Supramolecular control over protein assembly

D.A. Uhlenheuer

Research output: ThesisPhd Thesis 1 (Research TU/e / Graduation TU/e)

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Abstract

Supramolecular chemistry is the study of non-covalent interactions in and between molecules and the resulting multimolecular complexes. The interactions between molecules in biological systems, such as proteins, oligonucleotides, lipids and their complexes are supramolecular interactions. Even though the initial inspiration for novel synthetic supramolecular assemblies came from biology, it was not until only recently that supramolecular systems started to find application in the investigation and modulation of biological systems themselves. Highly relevant in this respect is the need for synthetic supramolecular systems to meet certain requirements in order to be applicable to biological systems. The applied supramolecular interaction should be bioorthogonal and selective and occur at low concentrations in aqueous medium compatible with the typically dilute concentrations of biomolecules. Furthermore, it must be possible to synthetically modify the supramolecular elements to enable their selective conjugation to target biomolecules. In the first chapter, different examples of host-guest systems as applied to the controlled assembly of proteins are discussed. In this thesis, the application of host-guest chemistry to controlled protein assembly was investigated. Cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) were used as protein model systems because of the ability to reliably monitor their dimerization in solution by fluorescence spectroscopy, as well as study their assembly on different surfaces by fluorescence microscopy. In chapters two, three and four, three different host-guest systems were explored as synthetic host-guest systems for protein assembly. The different supramolecular elements were conjugated to fluorescent proteins via expressed protein ligation. For this purpose, proteins were expressed as C-terminal thioesters using an intein-based system. The protein thioesters were then ligated to supramolecules modified with an N-terminal cysteine. In the second chapter, two sets of CFP and YFP – monomeric analogues and analogues with a weak affinity for dimerization – were functionalized with the hostguest molecules lithocholic acid and ß-cyclodextrin. The two protein pairs were studied as model systems for proteins with different intrinsic affinities in an effort to investigate the interplay between the host-guest interaction and the intrinsic protein-protein interaction. In this way, the synthetic host-guest system was shown to induce selective formation of a protein heterodimer for the monomeric analogues. In the case of the dimerizing analogues, the host-guest interactions and the intrinsic protein-protein interactions acted cooperatively, leading to heterodimer formation at lower protein concentrations. Additionally, the introduction of cysteine side chains next to the host- and guest-elements allowed for covalent locking of the supramolecular-induced protein complex through reversible disulfide bridge formation. The disassembly of the covalently-locked heterodimer could then be induced in a stepwise manner. The trajectory of the disassembly process was found to differ for the two protein pairs as a result of the interplay between the supramolecular host-guest system and the intrinsic protein affinity. The host-guest complex of lithocholic acid and ß-cyclodextrin enables induced protein heterodimerization at µM concentrations. To gain control over protein dimers at even lower concentrations, an advanced host-guest complex consisting of lithocholic acid in combination with heptakis-[6-deoxy-6-(2-aminoethylsulfanyl)]-ß-cyclodextrin was investigated in chapter three. This host-guest complex had been reported to possess a significantly higher affinity than the previously applied system with ß-cyclodextrin. For protein ligation of heptakis-[6-deoxy-6-(2- aminoethylsulfanyl)]-ß-cyclodextrin, conditions were developed for the synthesis and purification of a mono-cysteine intermediate. Expressed protein ligation to YFP and subsequent purification via ion-exchange chromatography yielded pure protein modified with the novel ß-cyclodextrin derivative. Protein dimerization of the newly modified protein with a lithocholic acid modified CFP was investigated in solution with fluorescence spectroscopy. First results indicated that the new host-guest complex is indeed applicable to supramolecular protein dimerization at submicromolar concentrations, in line with the original design. In chapter four, the use of cucurbit[8]uril as an inducer of protein heterodimerization between two modified fluorescent proteins was investigated. Cucurbit[8]uril is known to form a ternary host-guest complex with methylviologen and naphthalene derivatives. Therefore, the proteins CFP and YFP were ligated to methoxynaphthalene and methylviologen respectively and the selective formation of a protein heterodimer in the presence of cucurbit[8]uril could be observed. In addition to the normal enhanced fluorescent protein variant, a second pair of fluorescent proteins, carrying hydrophobic mutations, was investigated to study unspecific aggregation due to the methylviologen modification. In this way it could be shown that the presence of cucurbit[8]uril shields the methylviologen thereby enabling the use of the ternary host-guest complex for controlled protein assembly in solution. Chapters five and six of the thesis investigate the application of the SNAP-tag technology for the labeling of proteins with supramolecular elements. SNAP-tag labeling is compatible with intracellular labeling and labeling in cell lysates, possibly making it superior to expressed protein ligation for the use of such supramolecular systems to answer more biologically-relevant questions. First, different supramolecular ligands were attached to an O6-benzylguanine scaffold, which is required for reaction with SNAP-tag fusion proteins. Fluorescent proteins were expressed as SNAP-tag fusion proteins and then ligated to benzylguanine conjugates of lithocholic acid, ß-cyclodextrin and a supramolecular polymer. Compared to the previously used ligation technique, the SNAP-tag labeling is a fast and high yielding method for the ligation of proteins to different supramolecular elements. The last chapter deals with the application of SNAP-tag-labeled proteins for immobilization on different surfaces in collaboration with groups from the University of Twente and the Westfälische Wilhelms Universität Münster. Proteins were first conjugated to bisadamantane and a ferrocene respectively. Then the immobilization on ß-cyclodextrin surfaces and cucurbit[7]uril surfaces was studied. The binding of the functionalized proteins was influenced by the SNAP-tag fusion, such that individual optimization of binding conditions was required. Under optimized conditions, adamantane functionalized fluorescent proteins were specifically immobilized on ß-cyclodextrin surfaces and ß-cyclodextrin vesicles. Furthermore the attachment of ferrocene to a SNAP-tag fusion protein enabled immobilization on a cucurbit[7]uril surface. Taking into account the high protein labeling efficiency, the SNAP-tag is therefore a valid approach for constructing new protein modified materials.
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Biomedical Engineering
Supervisors/Advisors
  • Brunsveld, Luc, Promotor
Award date19 Sep 2011
Place of PublicationEindhoven
Publisher
Print ISBNs978-90-386-2536-2
DOIs
Publication statusPublished - 2011

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