- Christoph A. Bücherl (1)
- Marije Aan Toorn Den (1)
- W.J.H. Berkel Van (1)
- W.J.H. Berkel van (1)
- J.W. Borst (2)
- Janwillem Borst (1)
- Sacco C. Vries De (1)
- N.J.E. Dongen Van (1)
- Wilma Esse Van (1)
- R.G. Fokkink (1)
- Adrie H. Westphal (2)
- Johannes Hohlbein (1)
- Ellard Hooiveld (1)
- J.A. Hoop de (1)
- Stefan J. Hutten (1)
- Willem J.H. Berkel van (2)
- J.M. Kleijn (4)
- Antsje Nolles (4)
- A. Nolles (2)
- Danny S. Hamers (1)
- A.J.W.G. Visser (1)
- A.H. Westphal (2)
- Jan Willem Borst (2)
FRET Reveals the Formation and Exchange Dynamics of Protein-Containing Complex Coacervate Core Micelles
Nolles, Antsje ; Hooiveld, Ellard ; Westphal, Adrie H. ; Berkel, Willem J.H. van; Kleijn, J.M. ; Borst, Jan Willem - \ 2018
Langmuir 34 (2018). - ISSN 0743-7463 - p. 12083 - 12092.
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.
Colorful packages : fluorescent proteins in complex coacervate core micelles
Nolles, Antsje - \ 2018
Wageningen University. Promotor(en): Willem van Berkel, co-promotor(en): Jan Willem Borst; Mieke Kleijn. - Wageningen : Wageningen University - ISBN 9789463437226 - 175
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.
Colorful packages : Encapsulation of fluorescent proteins in complex coacervate core micelles
Nolles, Antsje ; Westphal, Adrie H. ; Kleijn, J.M. ; Berkel, Willem J.H. van; Borst, Jan Willem - \ 2017
International Journal of Molecular Sciences 18 (2017)7. - ISSN 1661-6596
Anthozoa - Chromophore - Circular dichroism - Diblock copolymer - Dynamic light scattering - Fluorescence correlation spectroscopy - Hydrozoa - Polyelectrolyte - Protein structure - Steady-state fluorescence
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).
Encapsulation into complex coacervate core micelles promotes EGFP dimerization
Nolles, A. ; Dongen, N.J.E. Van; Westphal, A.H. ; Visser, A.J.W.G. ; Kleijn, J.M. ; Berkel, W.J.H. Van; Borst, J.W. - \ 2017
Physical Chemistry Chemical Physics 19 (2017)18. - ISSN 1463-9076 - p. 11380 - 11389.
Complex coacervate core micelles (C3Ms) are colloidal structures useful for encapsulation of biomacromolecules. We previously demonstrated that enhanced green fluorescent protein (EGFP) can be encapsulated into C3Ms using the diblock copolymer poly(2-methyl-vinyl-pyridinium)41-b-poly(ethylene-oxide)205. This packaging resulted in deviating spectroscopic features of the encapsulated EGFP molecules. Here we show that for monomeric EGFP variant (mEGFP) micellar encapsulation affects the absorption and fluorescence properties to a much lesser extent, and that changes in circular dichroism characteristics are specific for encapsulated EGFP. Time-resolved fluorescence anisotropy of encapsulated (m)EGFP established the occurrence of homo-FRET (Förster resonance energy transfer) with larger transfer correlation times in the case of EGFP. Together, these findings support that EGFP dimerizes whereas the mEGFP mainly remains as a monomer in the densely packed C3Ms. We propose that dimerization of encapsulated EGFP causes a reorientation of Glu222, resulting in a pKa shift of the chromophore, which is fully reversible after release of EGFP from the C3Ms at a high ionic strength.
Visualization of BRI1 and SERK3/BAK1 nanoclusters in Arabidopsis roots
Hutten, Stefan J. ; Hamers, Danny S. ; Toorn, Marije Aan Den; Esse, Wilma Van; Nolles, Antsje ; Bücherl, Christoph A. ; Vries, Sacco C. De; Hohlbein, Johannes ; Borst, Janwillem - \ 2017
PLoS ONE 12 (2017)1. - ISSN 1932-6203
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
Nolles, A. ; Westphal, A.H. ; Hoop, J.A. de; Fokkink, R.G. ; Kleijn, J.M. ; Berkel, W.J.H. van; Borst, J.W. - \ 2015
Biomacromolecules 16 (2015)5. - ISSN 1525-7797 - p. 1542 - 1549.
fluorescence correlation spectroscopy - protein - dynamics - behavior - nanocontainers - purification - copolymers - lipase - tag
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-b-poly(ethylene-oxide)205 and poly(2-methyl-vinyl-pyridinium)128-b-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.