Niels Zijlstra

Nanophotonic control of the forster resonance energy transfer efficiency

We have studied the influence of the local density of optical states (LDOS) on the rate and efficiency of Förster resonance energy transfer (FRET) from a donor to an acceptor. The donors and acceptors are dye molecules that are separated by a short strand of double-stranded DNA. The LDOS is controlled by carefully positioning the FRET pairs near a mirror. We find that the energy transfer efficiency changes with LDOS, and that, in agreement with theory, the energy transfer rate is independent of the LDOS, which allows one to quantitatively control FRET systems in a new way. Our results imply a change in the characteristic Förster distance, in contrast to common lore that this distance is fixed for a given FRET pair.

Molecular composition of sub-stoichiometrically labeled α-synuclein oligomers determined by single-molecule photobleaching

Bleaching proteins: Single-molecule photobleaching approaches and sub-stoichiometric labeling with fluorophores give insight into the number of monomers that form a specific alpha-synuclein oligomer. The results show that this alpha-synuclein oligomer is present as a single, well-defined species consisting of 31 monomers.

Locally Resolved Membrane Binding Affinity of the N-Terminus of α-Synuclein

α-Synuclein is abundantly present in Lewy bodies, characteristic of Parkinsons disease. Its exact physiological role has yet to be determined, but mitochondrial membrane binding is suspected to be a key aspect of its function. Electron paramagnetic resonance spectroscopy in combination with site-directed spin labeling allowed for a locally resolved analysis of the protein-membrane binding affinity for artificial phospholipid membranes, supported by a study of binding to isolated mitochondria. The data reveal that the binding affinity of the N-terminus is nonuniform.

Elucidating the aggregation number of dopamine-induced α-synuclein oligomeric assemblies.

Conventional methods to determine the aggregation number, that is, the number of monomers per oligomer, struggle to yield reliable results for large protein aggregates, such as amyloid oligomers. We have previously demonstrated the use of a combination of single-molecule photobleaching and substoichiometric fluorescent labeling to determine the aggregation number of oligomers of human α-synuclein, implicated in Parkinsons disease. We show here that this approach is capable of accurately resolving mixtures of multiple distinct molecular species present in the same sample of dopamine-induced α-synuclein oligomers, and that we can determine the respective aggregation numbers of each species from a single histogram of bleaching steps. We found two distinct species with aggregation numbers of 15-19 monomers and 34-38 monomers. These results show that this single-molecule approach allows for the systematic study of the aggregation numbers of complex supramolecular assemblies formed under different aggregation conditions.

The number of α-synuclein proteins per vesicle gives insights into its physiological function

Although it is well established that the protein α-synuclein (αS) plays an important role in Parkinson’s disease, its physiological function remains largely unknown. It has been reported to bind membranes and to play a role in membrane remodeling processes. The mechanism by which αS remodels membranes is still debated; it may either affect its physical properties or act as a chaperone for other membrane associated proteins. To obtain insight into the role of αS in membrane remodeling we investigated the number of αS proteins associated with single small vesicles in a neuronal cell model. Using single-molecule microscopy and photo-bleaching approaches, we most frequently found 70 αS-GFPs per vesicle. Although this number is high enough to modulate physical membrane properties, it is also strikingly similar to the number of synaptobrevins, a putative interaction partner of αS, per vesicle. We therefore hypothesize a dual, synergistic role for αS in membrane remodeling.

Nanobiophotonics: Using the nanophotonics toolbox to manipulate biological fluorophores

We show that nanophotonic manipulation of biological fluorophores can alter the spectral characteristics of visible fluorescent proteins (VFP), and that the changed spectral properties quantitatively reflect the sub surface quality of photonic crystals. Nanophotonic manipulation of VFP fluorescence lifetimes gives access to the quantum efficiency of emitting states without biasing from dark states, a parameter inaccessible to conventional methods.