and SERK3/BAK1 nanoclusters in Arabidopsis roots

Brassinosteroids (BRs) are plant hormones that are perceived at the plasma membrane (PM) by the ligand binding receptor BRASSINOSTEROID-INSENSITIVE1 (BRI1) and the co-receptor SOMATIC EMBRYOGENESIS RECEPTOR LIKE KINASE 3/BRI1 ASSOCIATED KINASE 1 (SERK3/BAK1). To visualize BRI1-GFP and SERK3/BAK1-mCherry in the plane of the PM, variable-angle epifluorescence microscopy (VAEM) was employed, which allows selective illumination of a thin surface layer. VAEM revealed an inhomogeneous distribution of BRI1-GFP and SERK3/BAK1-mCherry at the PM, which we attribute to the presence of distinct nanoclusters. Neither the BRI1 northeSERK3/BAK1 nanocluster density is affected by depletion of endogenous ligands or application of exogenous ligands. To reveal interacting populations of receptor complexes, we utilized selective-surface observation-fluorescence lifetime imaging microscopy (SSO-FLIM) for the detection of Forster resonance energy transfer (FRET). Using this approach, we observed hetero-oligomerisation of BRI1 and SERK3 in the nanoclusters, which did not change upon depletion of endogenous ligand or signal activation. Upon ligand application, however, the number of BRI1-SERK3/BAK1 hetero-oligomers was reduced, possibly due to endocytosis of active signalling units of BRI1-SERK3/BAK1 residing in the PM. We propose that formation of nanoclusters in the plant PM is subjected to biophysical restraints, while the stoichiometry of receptors inside these nanoclusters is variable and important for signal transduction

Encapsulation of GFP in Complex Coacervate Core Micelles

Protein encapsulation with polymers has a high potential for drug delivery, enzyme protection and stabilization. Formation of such structures can be achieved by the use of polyelectrolytes to generate so-called complex coacervate core micelles (C3Ms). Here, encapsulation of enhanced green fluorescent protein (EGFP) was investigated using a cationic-neutral diblock copolymer of two different sizes: poly­(2-methyl-vinyl-pyridinium)₄₁-<i>b</i>-poly­(ethylene-oxide)₂₀₅ and poly­(2-methyl-vinyl-pyridinium)₁₂₈-<i>b</i>-poly­(ethylene-oxide)₄₇₇. Dynamic light scattering and fluorescence correlation spectroscopy (FCS) revealed a preferred micellar composition (PMC) with a positive charge composition of 0.65 for both diblock copolymers and micellar hydrodynamic radii of approximately 34 nm. FCS data show that at the PMC, C3Ms are formed above 100 nM EGFP, independent of polymer length. Mixtures of EGFP and nonfluorescent GFP were used to quantify the amount of GFP molecules per C3M, resulting in approximately 450 GFPs encapsulated per micelle. This study shows that FCS can be successfully applied for the characterization of protein-containing C3Ms

VAEM reveals a heterogeneous distribution of BRI1-GFP and SERK3-mCherry in the PM.

<p>Typical VAEM images of live root epidermal cells of 6 day old <i>A</i>. <i>thaliana</i> seedlings showing PM distribution of (<b>A</b>) BRI-GFP line1, (<b>B</b>) BRI1-GFP line 2 and (<b>C</b>) SERK3-mCherry, (<b>D</b>) BRI1-GFP in a <i>serk1serk3</i> mutant plant. Images taken are of epidermal cells in the early elongation zone. Scale bars represent 10 μm.</p

Quantification of clusters/ μm² and cluster size of BRI1 and SERK3.

<p>Quantification of clusters/ μm² and cluster size of BRI1 and SERK3.</p

BRI1-GFP nanoclusters imaged using VAEM and SSO-confocal imaging.

<p>BRI1-GFP line 2 expressed in root epidermal cells imaged using VAEM (A) or SSO-confocal imaging (B). Both imaging modalities show similar nanocluster distributions. Scale bars represent 10 μm.</p